Novel Approaches to Investigate Behaviors of
Bacteria by Atomic Force Microscopy and
Circulating Tumor Cells through Microfluidics
AMHh
~MASSACHUSETTS
INS
OF TECHNOLOGy
by
MAY 1E2014
David Steven Gray
LIBRARIES
S.B. Materials Science and Engineering
MIT, 2006
Submitted to the Department of Materials Science and Engineering
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2014
@ Massachusetts Institute of Technology 2014. All rights reserved
Signature redacted
Signature or Author
Department of Materials Science and Engineering
17ebruair" 1A 9014
Signature redacted
C ertifie d by ..............,1....... I........
r .. ....
............................ ........... ...
........... ,................... n. ..
...h...
.
Angela Belcher
W. M. Keck Professor of Materials Science and Engineering
and Biological Engineering
Accepted by..................
.Signature
redacted
Gerbrand Ceder
Chair, Departmental Committee on Graduate Students
E
Novel Approaches to Investigate Behaviors of
Bacteria by Atomic Force Microscopy and
Circulating Tumor Cells through Microfluidics
by
David Steven Gray
Submitted to the Department of Materials Science and Engineering
on February 14, 2014 in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy in Materials Science and Engineering
ABSTRACT
The adaptability and apparent ingenuity of renegade and intruding cells within the human
body present formidable challenges in warding off disease. As the longevity of humans
increases, cancer will afflict greater numbers, and if bacteria continue to grow resistant to
conventional antibiotics, new treatment approaches will need to be identified. Through the
use of two types of advanced instrumentation, a high-speed atomic force microscope (AFM)
and microfluidic devices, further insights into behaviors of bacteria and cancer cells were
sought, respectively. Although involving very different types of cells, the projects were
characterized by overarching similarities, including the aim of studying the cells at the
individual level and the need to attach the cells to a substrate to accomplish this. Ultimately,
these studies uncovered phenomenon that without the AFM and microfluidics may have
gone unnoticed. Specifically, a new, possible two-phase response of bacteria to an
antimicrobial peptide (AmP) was discovered by high-speed AFM, and very large clusters of
circulating tumor cells (CTCs) with platelets were captured on the microfluidic device albeit the mechanism by which this happens remains to be determined. These insights
were the result of seeking to understand the response of E. coli to CM15, a particular AmP,
and attempting to isolate platelet-CTC complexes with a herringbone microfluidic device
functionalized with antibodies that bind to surface markers on activated platelets.
Thesis Supervisor: Angela Belcher, PhD
Title: W. M. Keck Professor of Materials Science and Engineering and Biological Engineering
2
Acknowledgements
"... 0 Thou Who hast cast Thy splendor over the luminous realities of men, shedding upon
them the resplendent lights of knowledge and guidance, and hast chosen them out of all
created things for this supernal grace, and hast caused them to encompass all things, to
understand their inmost essence, and to disclose their mysteries, bringing them forth out of
darkness into the visible world..."
-'Abdu'l-Bahi
3
Chapter Contents
Chapter 1. Introduction...................................................................................................................15
1.1. Broad Overview ......................................................................................................................
1.2. Thesis Chapters.......................................................................................................................16
1.2.1.
1.2.2.
1.2.3.
1.2.4.
1.2.5.
Chapter 2.
Chapter 2...........................................................................................................................16
Chapter 3...........................................................................................................................17
Chapter 4...........................................................................................................................17
Chapter 5...........................................................................................................................17
Chapter 6...........................................................................................................................18
Significance of Circulating Tumor Cells and Platelets ...........................
2.1. Introduction..............................................................................................................................19
2.2. Cancer Biology.........................................................................................................................20
2.2.1. Types of Cancer ..............................................................................................................
2.2.2. Progression ......................................................................................................................
2.3. Significance of Early Cancer Diagnosis and Constant Monitoring of its
Evolution................................................................................................................................................24
2.4. Traditional Cancer Diagnostics ..................................................................................
2.5. Circulating Tum or Cells in the Context of Treatm ent.........................................
2.6. Approaches for Capture of CTCs...............................................................................
2.7. Significance of Platelets in Metastasis......................................................................
2.7.1. Features of Hum an Platelets...............................................................................
2.7.2. Interaction of Platelets with Circulating Tum or Cells ..............................
2.8. Motivation to Isolate CTCs by Means of Platelets ..............................................
2.8.1. The Epithelial-Mesenchymal Transition Has Been Implicated in
M etastasis .........................................................................................................................................
2.8.2. Signaling from Platelets to Cancer Cells Results in an EpithelialM esenchym al-Like (EMT-Like) Transition....................................................................
Chapter 3.
15
19
20
20
27
29
30
31
31
34
36
37
41
A Protocol to Produce Platelet-Tumor Cell Complexes..........................43
3.1. Introduction..............................................................................................................................43
3.2. Com ponents..............................................................................................................................43
3.2.1. Platelets..............................................................................................................................43
44
3.2.2. Cancer Cells ......................................................................................................................
3.3. Protocol to Form Complexes of Platelets and Cancer Cells...........44
45
3.3.1. Label Platelets with Calcein and Add to Cancer Cells ..............................
Cell
3.3.2. Label Platelets with Antibodies after Forming Platelet-Tumor
Com plexes.........................................................................................................................................45
3.4. Analysis by Fluorescence Microscopy and Fluorescence-Activated Cell
Sorting (FACS)......................................................................................................................................46
3.5. Results.........................................................................................................................................46
4
3.5.1. Verification of the Formation of Complexes Consisting of LS180s and
Platelets by Fluorescence Microscopy and FACS.......................................................
47
3.5.2. Verification of Complexes of 4T1s and Platelets by Fluorescence
M icro scop y .......................................................................................................................................
50
3.6. Significance of Results....................................................................................................
51
Chapter 4. Initial Approaches to Capture Platelet-Tumor Cell Complexes.......... 56
4 .1. In trodu ction ..............................................................................................................................
56
56
4.2. Magnetic Activated Cell Sorting (MACS)................................................................
56
4.2.1. Background on MACS..............................................................................................
4.2.2. MACS for Capturing Cancer Cells with Platelets ........................................
57
4.3. Protocol to Isolate Tumor Cells with Platelets Using MACS ........................... 59
60
4 .4. R esu lts .........................................................................................................................................
4.4.1. Apparent Enrichment of Platelet-Cancer Cell Complexes...................... 60
4.5. Limitations of MACS of a Technique to Capture Platelet-CTC Complexes...62
4.6. Interactions and Isolation of Platelet-Tumor Cell Complexes with Flow
63
C ham b er .................................................................................................................................................
4.6.1. B ackgroun d ......................................................................................................................
63
4.7. Rolling and Binding of Tumor Cells with Platelets on Functionalized
64
Substrates with Flow Chamber .............................................................................................
4.7.1. Cancer Cell Line, Whole Blood, and Formation of Platelet-Tumor Cell
C om plexes.........................................................................................................................................64
4.7.2. Patterning PSGL-1 on Petri Dish for Testing with Flow Chamber............64
4.7.3. Patterning PSGL-1 Asymmetrically on Gold-Coated Glass Slide for
65
Testing with Flow Chamber.................................................................................................
66
4.7.4. Instrum entation .............................................................................................................
6
4 .8 . R esu lts.........................................................................................................................................6
4.8.1. Interaction between Platelet-Tumor Cell Complexes on Uniformly
66
Coated PSG L-1 Surface ................................................................................................................
4.8.2. Attempt to Separate Complexes by Rolling on Asymmetrically Patterned
Slides 67
67
4.9. Summary of Initial Two Approaches ......................................................................
68
4.10. Significance of Results .................................................................................................
Chapter 5. Capturing CTCs with Platelets with Herringbone Microfluidic Device 72
72
5 .1. In tro d u ction ..............................................................................................................................
5.2. Rationale for Use of Microfluidics for CTC Capture............................................ 73
73
5.2.1. Description of Microfluidics and Applications............................................
5.2.2. Development of Microfluidic Devices Leading to and Including the
73
Capability of CTC Separation.............................................................................................
5.2.3. Seeming Limitations Associated with CTC Capture with Microfluidics
Are Not Insurm ountable.............................................................................................................77
5.3. Advantages of Herringbone Architecture for CTC Capture............................. 78
79
5 .4 . A pp ro ach ....................................................................................................................................
5
79
Experimental System ..............................................................................................
Testing Capture with Cancer Cells and Platelets in Buffer .................... 81
Testing Capture with Spiked Platelet-Cancer Cell Complexes into Blood
82
82
5.4.4. Testing Capture with Samples from Mouse Models .............
3
5 .5 . R esu lts.........................................................................................................................................8
5.5.1. Comparison of Antibodies (Anti-CD63 and Anti-CD62) - Relative
84
Binding With and Without Platelets...............................................................................
5.5.2. Capture Efficiency of 4T1-Platelet Complexes in Whole Blood With
84
Herringbone Device and Purity........................................................................................
5.5.3. Capture of CTCs in Blood from Mice with Tumors..............86
5.5.4. Capture of CTCs and Clusters Ranging Significantly in Size and Staining
Positively for Platelet Markers on Control and Standard Device ........................ 95
5.6. Sum m ary of Results...............................................................................................................99
5.7. Reflections and Outlook on Capturing CTCs by Means of Platelets............. 99
5.7.1. Implications of the Capture of Very Large Clusters ........................................ 99
100
5.7.2. Possible Mechanism of Capture on Control Device .............
5.7.3. Sensitivity of Capture................................................................................................100
5.4.1.
5.4.2.
5.4.3.
Chapter 6. Imaging Bacteria Treated with an Antimicrobial Peptide by High-Speed
107
Atomic Force Microscopy .................................................................................................................
107
6.1. Introdu ction ...........................................................................................................................
6.2. Atomic Force Microscopy (AFM)..................................................................................107
6.2.1. AFM Operating Principles.......................................................................................108
6.2.2. AFM Components and Functions.........................................................................108
6.2.3. Development of the AFM to Image Soft Materials........................................111
11
6 .2 .4. Lim itation s.....................................................................................................................1
112
6.2.5. H igh-speed A FM ..........................................................................................................
6.3. Antimicrobial Peptides (AmPs).....................................................................................114
6.3 .1. Sign ifican ce....................................................................................................................114
115
6.3.2. M odes of A ction ...........................................................................................................
6.3.3. Size, Structure, and Composition of AmPs.......................................................115
116
6.3.4. Frontier of Learning about AmPs ........................................................................
116
6.4. Experimental Details .........................................................................................................
and
for
Imaging
on
Slide
Attachment
and
6.4.1. Preparation of Bacteria
116
Interaction w ith Am P ................................................................................................................
6.4.2. Description and Handling of CM15.....................................................................117
6.4.3. Imaging with High-Speed AFM and Fluid Cell................................................117
6.4.4. Correlating Bacterial Surface Corrugation with Cell Death......................118
118
6.4.5. Bulk Cell Killing Assay ..............................................................................................
Peptides.....................................................................118
Control
with
Comparison
6.4.6.
18
6 .5 . An alysis....................................................................................................................................1
6.5.1. Phase Data as Choice for Evaluating Bacterial Surface Changes............118
6.5.2. Quantifying CM15 Kinetics on E. Coli.................................................................119
19
6 .6. R esults......................................................................................................................................1
6
6.6.1. Addition of CM15 Changes E. Coli from Smooth to Corrugated with
Variation of Onset of Roughening........................................................................................119
6.6.2. Effects on E. Coli from Control Peptide and Conventional Antibiotic
120
Com pared to CM 15 ....................................................................................................................
6.6.3. Connection between Corrugation and Cell Death.........................................121
6.6.4. Quantification of Cell Surface Corrugation Before and After Addition of
CM15 122
6.6.5. Assay on Bulk Cell Killing with CM15 and E. Coli..........................................124
1 24
6 .7 . D iscussion ...............................................................................................................................
7
List of Figures
Figure 1. Sites of Metastases from Cancer Types Causing Most Deaths in the United
States. From recent cancer statistics in the United States, the following types of
cancer result in the greatest number of deaths: lung cancer, breast cancer, colon
cancer, pancreatic cancer, melanoma, and ovarian cancer. Shown are common
23
sites of metastasis from these types of cancer. ...........................................................
Figure 2. Therapeutics for Tumors with Particular Genetic Mutations. Drugs listed
in this image have been found more efficacious in treating various types of
cancer characterized by the presence of absence of specific mutations.........26
Figure 3. Schematic of the Internal Structure of a Human Blood Platelet. In the
resting state, platelets are shaped as disks. In their alpha and dense granules
are stored numerous types of agents that can be released in response to the
environ m ent....................................................................................................................................32
Figure 4. Aggregation of Platelets Upon Addition of ADP. Platelet rich human blood
plasma (left) forms white flakes (right) after the addition of ADP, which
activates platelets and causes them to aggregate. A magnetic stirrer is at the
bottom of b oth vials......................................................................................................................34
Figure 5. Label Platelets with Calcein and Add to Cancer Cells. After isolating
platelets from whole blood through centrifugation, they can be labeled with
calcein AM, which esterases in the platelets convert to calcein, producing
45
fluorescence with a peak emission at 515 nm .............................................................
Figure 6. Label with Antibody After Adding Platelets to Cancer Cells. After isolating
platelets from whole blood through centrifugation and incubated with tumor
cells, they can be labeled with antibodies and imaged or analyzed by flow
46
cy to m etry..........................................................................................................................................
Figure 7. Fluorescence Microscopy with Platelets Labeled with Calcein and Tumor
Cells. Platelets labeled with calcein were incubated with LS180s grown in a
flask and subsequently imaged by fluorescence microscopy to qualitatively
47
determ ine binding. (Scale Bar: 20 pm) ...........................................................................
Figure 8. Verification of Platelet-Tumor Cell Formation by Fluorescent Microscopy.
Tumor cells are labeled with CellTrackerTM Red (red), and platelets are stained
with calcein (green). The overlap of these two colors is indicated in yellow. The
bright-field image to the left without color was captured from the same position
48
as the composite picture. (Scale Bar: 20 im)............................................................
Figure 9. Fluorescence Microscopy of Platelets Labeled with Anti-P-Selectin PE and
Tumor Cells. This composite image of platelets, LS180s, and anti-P-selectin PE
antibody (orange) further supports the conclusion that activated platelets are
49
binding to tum or cells. (Scale Bar: 20 pm ) ..................................................................
Figure 10. FACS with Platelets Labeled with Anti-P-Selectin PE and Tumor Cells.
Performing FACS with tumor cells and platelets and labeled with anti-P-selectin
PE provided further support that washed platelets bind to LS180s with the
protocol developed herein. Three different populations of tumor cells and
platelets were processed labeled and unlabeled to semi-quantitatively measure
the degree of binding. As an approximation for quantification, any events
8
falling outside of the green polygon in the FACS plot surrounding 98.3% of the
events from the sample of LS180s labeled with CellTracker GreenTM were
50
regarded as positive for PE. ...............................................................................................
PE
the
anti-P-selectin
of
The
presence
to
4T1.
Platelets
of
11.
Binding
Figure
antibody (red) in the fluorescence microscopy image at the top right provides
evidence that washed platelets isolated from whole blood bind to 4T1s (green)
in buffer, especially in comparison to the three images also shown, which
51
served as controls. (Scale Bar: 50 pm )...........................................................................
Figure 12. Attaching MicroBeads to Platelets Bound to Tumor Cells. Anti-PE
MicroBeads bind to antibodies conjugated with PE. Thus, the anti-P-selectin PE
antibody that targets activated platelets provides a site for the MicroBeads to
58
attach to the platelet-tumor cell complexes................................................................
Figure 13. Performing MACS and Collecting Effluent at Each Stage. The column is
placed in the MACS Separator where its magnetic field is concentrated. The cell
solution containing both labeled and unlabeled cells is added in the first step,
and the effluent is collected as the initial "flow through". The effluent from the
next three washes is either collected in a single tube or separate tubes
(pictured) for analysis yielding the "wash I", "wash II", and "wash III" samples.
In the final step, a plunger is used to forcefully expel the contents of the column
now removed from the MACS Separator, and this "collection" sample is
expected to contain the cells labeled with MicroBeads..........................................60
Figure 14. Fluorescence Microscopy with Platelets Labeled with Anti-P-Selectin PE
and Tumor Cells After Addition of MicroBeads. After forming platelet-tumor
cell complexes, adding anti-P-selectin PE, and MicroBeads that attach to PE, the
resulting sample was imaged by fluorescence microscopy to determine that the
addition of MicroBeads does not diminish the signal and the antibody remains
61
attached to its target. (Scale Bar: 20 pm )......................................................................
Figure 15. Fluorescence Microscopy of Effluents from MACS with Platelets Labeled
with Anti-P-Selectin PE and Tumor Cells. While performing MACS on platelettumor cell complexes labeled with anti-P-selectin and MicroBeads, effluent
from each step outlined in Figure 13. The PE signal was observed only in the
collection step, providing evidence that MACS could be used to isolate platelettumor cell complexes with P-selectin on the platelets as a marker. (Scale Bar:
62
2 0 p m ) ................................................................................................................................................
Figure 16. Configuration of Flow Chamber on Petri Dish to Test Interaction with
PSGL-1 Fc, Protein A, and a Combination Thereof. LS180s with and without
platelets were passed through a flow chamber placed on a petri dish that was
functionalized in separate regions with PSGL-1 Fc, Protein A, and a combination
thereof. A region of the petri dish that was not functionalized with these
molecules was also observed during flow to determine the degree of nonsp ecific interactions......................................................................................................................65
Figure 17. Schematic Depiction of Functionalization of PSGL-1 Fc on Gold-Coated
Glass Slide. By covalently attaching Protein A to a gold surface, PSGL-1 Fc can
also bind in an oriented fashion. The intent was to make available the PSGL-1
portion of the chimera for interaction with P-selectin displayed on activated
platelets, which were expected to be bound to tumor cells in solution...........66
9
Figure 18. Optical Microscopy Images Showing Rolling and Attachment of Tumor
Cells Coated with Platelets and No Apparent Interaction Without Platelets.
Tumor cells in clusters of various sizes roll and attach to a PSGL-1 coated
substrate (left) and appear not to interact in the absence of platelets but with
the same type of functionalized surface as evidenced by blurred objects in the
image indicating cells in motion (right). (Scale Bar: 20 pLm).................................67
Figure 19. Functionalization Chemistry to Coat Microfluidic Channels with
Biotinylated Antibody. Silane, N-Succinimidyl 4-maleimidobutyrate, and
NeutrAvidin were added sequentially to provide a binding site for biotinylated
antibodies. It should be noted that there is not necessarily certainty about the
location of biotin after conjugating it to antibodies for attachment to
NeutrAvidin, which means the site interacting with its target may not be
exposed as depicted above ...................................................................................................
80
Figure 20. Configuration of Herringbone Devices in Series or Parallel. Sample from
one syringe flows through several devices in series in the top two photographs
from an experiment. In the bottom pair of photographs, multiple syringes are
connected to the same number of devices. Each device is approximately 2.5 cm
w ide and 7.5 cm long....................................................................................................................81
Figure 21. Comparison of Anti-CD63 and Anti-CD62 Antibodies and Relative
Binding with and without Platelets. Four samples were processed through
microfluidic devices functionalized with either anti-CD63 or anti-CD62P
antibodies, and each pair of samples contained 4T1s with and without platelets.
The results from this experiment indicate higher capture in the presence of
platelets and many more cells isolated with anti-CD63 compared to anti-CD62P
an tib o d ies..........................................................................................................................................8
4
Figure 22. 4T1-Platelet Complexes in Whole Blood Captured with a Herringbone
Device. Blood spiked with platelet-4T1 complexes was processed in
herringbone devices, and 4Tls (FITC) were counted along with others captured
on the device labeled with Hoescht (DAPI) to measure the purity of capture.
(Scale B ar: 5 0 im ).........................................................................................................................8
5
Figure 23. Individual Images of Devices that Captured Cells from Blood from Mouse
with Tumor Formed After Mammary Pad Injection. Blood was processed in a
series of microfluidic devices from a mouse that had been injected with 4T1
cells in the mammary pad. The first device through which the blood flowed was
functionalized with an anti-CD63 antibody, and cells positive for ZsGreen, such
as shown in these images, were captured. (Scale Bar: 50 pLm)............................87
Figure 24. Order of Microfluidic Devices Each Functionalized with a Different
Antibody. Microfluidic devices were connected to the input and output and to
each other in series. The antibodies used in this experiment were anti-CD63,
anti-CD62P, and anti-EpCAM, and the devices coated with these antibodies
88
w ere ordered respectively....................................................................................................
Figure 25. Individual Images of Devices with Captured Cells from Blood from Mouse
with Tumor Formed after Tail Vein Injection. Multiple images from the same
location of the device coated with anti-CD63 were recorded using different
filters (in parentheses in the label below each image) to detect the presence of
10
antibodies (anti-EpCAM PE and anti-CD45 APC) and dyes (Hoescht and
89
ZsGreen). (Scale Bar: 50 pm ).............................................................................................
Figure 26. Composite Images of Anti-CD63 Device that Captured Cells from Blood
from Mouse with Tumor Formed After Tail Vein Injection. Several composite
images from different locations of the device coated with anti-CD63 were
captured at two different levels of magnification (10x and 20x) after blood from
a mouse that had 4T1 cancer cells injected weeks earlier through the tail vein.
In all of these images, blue represents signal from the DAPI channel (Hoescht)
and green from the FITC channel (ZsGreen), while light blue indicates the
overlap of the two signals. Red in the smaller two images on the right side
corresponds to the Cy5 channel (anti-CD45 APC), which as seen in Figure 25
was minimal in signal for that particular image and which was not included in
creating the composite image on the left side above. (Scale Bar: 50 p1m)............90
Figure 27. Composite Images of Anti-CD63 and Anti-EpCam Devices that Captured
Cells from Blood from Mouse with Tumors Formed After Tail Vein Injection.
Images from different locations on the devices coated with anti-CD62 and antiEpCAM antibodies, which were downstream from the anti-CD63 coated device
shown in Figure 26. In all of these images, blue represents signal from the DAPI
channel (Hoescht), green from the FITC channel (ZsGreen), and red from the
91
anti-CD45 APC antibody (Cy5). (Scale Bar: 20 pm).................................................
Tail
in
the
Injections
Mice
after
of
Figure 28. In Vivo Imaging System (IVIS) Pictures
Vein to Determine Qualitatively Tumor Burden. The autofluorescence signal
(middle image) is subtracted from the GFP signal (left image) to determine the
burden of tumors consisting of 4T1 cells (right image). The regions in the chest
of the mice that are associated with no autofluorescence are areas where fur
had been removed immediately prior to imaging with IVIS. From the composite
image, Mouse 2 and Mouse 3 appeared to have a higher tumor burden than
Mouse 1. In each of the three pictures, the boxes below the mice indicate
92
whether they are Mouse 1, Mouse 2, or Mouse 3 ......................................................
After
GFP
Filter.
with
Lungs
of
Resected
Images
Figure 29. Low Magnification
collection of blood from three mice to use in microfluidic devices to attempt to
capture CTCs, the lungs from the mice were resected and imaged by
fluorescence microscopy at a very low magnification with the GFP filter to
qualitatively assess tumor burden. The lungs from Mouse 1 shown in the two
images on the left side of this figure produced a lower signal than those
corresponding to Mouse 2 and Mouse 3. (Scale Bar: 2 mm)........................93
Figure 30. Photograph of Resected Lungs from Mice Injected with 4T1 Cells. Pieces
of resected lung from each of three mice used in one experiment were
photographed and examined to measure qualitatively the tumor burden. These
microscopic images supported the conclusion that Mouse 2 and Mouse 3 were
burdened with greater lung tumors than Mouse 1. (Scale Bar: 10 mm - applies
93
to the level at the bottom of w ell) ....................................................................................
on
Anti-CD63
2
Figure 31. Captured Cells after Processing Blood from Mouse
Antibody Coated Device. Both large clusters (left image and top right) and
smaller number of cells (bottom right) were captured while processing blood
from Mouse 2 with an anti-CD63 coated device. Red indicates the presence of
11
DAPI, and green corresponds to signal from the FITC channel, which would be
provided by ZsGreen in 4T1 CTCs..............................................................................94
Figure 32. Captured Cells after Processing Blood from Mouse 3 on Anti-CD63
Antibody Coated Device. Only smaller numbers of cells were captured while
processing blood from Mouse 3 with an anti-CD63 antibody coated device. Red
indicates the presence of DAPI, and green corresponds to signal from the FITC
channel, which ZsGreen in 4T1 CTCs provides.................................................................95
Figure 33. Large, Medium, and Small Clusters of CTCs Captured on the Herringbone
Microfluidic Device Coated with Isotype Antibody. Various sizes of clusters
were captured on the herringbone microfluidic device functionalized with an
isotype antibody. This chip was positioned first in series, and signals from
three channels were recorded: DAPI, FITC (ZsGreen in 4T1), and PE (Anti-CD62
antibody). Blue, green, and red correspond to these signals, respectively.
96
(Scale Bar: 50 pm)..........................................................................................................
Device
Functionalized
Microfluidic
Stitched
Images
from
Herringbone
Figure 34.
with Isotype Antibody. Large clusters were predominantly captured on this
H B-chip functionalized with an isotype antibody and positioned first in series
before the anti-CD63 antibody-coated device. Signals from three channels were
recorded: DAPI, FITC (ZsGreen in 4T1), and PE (anti-CD62 antibody). Blue,
green, and red correspond to these signals, respectively. Two large bubbles in
two channels distort the signal of several of the clusters (Scale Bar: 500 p1m)..97
Figure 35. Stitched Images from Herringbone Microfluidic Device Functionalized
with Anti-CD63 Antibody. Fewer and smaller clusters were captured on this
herringbone microfluidic device functionalized with anti-CD63 antibody and
positioned second in series after the isotype antibody-coated device. Signals
from three channels were recorded: DAPI, FITC (ZsGreen in 4T1), and PE (antiCD62 antibody), which likely was not washed completely from two channels
above. Blue, green, and red correspond to these signals, respectively. (Scale
8
Bar: 5 0 0 im )....................................................................................................................................9
Figure 36. Schematic of AFM Components. Typically an AFM measures the nanoscale
topography of a surface by rastering a tip over it. The tip and cantilever
respond to changes in the properties of the sample, and deflections are
measured by a photodiode's determination of the location of a laser spot that is
reflected of the tip. Feedback electronics direct the scanner according to realtim e changes in the tip deflection.......................................................................................109
Figure 37. Representations of Prototype Head Design and Photographs of
Fabricated Head with MultiMode V. The version of this optics head is based on
a design by Hansma, et al. and is described in detail in U.S. patent 6,871,527. In
sum, several novel features of this prototype head enable the AFM to operate
with a smaller focused spot of light that previously was impossible..................113
Figure 38. Change in Bacterial Cell Surface from Smooth to Corrugated After
Addition of CM15. This series of images from the high-speed AFM
demonstrates that the onset of cell surface corrugation after introducing CM15
(t = 0 s) varies from cell to cell. The difference in time between capturing each
image is 13 s, and the images are from the phase data (Scale Bar: 1 pm).........120
12
Figure 39. No Significant Cell Surface Corrugation after Introduction of Control
Peptide. The addition of 2K1 (A and C), which is the positively charged control
peptide with no known antimicrobial activity at this concentration, did not
result in significant change of surface variations after at least 43 min (B and D)
as supported by comparison of cross-sections before and after addition of 2K1
of norm alized phase (bottom)..............................................................................................121
Figure 40. Images from AFM and Fluorescence Microscopy Captured at the Same
Location Prior to and After Adding CM15. Comparing tapping-mode images (A
and C) and fluorescence images (B and D) at the same location shows that
corrugated bacteria are typically dead (red) and bacteria with smooth surfaces
are alive (green). The surfaces and health of the cells before addition of CM15
(A and B) were more often smooth and viable than 30 min after (C and D).
These AFM images were recorded at a scan rate of 0.5 Hz and a resolution of
512 x 256 pixels. (Scale Bar: 5 gm ).....................................................................................122
Figure 41. Quantification of Bacterial Roughness Prior to and after Addition of
CM15. The series of AFM images (left) shows bacteria at 105 s intervals after
injection of CM15. Images were recorded every 21 s (fifth image shown) at a
resolution of 1024 x 256 and a scan rate of 12.2 lines s-. From each phase
image in the time progression, cross-sectional data was extracted as shown for
bacterium 1 (bottom right). The average bacterial roughness as a function of
time before and after addition of CM15 is presented in the top right with
numbers corresponding to labels in the time series of AFM images...................123
Figure 42. Bulk Cell Killing Assay with CM15 and E. Coli. Behavior interpolated
through the first 5 min of CM15 antimicrobial activity in bulk correlates well
with measurements on single cells with the AFM........................................................124
13
List of Tables
Table 1. Imaging Techniques for Cancer and Their Strengths......................................27
Table 2. Types of Granules in Platelets and the Respective Agents They Contain.....32
Table 3. Contrasts in Characteristics between Epithelial and Mesenchymal Cells.....38
Table 4. Examples of Microfluidic Applications within a Selection of Fields and
73
In d u stries ..........................................................................................................................................
Table 5. Capture Efficiency of 4T1 with Anti-CD63 and Anti-CD62P ......................... 85
Table 6. Rough Capture Purity of 4T1s with Anti-CD63 and Anti-CD62P Antibody
85
Coated Herringbone Devices .............................................................................................
14
Chapter 1. Introduction
1.1. Broad Overview
Advances in instrumentation provide new opportunities to discover and further understand
scientific phenomena. Improvements in technology can provide insight into workings of the
world in ways otherwise impossible. This thesis reports on investigations involving two
types of cells - bacterial and mammalian - with two types of instruments, atomic force
microscopy (AFM) and microfluidic devices, respectively. Although these projects are
unique in their specific aims, they are nonetheless characterized by several overarching
similarities.
There are at least three parallels between these two areas of investigation. Very broadly,
the goal of both projects is to study the characteristics and behavior of a small population of
cells. Secondly, and more specifically, a necessary step in studying the cells of interest is the
attachment of these bacteria and mammalian cells to a surface to examine them by
microscopy. And, third, through capture and examination of the cells, a hope is that the
advancement in understanding about their behavior and characteristics could ultimately
assist in yielding successful approaches to eliminate the source of these diseases, which are
infection by bacteria and malignant tumors.
Increasing understanding in these areas of biology has the potential to significantly impact
human health. A parallel between bacteria and cancer cells beyond just being causes of
disease is their ability to adapt to their environment for survival and growth. In certain
circumstances, ineffective treatment of a bacterial infection or tumor will result in the
return of the disease to a level of even greater strength. On the societal scale, bacteria have
become more resistant to some common antibiotics, and new approaches might need soon
to be discovered to combat "superbugs" with multidrug resistances.
With regard to cancer, due to the extension of human life by prevention of other causes of
death and the mass adoption of lifestyle changes that involve behaviors that cause cancer,
the scourge of cancer is increasing globally 1 . In 2008, of the 57 million deaths total
3
worldwide 2, 7.6 million, or 13.3% were estimated to be a result of cancer . Ninety percent
4
of cancer deaths are due to tumors that are seeded by cells from the initial site of cancer .
15
The first project reported here utilized an advanced technology - the herringbone
microfluidic device - to investigate the feasibility of a new technique to capture circulating
tumor cells (CTCs) with platelets (Chapter 5). Background on the significance of cancer and
the relevance of CTCs to its study and treatment is presented in Chapter 2. After describing
the protocol developed to create samples for testing various methods towards capturing
CTCs by means of platelets (Chapter 3), in Chapter 4 are described the two initial studies
that involved magnetic activated cell sorting (MACS) and subsequently a flow chamber
device. These experiments led to testing this novel approach with the herringbone chip.
The second project, which is about the action of antimicrobial peptides (AmPs) on bacteria
observed by AFM, was made possible by an advance in this type of microscope's image
acquisition rate and required a protocol to attach the bacteria to the substrate for imaging
(Chapter 6).
Besides the type of cell examined, a major distinction between these two projects is their
starting number in the experiments. An abundance of bacterial cells can be grown within
hours and then used in experiments, while the cancer cells that are attempted to be studied
in the most impactful experiments are exceedingly rare, a tiny fraction of a percent of the
total number of various cells in the sample that is processed.
Detailed protocols and the specific materials used in experiments described herein are
included in the appendix following each chapter except Chapter 2, the content of which
provides background and motivation and does not report methods or results.
1.2. Thesis Chapters
1.2.1. Chapter 2
Amazing and disturbing as it is that one cell can randomly acquire mutations in its genetic
code to allow it to proliferate unchecked in the body, of greater wonder and concern is that
given sufficient time subsequently spawned cells continue to drift from normalcy towards
malignancy eventually yielding those that circulate in the blood stream to create new
tumors in distant locales. Eradicating the set of cells while confined and benign before such
16
a capability is acquired is a prime objective of cancer treatment. Traditional techniques of
diagnosing cancer and monitoring its progression rely largely on biopsies and imaging. A
promising new area of research is to isolate, enumerate, and analyze CTCs - an approach
described as a "liquid biopsy" - to detect and improve treatment of cancer by both further
understanding each patient's unique disease and the process of metastasis more generally.
The changes a tumor cell undergoes to become a renegade also can differentiate it from
peers within the body, and a multitude of techniques have been tested to determine their
respective abilities to isolate CTCs through such distinguishing features. The motivation to
capture CTCs by means of platelets is presented at the close of Chapter 2.
1.2.2. Chapter 3
Foundational to learning how to separate CTCs by using surface markers on platelets was
developing a protocol to consistently produce platelet-tumor cell complexes. These
complexes made in buffer could be tested for capture from that simple solution or spiked
into blood from a healthy donor animal or human and then processed through each of the
devices to test their efficacy. In creating these complexes in vitro, a balance must be reached
between the extremes of no platelet activation, which would yield no complexes, and such a
high degree of platelet activation that clumps of cells are produced of great size rendering
them untestable and not relevant to in vivo processes.
1.2.3. Chapter 4
Magnetic activated cell sorting (MACS) and functionalized flow chambers were the first two
approaches tested here as a means to isolate platelet-tumor cell complexes, and they served
as proof-of-concept methods. The potential of both techniques to accomplish this goal was
determined by trying to capture complexes of platelets and tumor cells generated according
to the protocol established in Chapter 3. Data from the experiments presented in this
chapter guided the subsequent studies with microfluidics discussed in Chapter 5.
1.2.4. Chapter 5
The advancements in recent decades of developing devices with channels that precisely
control the flow of fluid has had widespread utility, including providing new approaches to
process such a complex solution as blood, which contains at least a score of different types
17
of cells at a total concentration of approximately a billion cells per milliliter in humans.
Starting with only several CTCs in this volume of blood, these microfluidic devices have
shown the ability to capture at least one CTC from such a sample. With one of the latest
generation microfluidic devices during the time of the start of this project, a new approach
of capturing CTCs with platelet surface markers as a target was tested, and complexes of
platelets and CTCs were consistently captured in buffer and after spiking them into blood.
Experiments with mouse models using the same approach also demonstrated the ability to
isolate CTCs from blood.
Ultimately, further optimization is required before the most impactful application of this
specific technique of utilizing surface markers from platelets to pull away CTCs from human
blood will be realized. Recommended next steps include more extensive testing with
controls, measuring more precisely the capture efficiency and sensitivity, and optimization
of the device parameters to improve performance.
1.2.5. Chapter 6
The final chapter reports on a study enabled by advancements in a remarkable type of
microscope that under appropriate conditions can visualize individual atoms by touching
thems. Such an incredibly precise instrument might rightly be suspected of operating at a
burdensomely slow pace when characterizing areas with dimensions 100,000 times larger
(regions 0.1 nm in length for an atom and up to 10 gm for more typical scans). Amazingly,
through decades of incremental developments, these greater area images can now be
produced in seconds rather than minutes. This opens doors to investigating realities so far
unseen and otherwise impossible to analyze. High-speed AFM has demonstrated its
potential as an instrument capable of revealing insights into processes at the level of
biomolecules. The next stage has been to extend this to the study of individual cells.
Through the combination of fluorescence microscopy and high-speed AFM, the kinetics of
an AmP acting on E. coli was researched and is reported in this final chapter.
18
Chapter 2. Significance of Circulating Tumor Cells and Platelets
Abstract
The deadliness of cancer is largely due to its eventual acquisition of traits that enable it to
disseminate. Originally a component of the body but turned renegade, it is not completely
surprising that tumor cells would co-opt biological processes within an afflicted individual
for their own benefit. In this chapter is described a cell transition that circulating tumor
cells (CTCs) undergo as a response to interacting with platelets in circulation. Although
these processes are helpful to the CTCs along their journey through the hostile bloodstream,
the hope is that this affinity to platelets can be turned against CTCs as a means to capture
them from circulation to study, which may ultimately yield further insights into the process
of metastasis and methods and approaches to treat cancer.
2.1. Introduction
Starting in the early 1970s and with the subsequent improvement in understanding of
molecular and cellular biology due to cancer research, there have been especially significant
advances in understanding this broad category of hundreds of diseases 6. Since the "War on
Cancer" was declared in 1971, the National Cancer Institute alone has spent at least $90
billion on research and treatment. 7 Despite the tremendous amount of resources devoted
to this endeavor and the learning that has led to strategies to prevent and treat cancer,
there has been only a five percent decrease in the rate of cancer deaths between 1950 and
2005 when adjusting for population size and age 8 . This modest drop bespeaks the need for
more effective, novel approaches to cancer therapy and prevention 9, which will result in
and from a fuller knowledge about incompletely understood causes of cancer.
Beginning inside a single cell is the progression of events that ultimately produces cancer.
This multistage process typically requires a prolonged period of time. For instance, young
people exposed to the carcinogens released from smoking cigarettes often develop cancer
only after two or three decades10 . Increased knowledge about cancer and the development
of new technologies have yielded a suite of cancer diagnostics, which are used in screening
tests and are increasingly sensitive in detecting tinier tumors 1 . While there are actually
19
potential harms in screening for cancer, such as risks for serious complications arising
particularly from invasive tests, false-positive test results, and overdiagnosis, yet 3 - 35% of
premature deaths due to cancer are estimated as being able to have been avoided through
screening12.
Relatively recently, devices have been fabricated that enable the capture, counting, and
study of the biology of circulating tumor cells (CTCs), and this, collectively, is a promising
approach to advance understanding of cancer. New insights have already been discovered,
such as the implication of WNT signaling in the spread of cancer (metastasis)13. Although
there has been clear progress in this area, advances are still necessary to utilize fully this
approach. For instance, how can it be determined which cells captured are relevant to the
progression of cancer, and how can all relevant cancer cells be captured?
2.2. Cancer Biology
The starting point of cancer has been described as the transformation of a healthy cell into a
"renegade cell"14. The nature of such a cell that qualifies it as being "renegade" is its
inability to restrain properly its own growth at the potential cost to the entire organism.
Essentially, cancer cells harbor genetic aberrations that enable capabilities, which, in
combination, result in unregulated cell proliferation and eventual invasion into nearby and potentially distant - parts of the body.
2.2.1. Types of Cancer
Cells constituting nearly every type of tissue in the body are capable of acquiring the ability
to give rise to a tumors. The organ in which cancer is first formed or its cell type of origin is
often the basis for the name of that type of cancer16. In Figure 1 is shown types of cancer
that cause the greatest number of deaths in the United States: lung cancer, breast cancer,
colon cancer, pancreatic cancer, melanoma, and ovarian cancer 7 .
2.2.2. Progression
Fundamentally, the cause of cancer is abnormality at the genetic level that results in
8
capabilities that have been described as "hallmarks of cancer"1 :
20
*
sustaining proliferative signaling
*
evading growth suppressors
*
resisting cell death
*
enabling replicative immortality
*
inducing angiogenesis
9
activating invasion and metastasis
It should be noted that the first five of the above "hallmarks" are said to be characteristic
also of benign tumours.19 The unique hallmark of malignant disease in this list is the ability
20
of cancerous cells to invade and metastasize.
Reported to underlie these hallmarks are genome instability and inflammation. The former
helps create genetic diversity that quickens the rate of acquiring the hallmarks. Two
additional "emerging hallmarks" that may be added due to their potential generality to
many types of cancer are reprogramming of energy metabolism and evading immune
destruction. 21
Formation
Although there are very effective systems inside the cell that maintain the integrity of the
genetic code, a consequence of the fundamental limitations of DNA replication is that
mutations cannot be completely avoided 22. While about 5% to 10% of cancers result
directly from mutations inherited from a parent
23
, numerous unrepaired genetic changes
formed during the life of an organism can cause, cumulatively, the formation of a tumor.
Several sources of these alterations include radiation, chemicals, and viruses, and mutations
giving rise to abnormalities in gene function can result both in proteins functioning
abnormally and the improper regulation of the construction of new proteins.
Since much of a cell's behavior is a result of the activity of its constituent proteins and their
number in that cell, aberrant abilities may be gained from changes in gene function,
including the above-mentioned "hallmarks of cancer", which are shared by most - and
perhaps all - cancers 24. Multiple genetic alterations are necessary in order to empower the
cell with such capabilities that can cause cancer, and each successive step may confer a
21
particular type of growth advantage, which results in the progressive change of cells from
normality to cancer-enabling 2s.
Growth
While the origin of practically all malignant tumors is monoclonal (coming from a single
ancestral cell) 2 6, cells within a single tumor can be heterogeneous functionally and
phenotypically due to genetic change and environmental differences 27. As the cells
constituting an incipient tumor continue to divide and grow, their genetic material
undergoes further random mutations, which result in cellular changes that can confer
capabilities enabling the cell to meet the challenges associated with multiple selective
pressures, 28 such as the lack of space, oxygen, and glucose. In this process analogized to
Darwinian evolution, such a cell that becomes increasingly and sufficiently fit survives and
generates a greater number of daughter cells than do those of the same tumor that lack that
beneficial mutation. Accordingly, this population of mutant cells expands (clonal
expansion) 29.
Metastasis
Even after an initially normal cell's process of profound genetic change traversed in its
journey to become neoplastic, without developing the further capability of travelling away
from the site of disease, tumors consisting of such cells are designated as benign. Malignant
tumors are capable of spreading from its starting location by moving through the
bloodstream (haematogenous spread), via the lymphatic system (lymphatic spread), or by
transcoelomic routes 30.
In the metastatic process, after a series of divisions by the initial malignant cell and its
daughter cells that yields a tumor the size of dozens of cells, the tumor cell mass releases
angiogenic signals, which are able to lead to the growth of blood vessels inward to the
31
tumor. To complete the formation of a secondary tumor, metastatic cells :
.
are freed from the tumor mass
22
*
degrade the neighboring extracellular matrix, which includes the basement
membrane
*
enter the blood vessels
*
survive within the circulation
*
attach to the endothelium near the site of the future secondary organ
*
migrate through the endothelium
*
divide in the target organ (Figure 1)
Figure 1. Sites of Metastases from Cancer Types Causing Most Deaths in the United
States. From recent cancer statistics in the United States, the following types of cancer
result in the greatest number of deaths 32 : lung cancer, breast cancer, colon cancer,
pancreatic cancer, melanoma, and ovarian cancer. Shown are common sites of metastasis
from these types of cancer. 33
Dr. Thomas Ashworth was an Australian physician who made the discovery by microscope
34
in 1869 of the existence of tumor cells in the blood of a patient with cancer . He
postulated:
"...cells identical with those of the cancer itself being seen in the blood may tend to
throw some light upon the mode of origin of multiple tumours existing in the same
person. 35 "
23
Figure 1 shows typical locations of secondary tumors from particular types of cancer. That
CTCs are as seeds is a simile from at least the 19th century when Stephen Paget proposed
that "seeds" produce metastasis in organs with compatible microenvironments, or "soils"36.
This idea from 1889 that cross-talk and compatibility between the cancer cell and the
milieu of a particular organ play a large role in metastasis is still influential in explaining the
patterns of metastases 37.
Research over scores of years has yielded significant experimental support that there is
indeed a role of CTCs in the metastatic spread of cancer. The presence of CTCs is indicative
of great progress along the pathway of a tumor to generate metastases, and, as such,
technologies with high sensitivity and reproducible results have shown recently the
potential diagnostic value of these cells 3 8.
While the spread of cancer through metastasis is believed to happen mostly though blood
by hematogenous dissemination 39, this process is regarded to be vastly inefficient in that
the formation of a secondary tumor by one of the up to 4 x
106
tumor cells released into the
circulation daily per gram of primary tumor 40 is a relatively rare event.
Despite this seemingly massive number of tumor cells that may regularly be released into
the bloodstream, the actual fraction of CTCs in the circulation relative to all the other blood
cells is exceedingly small - estimated to be one CTC per billion normal blood cells in
advanced cancer patients 41 - which poses a great engineering challenge to isolate these
seeds of cancer.
2.3. Significance of Early Cancer Diagnosis and Constant Monitoring of
its Evolution
In the same way that it is preferable to remove a dandelion before its seeds develop and
disseminate to avoid damage in the production of crops, if a tumor can be identified before
it metastasizes, there is a much greater chance that a patient will survive. For example,
there is a dramatic difference in the five-year survival rate depending on the timing of
diagnosis of colorectal cancer, the type of cancer that causes the second largest number of
24
cancer deaths 42. These are the rates of survival according to the timing of detection and
treatment 43:
*
90% if diagnosed while localized (i.e., it does not extend beyond the bowel wall)
*
68% if there is regional disease (ie, lymph node is involved with the disease)
*
10% if distant metastases are present
Considering the high frequency of diagnosing common types of cancer only at a late stage
and that the survival rate would be higher than 85% if the cancers listed below were
diagnosed at an early stage, identifying the disease earlier is a challenge of critical
importance in the treatment of cancer 44. Three common types of cancer are diagnosed at
45
the following rates in the US at a late stage :
*
72% - lung cancer patients
*
57% - colorectal cancer patients
*
34% - breast cancer patients
While the existence of some tumors is visually evident, the majority are deep inside the
body and unseen during a typical exam, and even for those that can eventually be detected
by eye, determining their incipience early improves treatment outcomes46.
The spectrum of options to treat tumors ranges from no action to a combination of surgery,
radiation, and chemotherapy 47 . Most influential factors determining treatment include the
48
benignity or malignancy of the tumor and its type and location . If the tumor is not able to
spread and will not result in symptoms or changes in organ function, nothing may need to
be done 49. Cancers that are located in only a single place are often treated by surgery to
remove the tumor, and radiation and chemotherapy may be added if surgery cannot remove
the entirety of the cancers0 .
Clearly, a core aspect of cancer that is so frightening is its essentially inevitable result in
death to the patient harboring the tumor provided sufficient time, and the unpredictability
of its response to treatment is a factor contributing to its persistently menacing character
whenever present. In like manner, cancer is a category of diseases in which the same organ
25
can be afflicted in two different people and the same administration of treatment to both
could yield remission in one and not prevent death in the other. The cause of this seeming
fickle and capricious nature of cancer is, fundamentally, the process by which neoplasias are
initially formed and progress to malignancy. Not only is every tumor unique broadly
speaking, but also the cells within tumors are heterogeneous. Furthermore, each of the
cancerous cells is dynamic in time, subject to continued genetic change and responding in a
unique way to its environment, including the drugs and radiation that may be used as
agents against its existence.
In the mind of many searching to cure cancer, personalized medicine holds great promise in
enhancing treatment efficacy, and the ability to sequence entire human genomes at an
increasingly economical cost is foundational to the confidence that characterizing every
cancer patient's unique tumors will be feasible. An important demonstration of how
understanding the features of a tumor can increase the likelihood of survival given
appropriate treatment options is the finding that administering doses of a certain
monoclonal antibody to patients afflicted with a subset of breast cancer improves disease
free survivals1 . Further examples of therapeutics that are more effective in treating tumors
with particular genetic mutations are shown in Figure 2.
Breast Cancer
ER/PIR-positive: Tamoxifen
HER2-posiive: Herceptin
Chroi
ylgnu
BCR-ABL:BRAF
LnCacr
EGFR mutation: Erlotinib
ekmia
Melanoma
mutation: Vemurafenib
Colon Cancer
KRAS mutation-negative: Cetuximab
Figure 2. Therapeutics for Tumors with Particular Genetic Mutations. Drugs listed in
this image have been found more efficacious in treating various types of cancer
characterized by the presence of absence of specific mutations.5 2
26
2.4. Traditional Cancer Diagnostics
The goal of cancer treatment is to remove all the cells in the body that constitute the tumor
so as to prevent its continued growth and presence. Before, during, and after this attempt
to eliminate the tumor, various techniques can be used to plan and monitor treatment.
The capability to invade distinguishes a benign tumor from cancer, and a biopsy, which
typically involves removing a small amount of tissue for microscopic examination, can be
performed to determine if that tumor is noncancerous or malignant (cancerous). The
location of neoplasia determines if the biopsy procedure or operation is simple or serious.
To examine directly a tumor that is inside the body and even collect a tissue sample from it,
endoscopy is utilized. An endoscope consists of a flexible or rigid tube possibly with a light,
lens, and channel for additional medical instruments.
While an endoscope is inserted into an organ for imaging, there are numerous other
medical imaging devices that operate externally with respect to the patient. All these
modalities rely on detecting and interpreting the interaction of various inputs, such as
electromagnetic radiation and sound, with the body while passing through it. A description
of common diagnostic imaging techniques with each of their strengths is provided in Table
1s3.
While current imaging diagnostic techniques serve great purposes in detecting initial
tumors, determining their spread, and deciding on treatments, there is presently a
fundamental limitation in sensitivity that must be addressed through revolutionary
advances in tumor detection5 4. Specifically, typical imaging technologies detect tumors only
after they reach about 109 cells in size, or 1 cm 3 , which means that a patient classified as
being cured of cancer based on results of imaging instruments alone could still harbor a
single mass of up to a billion malignant cells 5 s.
Table 1. Imaging Techniques for Cancer and Their Strengths
X-ray Imaging
X-rays absorb differently according to
27
I Rapid result5 6
I
tissue, which enables determining
spread of cancer or suspicious areas.
Contrast agents drunk by or injected
into the patient can facilitate
distinguishing between organs and the
tumor.
Computed
Tomography
(CT or CAT)
scan
Utilizing computer-controlled X-rays, a
3D image of the body is produced.
Relative to radiographs,
easier to determine 3D
location of tumor
(Magnetic
Resonance
Imaging) MRI
The combination of external radio waves
and a strong magnetic field causes
tissues to themselves emit
radiofrequency signals, the intensity of
which is based on their chemical
composition, which enables producing a
3D image of the body's organs.
Can be better at
distinguishing soft tissues
than CT scans
Ultrasound
Send high frequency sound waves into
body and process those that are
collected after reflecting off organs and
tissues to produce picture of internal
organs and determine if a suspicious
mass is liquid or solids 7.
Shows tumors and can be
used as guide during
biopsies and treatment
Positron
Emission
Tomography
(PET scan)
Measure chemical changes in tissue,
such as increased sugar metabolism, by
nuclear imaging with small amounts of
radioactive substance connected to
compounds that cells in the body use or
that stick to tumor cells.
*
*
Single Photon
Emission
Computed
Tomography
Antibodies with attached radioactive
substances are injected into the
bloodstream and can adhere to tumors
they recognize. Similar to PET, a
28
Helps determine if a
growth is cancerous
and detects cancer
even when other
techniques indicate
normality.
More accurate in
detecting tumors
larger than 8 mm
and more aggressive
tumors. Potentially
helpful when
evaluating and
staging cancer that
has returned. Check
efficacy of treatment
by seeing if tumor
cells are dying.
Provide information about
blood flow to tissues and
the body's metabolism
(SPECT) scan
scanner and computer then together
produce a 2D or 3D image showing the
distribution of the radioactive tracer to
reveal the location of tumors.
A second broad method of understanding a patient's cancer condition is through blood tests
although additional ways of confirming the diagnosis are necessary since these alone, at
least historically, can be inconclusive. Blood tests to diagnose cancer are based on the
release of tumor markers, which are usually proteins that when at a certain threshold
58
concentration in the blood can be indicative of cancer . Despite the need to validate the
results, the test for markers can be used in diagnosis, staging, and population screening and
60
to detect metastasis and recurrent disease and predict and monitor treatment response9, .
61
Currently, at least twenty tumor markers are in use , and they have varying levels of
specificity. For instance, some tumor markers are released only by tumor cells; some are
produced by a variety of types of tumors; and some are simply expressed in tumor cells
62
above the normal level of the corresponding differentiated cells .
2.5. Circulating Tumor Cells in the Context of Treatment
There are several potential advantages to treating cancer with the aid of understanding
gleaned from studying an individual patient's CTCs. The essential benefit is additional
information available frequently in a manner that is not highly invasive. In at least one
instance, CTCs were shown to be superior to a particular serum marker, carcinoembryonic
antigen (CEA), in their ability to predict treatment responses as CEA did not have any
63
correlation with the outcomes of the rectal cancer patients in this investigation . The
number of CTCs captured may provide information beyond just tumor volume as measured
by traditional diagnostics
64
, and the genetic information they carry could provide a new
window into the status of metastasiss.
The first FDA-cleared CTC test to monitor patients throughout treatment is the
CELLSEARCH® CTC Test, and there are at least ten laboratories that currently offer this
specific service 66. This test has demonstrated the connection between the isolation of CTCs
67
from the peripheral blood with decreased overall survival . Data from a longitudinal
clinical trial between 2001 and 2003 led the FDA to clear CELLSEARCH® in 2004 as an
68
assay for capturing and enumerating CTCs in patients with metastatic breast cancer .
29
Monitoring CTCs while treating cancer has been shown to give further prognostic
information 69 and enumerating CTCs can help predict overall survival 70.
But there are currently significant limitations to this test, including only being able to
enumerate CTCs of epithelial origin and it only to be used to monitor patients with
metastatic breast, colorectal, or prostate cancer 71 . Furthermore, the test determines the
number of CTCs in a blood sample but does not analyze them beyond recording a
microscopic image to confirm their cell type 72.
While not yet FDA approved and currently only a Laboratory Developed Test (LDT),
physicians can already order blood tests from a company, Biocept, that beyond just
enumerating CTCs will also analyze them by fluorescence in situ hybridization (FISH) to
determine the number of copies of HER2 (human epidermal growth factor receptor 2) in a
CTC 73 . While the monoclonal antibody, Trastuzumab (Herceptin), has been shown to
improve survival of stage 1-3 HER2+ breast cancers, Biocept disclaims that a positive result
from its HER2 (FISH): OncoCEE-BR' predicts better clinical outcomes after subsequently
switching to a new treatment regimen 74.
2.6. Approaches for Capture of CTCs
Given that cells released from solid tumors reside normally in a different location and
belong to a separate category of cells than those types typically in the blood, it is
unsurprising that there are numerous distinguishing characteristics between these cells. A
multitude of approaches have been tested to try to separate CTCs from blood based on one
or more of their unique features. Enrichment by means of physical properties, including
size and density, has not yet been demonstrated to be as effective an approach for isolating
CTCs as compared to utilizing unique antigenic properties of the tumor cells.
At the core of many approaches for separating CTCs from blood are magnetic particles
coupled to antibodies, and this is in fact a component of the CELLSEARCH® test. A
commonly cited disadvantage of techniques that enrich CTCs by utilizing antibodies is that
the only cancer cells that were captured were expressing EpCAM, but this is not an intrinsic
limitation to employing antibodies. Further tests would be necessary to demonstrate the
applicability of employing antibodies targeting other surface markers to capture CTCs.
30
With the many possibilities afforded by microfluidics, a variety of approaches have been
tested with this technology and have been shown to hold tremendous promise. A sampling
of microfluidic devices employing antibodies to capture CTCs includes the "high-throughput
microsampling unit" (HTMSU) and the "Nano-Velcro" microfluidic chip, and the CTC-Chip
and Herringbone-Chip (HB-Chip) are discussed in greater detail in Chapter 4. These four
75
technologies all require no pre-processing yet are still very sensitive . Although research
with microfluidic devices and CTCs has demonstrated potential to assist in clinical settings,
there is not yet an FDA approved microfluidic device for this purpose.
In one recent study with the HB-Chip, circulating breast tumor cells were isolated as
76
individual cells, in clusters, and with platelets attached . This association of platelets with
CTCs on a microfluidic device is an important finding but also just another step forward in
demonstrating the potentially significant impact of platelets on tumor cells in transit.
2.7. Significance of Platelets in Metastasis
Since at least the 1960s, antimetastatic effects have been reported from treatments that
77
apparently interfere with the influence of platelets on CTCs , and subsequent decades of
research continued to implicate platelets as playing a role in the dissemination of tumor
cells 78. It is fascinating how healthy and normal platelets, which are present in such high
number in the blood stream, could be intrinsically wired to respond to CTCs in ways that
7
have been demonstrated through experiments to facilitate metastasis
980
. While this
natural interaction is, unfortunately, inherently difficult to disrupt, the attachment of
platelets to cancer cells and the platelets ensuing activation in response to these cancer
cells 81 presents a possible approach for isolating a particular subpopulation of CTCs.
2.7.1. Features of Human Platelets
Individual Platelet Characteristics and Traits
Platelets circulate in the blood stream with a lifetime of five to nine days and are formed
from much larger megakaryocytes, which are bone marrow cells 50 Rm - 100 Im in
diameter. An individual platelet is a cell fragment only about 2 gm in size and without a
31
nucleus. Its most well known function is its contribution to hemostasis (the process
that
can stop bleeding) that leads to blood clots.
microtubules
dense tubules
Surface-connecting
tubule
coat
glycogen
mitochondria
alpha granule
dense granule
Figure 3. Schematic of the Internal Structure of a Human Blood Platelet. In
the resting
state, platelets are shaped as disks. In their alpha and dense granules are stored
numerous
types of agents that can be released in response to the environment 2.
Though relatively simple in shape and structure (Figure 3), platelets present
a more
complex reality due to their ability to respond to specific signals in the blood,
become
activated, and release agents that stimulate both neighboring platelets and also
a range of
other cells. Amazingly, in response to the presence of a common enzyme - thrombin
- over
three hundred types of proteins are released by platelets8 3. A list of the many agents
contained internally in four types of platelet granules is in Table 2.
Table 2. Types of Granules in Platelets and the Respective Agents They Contain
Alpha granules
Fibrinogen, Factor V, Platelet derived growth factor (PDGF), Platelet
factor 4, Beta thromboglobulin, Albumin, Fibronectin,
Thrombospondin84
Dense (or delta)
granules
Calcium, ADP, ATP, GTP, thromboxane and serotonin 85
Lambda granules
Hydrolytic enzymes86
32
Peroxisomes
Catalase 87
In addition to the release of its alpha and delta granules, platelets upon activation also
dramatically change shape. The profound shape conversion from bioconcave disks to fully
spread cells results from the cortical actin of platelets rapidly reorganizing upon platelet
activation 88. A further increase in surface area is achieved by the extension of the platelets'
filopodia and the generation of lamellipodia 89. The ability to change shape and release the
contents of its granules empower the cell to produce a collective response with other
platelets to contribute to hemostasis and also promote other processes within the
bloodstream and even beyond
90.
Especially worthy of note is the difference in proteins expressed on the surface of activated
platelets as compared to quiescent platelets. For instance, the plasma membrane
expression of P-selectin is increased upon platelet activation through redistribution from
the cell fragment's alpha granule membrane 91 .
Collective Capabilities of Platelets in Healthy Humans
Platelets play critical roles in preventing blood loss in case of injury but also have a
considerable range of further responsibilities in routine human health maintenance. For
platelets to elicit action at the correct moment both as individual cell fragments and
collectively with the support of neighboring platelets, the signals from events demanding
changes in operation must be received, processed, and amplified, and there must be a
sufficient number of platelets available to produce the desired effect, for instance, to
maintain integrity of the vasculature and to help form a thrombus where there is vascular
injury 92. The normal concentration range of platelets in humans is from 15Ox 109/L to
400x109/L93. Lower or higher counts can result in the conditions termed
94
thrombocytopenia and thrombocytosis, respectively . In the latter case, there is an
increased risk of thrombosis 95 .
The formation of a platelet plug is one of the mechanisms by which hemostasis is achieved,
and the process required to construct such a plug requires multiple steps and numerous
platelet receptor-ligand interactions 96. Initially, at the location of vascular injury,
33
subendothelial components, such as collagens and laminins, can become exposed and
contacted by platelets 97. Subsequently activated signaling pathways collectively result in
complex responses from cells, including the integrin activation, granule content release, and
coagulant activity 98. While the precise sequence of steps involved in thrombosis is beyond
the scope of this thesis, the broad outline of the platelets' role provides insight into the
power of these small components of blood through their prevalence, sensing and signaling
ability, and recruitment (Figure 4).
Figure 4. Aggregation of Platelets Upon Addition of ADP. Platelet rich human blood
plasma (left) forms white flakes (right) after the addition of ADP, which activates platelets
and causes them to aggregate. A magnetic stirrer is at the bottom of both vials. 99
Platelets contribute not only to hemostasis and thrombosis but may also have roles in
inflammation, atherosclerosis, angiogenesis, wound healing, and antimicrobial host
defense
"
00
. Specific further functions of platelets include1
01
:
recruiting leukocytes and progenitor cells to places where there is vascular injury
and thrombosis
*
storing, producing, and releasing proinflammatory, anti-inflammatory, and
angiogenic factors and microparticles into the circulation
"
controlling thrombin generation to lead to fibrin clot formation
2.7.2. Interaction of Platelets with Circulating Tumor Cells
34
It is jarring to realize that a component of the body in a healthy state can interact with a
source of disease in such a way that increases its deadliness. Such is the potentiality when
platelets interact and attach to CTCs. Two immediate questions one might have after
learning that platelets directly contribute to metastasis are (1) how they bind to CTCs and
(2) how this previously innocuous entity imparts new abilities to the CTCs.
Mechanism of Attachment
At least some malignant tumor cells are able to aggregate platelets, and this process is
termed tumor cell-induced platelet aggregation (TCIPA)102. P-selectin, which is expressed
on the surface of activated platelets, has been implicated in mediating adhesion of platelets
to cancer cells in metastasis103, and these interactions are reported to be based mostly on
mucin- and glycosaminoglycan-type selectin ligands104.
Role in Facilitating Metastasis
That platelets play a role in facilitating metastasis is supported by the observation that
defective platelet function or lower platelet counts seems to result in lower metastasis
formation,105 and the ability of cancer cells to aggregate platelets correlates with the cancer
cells' metastatic potential1 06. The wide range of ways that platelets facilitate metastasis is
perhaps as remarkable as the sum effect of these conferred advantages to CTCs. Just some
of the benefits that platelets impart to CTCs are described below within three headings:
1. PlateletsProtectTumor Cells in Transit:The environment of the bloodstream is
hostile to CTCs in both passive ways - the high shear forces from the flow of blood and active ways, and effectors of the host immune response are elements that pose a
particular danger to CTCs1 07 . One component of the innate immune system is the
natural killer (NK) cell, which can recognize and eliminate tumor cells through
direct contact with them108. Studies since 1999 indicate that platelets can provide a
physical barrier between tumor cells and NK cells that protects CTCs from NK lysis
and may also inhibit some functions of NK cells by releasing suppressive or tumor
cell protective factors' 09. In addition to components in the bloodstream that actively
35
seek out CTCs, another hostile factor any cell in vascular transit experiences is high
shear force. These forces could damage CTCs, but they may be shielded from the
forces of flowing bloodilo by aggregated platelets that surround them.
2. PlateletsAssist in CTC Escapefrom the Vasculature: Platelets appear to play a role in
the process of tumor cells exiting the bloodstream and entering organs in
metastasis"1 . Most simply, this could be due to the increased overall size of CTCs
after platelet aggregation with the tumor cells to form complexes. This is thought to
provide a survival advantage by increasing the likelihood of embolizing the
microvasculature11 2. Furthermore, particular surface proteins on platelets
3
aggregated with CTCs facilitate adhesion to vascular endothelium11 . A study
suggesting that there is increased metastatic potential as a function of the number of
CTCs in a cluster showed that injected tumor cell clumps consisting of six or seven
cells produced a higher number of metastatic foci than the same total number of
tumor cells injected individually1 4. A hypothesis yet to be examined is whether
heteroaggregates of CTCs, platelets and leuokocytes, once trapped in the vasculature,
create a microenvironment that increases the ability for the CTCs to extravasate and
invade tissue"s.
3. PlateletsProvideSignals Advantageous to Tumor Cells: There is most often two-way
communication between any particular cell and its environment. In the specific case
of the site of a primary tumor, interactions of cancer cells with the
microenvironment can cause progression towards metastasis, and, more recently, it
has been shown that prometastatic signals are also provided during travel of a CTC
intravascularly. In addition to the many capabilities imparted by platelets to CTCs,
platelets release growth factors that could be used by tumor cells for growth, and
platelets provide a synergistically interacting set of signals to tumor cells that cause
a transition in CTCs"16. There are several aspects of the resulting changes in the
CTCs' characteristics that motivate trying to capture CTCs with platelets attached.
2.8. Motivation to Isolate CTCs by Means of Platelets
While scientists suggested years ago to consider platelets in the management and detection
of human cancer, the approach of capturing CTCs by means of platelets had yet to be
demonstrated. Fundamentally, the rationale to pursue this approach is to capture CTCs that
are undetected by other techniques. Given the evidence that these platelet-CTC complexes
36
are characterized by a greater metastatic potential, this capability to capture them could
enable further studies into whether measurements on their concentration in the blood
provide useful, additional information that can help to prognose cancer more accurately.
Also, the isolation of these complexes from blood could enable direct study to determine, for
instance, differences between cancer cells that bind platelets and those that don't, the
knowledge of which could be particularly impactful if platelet-CTC complexes are, in fact,
more deadly than either individual CTCs or clusters of CTCs without platelets attached
17
.
Many of the most deadly types of cancer are epithelial in origin and, thus, express a
particular glycoprotein, the epithelial cell adhesion molecule (EpCAM). The ubiquity of this
marker has been part of the rationale for developing techniques that rely on detecting
EpCAM to capture CTCs. The CELLSEARCH® assay, approved by the US Food and Drug
Administration for clinical use, involves capturing CTCs that express EpCAM, but not only
8
might this assay underestimate the number of EpCAM-expressing cells"1 , currently
approved CTC detection systems have been shown also to miss at least some CTCs that are
negative for EpCAM11 9. And CTCs that are epithelial in origin but are not expressing EpCAM
may have characteristics that make them more dangerous.
2.8.1. The Epithelial-Mesenchymal Transition Has Been Implicated in
Metastasis
Why would tumor cells that are epithelial in origin not express EpCAM? The answer lies in
the existence of a process, termed the epithelial-mesenchymal transition (EMT), by which
cells alter many of their characteristics. Epithelial-mesenchymal transitions happen in
0
three different biological settings12
1
12 :
"
Type 1: Developmental (implantation, embryogenesis, and organ development)
"
Type 2: Fibrosis and wound healing (tissue regeneration and organ fibrosis)
"
Type 3: Cancer (cancer progression and metastasis)
22
The functional consequences of an EMT depend on the context1 . Specifically, the genetic
and epigenetic changes in neoplastic cells that work in harmony with the EMT regulatory
circuitry result in outcomes that are very different from what is seen to occur in the first
two types of EMT12 3. Not only do the resulting properties from a type 3 EMT help enable the
37
carcinoma cells to move from the site of the tumor into the bloodstream, but these new cell
traits further increase their tumorigenic and proliferative potential124.
Epithelial and Mesenchymal Cells and the Spectrum Between These Cell Types
These two main cell types, epithelial and mesenchymal, were recognized in the late
nineteenth century based on their shape and organization during the development of the
embryo125 . But it was only in 1982 that EMT was recognized as a distinct process, and,
again, after a long time, EMT was recognized as a mechanism that could possibly be
involved in carcinoma progression.126 Carcinomas lose many of their epithelial
characteristics during progression, and tumors are partially staged accounting for this
phenomenon127. Some of the numerous changes that result from EMT are listed in Table 3,
at least many of which assist in an essential first step of cancer dissemination - local
invasion through the basement membrane128 :
Table 3. Contrasts in Characteristics between Epithelial and Mesenchymal Cells
Cell-cell interactions
Strong cell-cell
contacts (E-cadherin)
Low cell-cell interactions and cohesion
(switch from E-cadherin to Ncadherin)
Cell-matrix interactions
Low cell-matrix
interactions
High cell matrix interactions (integrin
up-regulation)
Extracellular matrix
remodelling
N/A
Expression of proteases that degrade
the ECM
Cytoskeleton
modifications
N/A
9
"
"
Cytoskeletal elements
reorganized
Peripheral actin cytoskeleton
replaced by stress fibers
Cytokeratin intermediate
filaments replaced by vimentin
Cell motility
enhancement
Low migration
capacity (basoapical
polarization)
High migration capacity (front-rear
polarization)
Resistance to anoikis129
Susceptibility to
anoikis
Resistance to anoikis
38
As the transition of cell characteristics from epithelial to mesenchymal covers a spectrum of
changes that are not always observed during EMT, a cell undergoing EMT does not always
switch lineage130. It is thought that the specific spectrum of changes that happen during a
13 1
particular EMT are an outcome of the extracellular signals received by the cell .
Characteristics Conferred by EMT that May Facilitate Metastasis
While the EMT may be just one potential mechanism that contributes to the advancement of
32
malignancy in invasive epithelial tumors that spread to new locations by metastasis1 , EMT
does confer an impressive range of characteristics that could make a cancer cell more suited
to be the seed of a new tumor. To be sure, though, there are differences between a
physiopathological and normal EMT133. With very variable environmental signals and
genetic heterogeneity in a tumor, the induction of an EMT in cancer, which is prompted by
34
intrinsic and extrinsic signals (e.g. gene mutations and growth factor signaling)1 , typically
is not coordinated and orderly and may not include particular events that happen in an EMT
during normal development3s, and it is theorized, as a result, that there is indeed a
spectrum of epithelial and mesenchymal properties that result from an EMT in cancer
cells13 6. It seems that an EMT associated with tumor progression may result in just some of
37
the aspects associated with EMT in normal development1 .
The achievement of a primary tumor to produce metastases, as noted above, is the result of
successfully traversing numerous and significant biological barriers. Cellular capabilities
conferred by an EMT facilitate the movement along the metastatic path and surmount
obstacles during the journey.
Various cell adhesion molecules (CAMs) are critical in preventing progression towards
metastasis1 38. The epithelial nature of some solid tumors causes the constitutive cancer
39
cells to maintain tight, homologous cell-to-cell contacts1 . Abundant junctions, including
adherens junctions, tight junctions, desmosomes and hemi-desmosomes, between cells in
epithelial tissue formed from multiprotein complexes anchor cells to each other and to the
ECM1 40. A step seen to facilitate cell escape from the primary tumor is the down-regulation
of proteins that create these junctions
41
.
39
After EMT, some types of proteins at the cell surface that are involved in epithelial cell
connections and attachment to the basement membrane, including E-cadherin and some
integrins, are not as highly expressed, and proteins, such as N-cadherin and other integrins,
are more highly populated at the surface of the cell, which, in sum, results in more transient
adhesion 142. Loss in E-cadherin function is thought to promote not only a cell's passive
dissemination but also its invasiveness143.
After a normal epithelial cell separates from its matrix, programmed cell death, anoikis,
results'44. Through EMT, though, epithelial cells can be reprogrammed so as not to require
such attachment for survival145 .
The initiation of enhanced motility of cancer cells that have undergone an EMT is reportedly
by binding between integrin and molecules on the extracellular matrix (ECM), which results
in an interaction among the actin cytoskeleton and intracellular molecules with focal
adhesions. These focal adhesions, in turn, create mechanical forces mediated by the actin
cytoskeleton enabling migration of cancer cells and their adhering to underlying
substrates146.
While cancer cells are able to invade without undergoing an EMT through other means,
such as amoeboid or collective cell invasion, during an EMT, non-motile epithelial cells are
7
converted to invasive mesenchymal cells14 . Part of the EMT process is the expression of
matrix metalloproteinases (MMPs)1 4 8, which are proteases that can degrade the ECM, cleave
a number of different groups of substrates, and, more broadly, increase the growth,
49
migration, invasion, metastasis, and angiogenesis of cancer cells1 . Matrix
metalloproteinases are thought to facilitate invasion perhaps in part by promoting both
extravasation and intravasation' 5 0 .
One final trait worthy of note that is conferred by an EMT to tumor cells is resistance to
radiation treatment and chemotherapy
1
. For instance, radioresistance in prostate cancer
2
is associated with EMT and other mechanismss .
A Possible Link: Stem Cell-Like Characteristics and EMT
40
Stem cells are undifferentiated cells capable of differentiating into specialized cells or
produce more stem cells by dividing. This ability for self-renewal, which is a trait of stem
cells, seems to be required of disseminated cancer cells to create metastases s3. Considering
that the EMT process is a mechanism by which cancer cells are enabled to spread, there is a
possibility that EMT also provides a self-renewal capability15 4. Interestingly, there are
parallels between the metastatic process and the steps involved in tissue repair and
regeneration, and these include the following actions of adult stem cells' 55 :
"
exiting tissue reservoirs
"
enter the circulation and survive
"
exit the circulation into secondary sites
"
proliferating, differentiating, and being involved in tissue reconstruction
Additionally, that cancer cells characteristic of stem cells are enriched in tumors after
standard chemotherapy furthermore implies a connection between EMT and cancer stem
cells (CSCs) given that there is increased therapeutic resistance of cancer cells that have
undergone an EMT1s6. Investigating this potential relationship between EMT and CSCs, it
has been found that there is a direct link between EMT and the acquisition of stem cell
properties15 7.
2.8.2. Signaling from Platelets to Cancer Cells Results in an EpithelialMesenchymal-Like (EMT-Like) Transition
58
Some of the numerous inducers of EMT in cancer cell lines include1 :
"
Transforming Growth Factor-p (TGF-)
"
Wnt
*
Snail/Slug
"
Twist
"
Six1
Within platelets are concentrations of TGF-P many times higher than most cell types and, by
the secretion of TGF-P and by activation through direct platelet-tumor cell contact of the
41
NF-KB pathway, platelets induce an epithelial-mesenchymal-like (EMT-like) transition in
CTCs and invasive behavior in vitro and promote the seeding of cancer in the lungs in vivols9.
Direct support for this proposal that EMT is mediated by TGF-P secreted by platelets comes
from observing strong TGF-P signatures through RNA sequencing after capturing with a
60
microfluidic device mesenchymal CTC clusters that often include attached platelets1 .
The fact that CTCs at least at times express epithelial markers, such as EpCAM and
cytokeratins, suggests that EMT is not an absolute prerequisite of tumor cells to access the
blood flow1 61. This suggests a mechanism by which tumor cells could become more
mesenchymal after leaving the site of the primary tumor and develop further metastatic
capacities162. The fact that potential significant advantages are conferred to CTCs once they
encounter platelets in the blood stream supports investigating whether enumerating and
studying platelet-CTC complexes in patients could provide valuable information in the
application of present treatments and for enhancing therapy for the future.
42
Chapter 3. A Protocol to Produce Platelet-Tumor Cell Complexes
Abstract
Testing the hypothesis that circulating tumor cells (CTCs) could be captured by means of
platelets required developing new protocols for sample preparation. Starting with whole
blood and flasks of cancer cells, tumor cells associated with activated platelets could
consistently be produced in buffer within hours. The work in this chapter served as a
foundation for further studies with techniques designed to isolate these complexes by
means of certain surface markers displayed by activated platelets.
3.1. Introduction
It is not apparent a prioriwhich of the numerous techniques and technologies shown
capable of capturing CTCs is most suitable for isolating CTCs by means of platelets. To
evaluate through rapid testing the capability of different approaches to capture platelet-CTC
complexes, there was a need to develop a method of producing samples of such clusters
reliably and from quickly replenishable sources. Lines of cancer cells can be grown in flasks
in incubators with appropriate media, and samples of blood can be collected from mice and
even purchased from vendors.
A fundamental first step to test these approaches was to identify cancer cell lines to which
platelets consistently attach, and evaluating the effectiveness of each approach required
isolating platelets from the massive number of background blood cells, such as white blood
cells and red blood cells, to drastically reduce the complexity of the system being studied.
While there are established protocols in the literature for this purpose, methods for
subsequently complexing platelets with cancer cells for the purposes of testing these
methods had not been published.
3.2. Components
3.2.1. Platelets
Given the complexity of blood and the great potential for interactions beyond those
between tumor cells and platelets, separating platelets from whole blood is critically
43
important for simplicity at least in the initial experiments of this study. Fortunately,
repeated centrifugation, aspiration, and resuspension can remove many of the components
of blood, such as the great number of red blood cells (RBCs), leaving platelet rich plasma
(PRP) or, if desired, washed platelets. In the latter case, the choice of buffer in the final
resuspension impacts the likelihood of the platelets to aggregate, which at a high level can
lead to clumping that interferes with experiments. The recipe for the buffer used in this
work for washing platelets and resuspending them after the final centrifugation step is in
the Chapter 3 Appendix and referred to here as the "platelet wash buffer". The protocol to
isolate PRP and washed platelets is also described in the appendix.
3.2.2. Cancer Cells
Given that lines of cancer cells have been shown to bind to platelets to varying degrees,
selecting one with proven affinity for platelets helps ensure that developing a protocol to
form platelet-tumor cell complexes is not impeded by the impossibility of binding. The first
cancer cell line tested to determine a protocol to produce platelet-tumor cell complexes was
LS180 (ATCC® CL-187'), which derives from a human colorectal adenocarcinoma
63
.A
mouse mammary cancer cell line, 4T1, was also tested. The growth of 4T1 cells in BALB/c
164
mice and subsequent metastatic spread is very similar to human breast cancer .
3.3. Protocol to Form Complexes of Platelets and Cancer Cells
A moderate degree of binding among platelets and cancer cells is necessary in order to
avoid forming large aggregates that are not useful in experiments intended to learn about
capturing CTCs with platelets as a marker. Starting with cancer cells cultured in a flask and
whole blood, it is possible to produce platelet-tumor cell complexes. The cancer cells must
be released from the flask and suspended in a buffer before adding them to washed
platelets or PRP. The mixture of these solutions is gently rocked before a final
centrifugation step to remove platelets not bound to the cancer cells. In order to determine
the location of platelets relative to tumor cells by microscopy, it is necessary to stain the
platelets, which was accomplished here with either calcein or an antibody tagged with a
fluorochrome. In addition to labeling platelets, the cancer cells can optionally also be
labeled with a dye that emits at a distinct wavelength since the platelets and cancer cells are
processed separately before interacting.
44
Details on the following experimental methods are described in this chapter's appendix, and
results from experiments using these methods are reported in Section 3.5 of this chapter.
3.3.1. Label Platelets with Calcein and Add to Cancer Cells
Calcein acetoxymethyl ester (calcein AM) permeates cells and only fluoresces after their
acetomethoxy groups are cleaved by intracellular esterases, which are active in live cells,
enabling the calcein portion of the original compound to fluoresce and to be trapped inside
the cell due to higher hydrophilicity. Platelets also contain esterases and, therefore, can be
labeled with calcein AM. After incubating platelets with calcein AM, at least one additional
centrifugation step is necessary to remove residual calcein AM that could label the cancer
cells when they are added to the platelets. A procedure similar to that of labeling platelets
is used optionally to label the cancer cells with a different dye, such as CellTrackerTM Red.
The set of steps to form clusters of tumor cells and platelets labeled with calcein is shown in
Figure 5.
whole blood
transfer
spa
Incubate
with Calcein,
TO
spin
AM
spin
;
transfer
LS180a
transfer
spin
[1eF
or run flow cytomneter
Figure 5. Label Platelets with Calcein and Add to Cancer Cells. After isolating platelets
from whole blood through centrifugation, they can be labeled with calcein AM, which
esterases in the platelets convert to calcein, producing fluorescence with a peak emission at
515 nm. 165
3.3.2. Label Platelets with Antibodies after Forming Platelet-Tumor Cell
Complexes
The steps to utilize an antibody in place of a cell permeable dye to test for the formation of
tumor-platelet complexes were very similar to the aforementioned protocol using calcein
45
AM. Rather than add calcein AM to isolated platelets, a fluorochrome-conjugated antibody
targeting activated platelets (anti-P-selectin PE) is added in the last set of steps (Figure 6).
Unbound antibody is removed through a final centrifugation.
LS180 or 4T1
sp
n
add w-oi.,
m., orr n flow *Vbom.Wr
Figure 6. Label with Antibody After Adding Platelets to Cancer Cells. After isolating
platelets from whole blood through centrifugation and incubated with tumor cells, they can
be labeled with antibodies and imaged or analyzed by flow cytometry.
3.4. Analysis by Fluorescence Microscopy and Fluorescence-Activated
Cell Sorting (FACS)
Fluorescence microscopy provided a quick, qualitative confirmation of the colocation of
platelets and tumor cells, and FACS served as a semi-quantitative measure of the percentage
of tumor cells bound to platelets. Samples of platelet-tumor cell complexes required no
additional processing prior to analysis by flow cytometry. Parameters on the FACS
instrument were set prior to acquisition to provide a proper visualization of different
populations, inlcuding unstained cancer cells, stained cancer cells, and stained cancer cells
attached to stained platelets. The population of stained cancer cells was gated, and the
percentage of tumor cells falling outside of this gate was calculated to provide a rough
determination of the number of tumor cells bound to platelets.
3.5. Results
In this chapter, a protocol is described that consistently results in the formation of platelettumor cell complexes, and it involved isolating and washing platelets from whole blood and
interacting them with two different types of cells, LS180 (human colon adenocarcinoma)
46
and 4T1 (mouse mammary tumor cell line), which have been shown in the literature to bind
to platelets. A variety of dyes and antibodies were used in experiments to verify that
platelets were indeed bound to tumor cells and that the platelets were activated, which is a
necessary feature to distinguish them from the resting platelets circulating abundantly in
the blood stream.
3.5.1. Verification of the Formation of Complexes Consisting of LS180s and
Platelets by Fluorescence Microscopy and FACS
Fluorescent microscopy and FACS provided evidence that the protocol developed to form
platelet-tumor cell complexes form whole blood and LS180s was effective.
Fluorescence Microscopy with Platelets Labeled with Calcein and LS180s
Platelets isolated form whole blood and labeled with calcein were incubated with LS180
tumor cells before imaging with fluorescent microscopy. The two images presented in
Figure 7 support that the platelets (green) are attached to the unlabeled tumor cells, which
are consistent with the size of LS180s observed in the tissue culture flask.
Figure 7. Fluorescence Microscopy with Platelets Labeled with Calcein and Tumor
Cells. Platelets labeled with calcein were incubated with LS180s grown in a flask and
subsequently imaged by fluorescence microscopy to qualitatively determine binding. (Scale
Bar: 20 im)
Fluorescence Microscopy with Platelets Labeled with Calcein and Tumor Cells
Labeled with CellTrackerTM Red
47
While the cells in initial experiments were of the expected size of LS180s and platelets, a
further experiment was performed with a second dye, CellTrackerTM Red, to distinguish
more conclusively between LS180s and platelets. In Figure 8, the LS180s are red, the
platelets are green, and the colocation of the two dyes is indicated by yellow, which
provides further qualitative support that platelets are binding to LS180s.
Figure 8. Verification of Platelet-Tumor Cell Formation by Fluorescent Microscopy.
Tumor cells are labeled with CellTrackerTM Red (red), and platelets are stained with calcein
(green). The overlap of these two colors is indicated in yellow. The bright-field image to the
left without color was captured from the same position as the composite picture. (Scale Bar:
20 prm)
Fluorescence Microscopy with Platelets Labeled with Anti-P-Selectin PE and Tumor
Cells
Through a series of experiments with several antibodies that target platelets, a particular
antibody, eBiosciences Anti-Human/Mouse CD62P (P-Selectin), was found that provides a
sufficiently strong signal to determine binding between platelets and tumor cells by
fluorescence microscopy and FACS. Figure 9 is a composite image of a sample prepared by
following many of the same steps in the protocol used with cell permeable dyes but with a
final step of adding an antibody. The results from an additional experiment (not shown)
that involved an isotype antibody also conjugated with PE did not provide a signal,
supporting that the anti-P-selectin antibody bound specifically.
48
Figure 9. Fluorescence Microscopy of Platelets Labeled with Anti-P-Selectin PE and
Tumor Cells. This composite image of platelets, LS180s, and anti-P-selectin PE antibody
(orange) further supports the conclusion that activated platelets are binding to tumor cells.
(Scale Bar: 20 [tm)
FACS with Platelets Labeled with Anti-P-Selectin PE and Tumor Cells
The final type of experiment to test binding of platelets to tumor cells involved FACS. In this
particular experiment - the results of which are shown in Figure 10 - three populations
of
cells were processed: unlabeled LS180, LS180 with CellTrackerTM Green, and LS180 with
CellTrackerTM Green and platelets with anti-P-selectin PE antibody. The spreading of
platelets and tumor cells to a higher PE signal while maintaining a strong FITC signal
strongly suggests that activated platelets are attached to tumor cells. Further, the small
number of events positive for PE and low in FITC is evidence of the relative lack of unbound,
activated platelets in solution.
49
Anti-P-Selectin
PE
Platelet
105
Platelet-
-0s
4) 03
10
<
Human Colon
Cancer Coll
-
d
0 102
103
10
105
CeNTracker
Sample
#PE+
#FITC+
%PE+
LS180(nodye)
0
0
0
LS180 CellTracker
GreenTM
164
9572
1.7
LS180 CellTracker
GreenTM, platelets,
anti-P-selectin PE
5062
7806
64.8
Figure 10. FACS with Platelets Labeled with Anti-P-Selectin PE and Tumor Cells.
Performing FACS with tumor cells and platelets and labeled with anti-P-selectin PE
provided further support that washed platelets bind to LS180s with the protocol developed
herein. Three different populations of tumor cells and platelets were processed labeled and
unlabeled to semi-quantitatively measure the degree of binding. As an approximation for
quantification, any events falling outside of the green polygon in the FACS plot surrounding
98.3% of the events from the sample of LS180s labeled with CellTracker GreenTM were
regarded as positive for PE.
3.5.2. Verification of Complexes of 4T1s and Platelets by Fluorescence
Microscopy
Combinations of 4T1s, platelets, and antibodies were used to determine whether washed
platelets bind to cells from this line as a result of essentially the same protocol outlined in
Chapter 3. Some of the cells did not strongly fluoresce, but, with the signal from the
antibody only apparent in the sample with 4T1s and platelets, images from this set of
50
samples strongly supported that conclusion that activated platelets were bound to the
tumor cells (Figure 11).
4T1
4T1 + Oatelets + antibody
4T1 + platelets
4T1 + antibody
Figure 11. Binding of Platelets to 4T1. The presence of the anti-P-selectin PE antibody
(red) in the fluorescence microscopy image at the top right provides evidence that washed
platelets isolated from whole blood bind to 4T1s (green) in buffer, especially in comparison
to the three images also shown, which served as controls. (Scale Bar: 50 Im)
3.6. Significance of Results
Much of the research into methods to isolate platelet-tumor cell complexes that is described
in the following three chapters depended on the development of the reliable method
presented herein to produce such clusters of cells with whole blood and a cancer cell line as
starting materials.
51
Chapter 3 Appendix
Reagents
Platelet Washed Buffer:
0
-
5 mM HEPES
145 mM NaCl
4 mM KCl
0.5 mM monosodium phosphate
5.5 mM glucose
0.5% BSA
After adding all the constituents to filtered water, the pH of the buffer was approximately
6.8, which is the pH at which the buffer was used.
Calcein:
BD Biosciences 354216
Calcein AM 10 x 500 pg
Resuspend in 200 VI DMSO to yield a molarity of 251 gM to create the stock solution
Dilute the stock solution to a concentration of 5 [tM in 1 ml of DMSO by adding 19.9 ptL.
CellTrackerTM Red:
Invitrogen
CellTrackerTM Red CMTPX
Add 14.6 ul of DMSO to vial of CellTrackerTM Red to reach a concentration of 5 mM for the
stock solution
Antibodies
*
eBiosciences Anti-Human/Mouse CD62P (P-Selectin), (eBiosciences 17-0626) - 1:20
dilution
Santa Cruz PSEL PE (sc-101336 PE) - 1:5 dilution
Mouse IgGi (PE) isotype antibody (abcam ab81200) - 1:10 dilution
Biological Materials
Blood
Whole human blood and mouse blood purchased from Bioreclamation in 10 mL quantities
and with either of the following anticoagulants:
52
*
-
Sodium EDTA
ACD
Tumor Cells
*
LS180 human colorectal adenocarcinoma
4T1 mouse mammary carcinoma constitutively expressing ZsGreen (gift from
Myriam Labelle of the Hyne's Lab at the MIT Koch Institute)
Protocols
Isolate Platelet Rich Plasma (PRP) from Whole Blood
e
e
Centrifuge whole blood at 1500 rpm, 7 min
Transfer PRP (yellowish solution on top layer) to new tube
Isolate Washed Platelets from Platelet Rich Plasma and Optionally Label Using
Calcein AM
*
e
-
e
e
Centrifuge PRP at 2200 g, 4.5 min
Discard the supernatant
Resuspend with Platelet Wash Buffer to desired concentration
o A typical concentration in this work was 1 mL of washed platelets per 1 mL
starting whole blood
Optionally, add calcein AM to the tubes
o Typically, 4.95 g1 calcein AM in DMSO at a concentration of 5 pM was added
per 1 mL washed platelets
Incubate the tube with washed platelets with calcein AM at 37 C for 1 hour
Spin platelets with or without calcein at 1900 g, 4 min and discard supernatant
Resuspend in buffer of choice with our without cancer cells for complexing
Complexing Platelets with Optionally Labeled LS180 Cancer Cells
*
e
*
-
Remove media from flask of LS180
Add 5 ml trypsin to flask
Allow to incubate 15 min at 37 0 C
Transfer cells to tube
Add 5 ml DMEM to cells with trypsin
Spin cells at 1200 rpm, 5 min
Aspirate trypsin and DMEM and add 10 ml DMEM
Count cells
Distribute cells into desired number of tubes
o Typical number of cells used is 1E6
Spin the tubes of LS180 cancer cells at 1200 rpm, 5 min
Remove media from tubes
Resuspend in buffer
53
Typically 1 mL of HBSS with calcium and magnesium added to reach
concentration of 1E6 / 1 mL
Optional labeling of LS180s with CellTrackerTM Red:
o Add 1 il CellTrackerTM Red
o Incubate for 20 min in water bath at 37 0 C
o Centrifuge at 1200 rpm, 5 min
o Aspirate supernatant
o Add 1 ml of DMEM with FBS and P/S to all three tubes of LS180s with
CellTrackerTM Red
o Incubate in 37*C incubator for at least an hour
o Centrifuge LS180s at 1200 rpm, 5 min
o Aspirate the supernatant
o Resuspend six tubes with buffer (typically 1 ml HBSS with calcium and
magnesium
Add LS180s in buffer to tubes with spun platelets
Place tubes of LS180s with platelets on rocker for 1 hour
After gentle rocking, spin the samples of platelets with LS180s at 1200 rpm, 5 min
Discard the supernatant of both tubes and resuspend tube according to
experimental goals
o Platelet-tumor cell complexes were typically resuspended in buffer, such as
HBSS with calcium and magnesium, or whole blood
o
e
-
Labeling Platelets with Antibodies After Forming Complexes
*
e
e
*
e
After centrifuging, remove the supernatant
Add 100 pl of the dilution solution of antibody to platelet-tumor
Place samples on ice for 1 hour
Remove the samples from ice that had been incubating with the antibodies
Add 1mlHBSS
Centrifuge 1200 rpm, 5 min
Remove supernatant
Add 1 ml buffer
Spin 1200 rpm, 5 min
Remove supernatant
Resuspend with buffer for further analysis
Complexing Platelets with 4T1 Cancer Cells
*
-
Remove media from flask of 4T1 and add 5 mL trypsin.
Allow to incubate for about 5 min in the 37*C incubator
Remove cells from incubator, remove trypsinized cells, and place in tube
Add 5 mL media (RPMI) to cells with trypsin
Spin cells at 1200 rpm, 5 min
Remove media and trypsin and add 4 mL media
Count cells and dilute typically to 1 x 106 / mL in centrifuge tubes
Spin tubes at 1200 rpm, 5 min
Remove media from tubes and add 1 mL HBSS with calcium and magnesium to
tubes
Place in 37*C incubator
54
-
Wash PRP by spinning at 1900 g, 4 min, and discard supernatant
Add solution of 4T1s to spun down platelets and rock 1 hour
Spin samples that contain platelets and 4T1s at 1200 rpm, 5 min
After spinning, remove supernatant and add HBSS with calcium and magnesium to
both tubes
Intrumentation
Microscope
Olympus IX51
Flow Cytometer
Performed at the MIT Koch Institute Flow Cytometry Core Facility:
-
BD FACScan running CellQuest Pro
BD FACS LSR HTS running BD FACSDIVA
Analysis
Analyze Samples by Fluorescence Microscopy
e
-
Add small volume of the samples (typically 100 gl) to a 96-well plate (Falcon ProBind flat bottom).
TRITC and FITC filters were used to image PE and calcein-stained cells.
Brightfield images were also captured.
55
Chapter 4. Initial Approaches to Capture Platelet-Tumor Cell
Complexes
Abstract
Proof-of-concept experiments to test the hypothesis that circulating tumor cells (CTCs) can
be captured by means of platelets first involved magnetic activated cell sorting (MACS).
Enrichment of tumor cells with platelets was evident, but the constraints associated with
MACS motivated testing a second approach - a flow chamber with functionalized substrates
to capture or otherwise direct the movement of target cells. The results from both
approaches confirmed the merit of further testing the degree to which CTCs could be
captured by means of attached platelets, and the learning generated helped guide the next
set of experiments with microfluidics (Chapter 5).
4.1. Introduction
The possibility of using activated platelets as targets to capture cancer cells was tested
initially with two technologies: magnetic activated cell sorting (MACS) and a functionalized
flow chamber. Samples of platelet-tumor cell complexes were created according to the
protocol generated in the previous chapter.
4.2. Magnetic Activated Cell Sorting (MACS)
Magnetic activated cell sorting as marketed by Miltenyi Biotec (MACS® Technology and
referred hereafter simply as MACS) is a specific technology that falls within a category of
cell separation techniques that operate by first attaching magnetic particles to cells of
interest. The cells are then isolated by applying a strong magnetic field. Proven to be an
effective approach for cell separation, including the isolation of extremely rare cells from a
much larger population of background cells 166, MACS has also been utilized in previous
studies to separate CTCs167.
4.2.1. Background on MACS
Fundamentals of Technology
56
As a first step in MACS, magnetic nanoparticles coated with antibodies that target a
particular surface antigen are incubated in a solution containing the cells of interest. After
sufficient time, the magnetic nanoparticles bind to the target cells, and the entire solution is
then passed through a column that is placed within a strong magnetic field. During this step,
cells coated with magnetic nanoparticles are collected within the column, and background
cells pass through. After removing the column from the magnetic field, the strong attractive
force in the column essential to retaining the cells no longer exists, and the target cells are
flushed out with a plunger and amassed in a collection tube.
There are two broad strategies for MACS - positive selection and negative selection - and a
method for vastly increasing the number of cell surface markers that can be utilized in
MACS. In positive selection, magnetic nanoparticles are coated with an antibody that
attaches to an antigen present on the target cell, while, in negative selection, the
nanoparticles coat cells that need to be removed from the solution containing cells desired
to be captured.
Applications of MACS
Given the wide applicability of MACS, the number of this technology's potential applications
is extensive. Broad research areas where MACS is used include: immunology, stem cell
research, neuroscience, and cancer research168.
4.2.2. MACS for Capturing Cancer Cells with Platelets
Using MACS as a technique to capture cancer cells with platelets has not been reported, and
identifying the most appropriate type of MACS MicroBeads (Miltenyi Biotec), size of MACS
Cell Separation Columns (Miltenyi Biotec), cancer cell line, source of platelets, and
procedures related to using all these materials was necessary.
MACS MicroBeads
MACS MicroBeads are 50 nm superparamagnetic particles coated with a particular type
among the vast array of antibodies169. Targeting two different antigens on platelets almost
or entirely unique to that cell when activated was sought as a potential means of isolating
57
platelet-CTC complexes from the abundant background cells in blood. One of the platelet
markers hypothesized to fulfill these requirements was P-selectin, a cell adhesion molecule
(CAM) that is stored in a-granules and translocated to the plasma membrane upon platelet
activation 17 0 .
Many types of MicroBeads are sold that are conjugated to antibodies that bind targets on
cells of great interest. For research into cells that are not researched as commonly,
MicroBeads can still be employed. In this case, the surface of the magnetic nanoparticles is
coated with antibodies that bind specifically to particular fluorochromes, such as
fluorescein isothiocyanate (FITC) or allophycocyanin (APC). Since fluorochromes are often
attached to commercial antibodies or could be conjugated by an individual researcher by
use of a kit, an even vaster number of cell types can be isolated by MACS by using magnetic
nanoparticles that have been coated with anti-fluorochrome antibodies.
Since antigens can be sufficiently large in size to contain multiple regions, or epitopes,
recognizable by as many antibodies and since these pairs of epitopes and antibodies can
vary in their affinity, determining which antibody is adequate for an application can be
critical. In this application, anti-phycoerythrin (anti-PE) MicroBeads were used to bind to
an antibody conjugated with phycoerythrin (PE) that targets P-selectin on platelets - anti-Pselectin PE antibody (Figure 12).
Anti-PE
MicroBeads
Anti-P-Selectin
PE Antibody
Human Colon Cancer Cogl
0L180)
-
Platelet
Figure 12. Attaching MicroBeads to Platelets Bound to Tumor Cells. Anti-PE
MicroBeads bind to antibodies conjugated with PE. Thus, the anti-P-selectin PE antibody
that targets activated platelets provides a site for the MicroBeads to attach to the platelettumor cell complexes.
MACS Cell Separation Columns
58
The columns through which the solution of cells and MicroBeads passed in order for the
target cells to be captured include a matrix containing ferromagnetic spheres coated with a
material that is not harmful to cells171. The spheres in the column increase the magnetic
field 10,000 times producing a gradient large enough to work with cells that are labeled
with magnetic nanoparticles to a degree that does not cover all epitopes1 72. Further, there is
sufficient space in the column between spheres to allow cells to flow freely. Among the
various models of MACS Cell Separation Columns, which accommodate different input
volumes and selection types, the MS Column appeared most suitable, for it could
accommodate up to 2x10 8 total cells and is designed for positive selection.
4.3. Protocol to Isolate Tumor Cells with Platelets Using MACS
After forming the platelet-tumor cell complexes and attaching the antibody, the MicroBeads
are added and incubated before centrifuging and washing to remove unbound MicroBeads.
The column through which the cell solution must pass is first placed in the MACS Separator
in there is a strong magnetic field, and buffer is passed through in an initial step to remove a
73
hydrophilic coating contained in the column that allows rapid filling1 . The cell suspension
is then applied to the column, and unlabeled cells pass through and are collected. Three
additional washes are performed to remove additional unlabeled cells. The total effluent is
collected either in one tube, or one tube is used for each of the three washes to allow for
finer analysis. In the last step, the column is removed from the separator and its
concentrated magnetic field to perform a final flushing of the contents of the column, which
are expected to be magnetically labeled cells (Figure 13).
59
flow through
wash I
wash II
wash I
collection
Figure 13. Performing MACS and Collecting Effluent at Each Stage. The column is
placed in the MACS Separator where its magnetic field is concentrated. The cell solution
containing both labeled and unlabeled cells is added in the first step, and the effluent is
collected as the initial "flow through". The effluent from the next three washes is either
collected in a single tube or separate tubes (pictured) for analysis yielding the "wash I",
"wash II", and "wash I1" samples. In the final step, a plunger is used to forcefully expel the
contents of the column now removed from the MACS Separator, and this "collection" sample
is expected to contain the cells labeled with MicroBeads.
4.4. Results
4.4.1. Apparent Enrichment of Platelet-Cancer Cell Complexes
After demonstrating that platelet-tumor cell complexes can be formed using cultured
LS180s and washed platelets from whole blood, the next step was to use them to test the
approach of isolating the complexes with MACS. First, the fluorescence of the antibodies
attached to the cells was tested after addition of MicroBeads to examine whether binding of
the magnetic nanoparticles to the fluorochrome on the antibody affected the interaction of
the antibody to its target or quenched its signal. Then, MACS was performed and effluent
from each step analyzed to determine whether the complexes were retained until the final
collection step.
Fluorescence Microscopy with Platelets Labeled with Anti-P-Selectin PE and Tumor
Cells After Addition of MicroBeads
60
The similarity of Figure 14 to Figure 9 indicates that any binding of the MicroBeads to the
fluorochrome on the antibody does not significantly diminish its signal due either to
potential release of the antibodies from the target or quenching.
Figure 14. Fluorescence Microscopy with Platelets Labeled with Anti-P-Selectin PE
and Tumor Cells After Addition of MicroBeads. After forming platelet-tumor cell
complexes, adding anti-P-selectin PE, and MicroBeads that attach to PE, the resulting
sample was imaged by fluorescence microscopy to determine that the addition of
MicroBeads does not diminish the signal and the antibody remains attached to its target.
(Scale Bar: 20 pim)
Fluorescence Microscopy of Effluents from MACS with Platelets Labeled with Anti-PSelectin PE and Tumor Cells
To examine the retention of platelet-tumor cell complexes during MACS, effluent from each
step was collected and examined by fluorescence microscopy. Images from the first flow
through and subsequent washes, which show no fluorescence, and the final collection,
which shows abundant signal from PE colocated with the tumor cells, shows the potential of
using MACS as a method to isolate platelet-tumor cells complexes from solution (Figure 15).
61
Step 1: Flow Through
Step 2: First Wash
Step 4: Third Wash
Step 3: Second Wash
Step 5: Collection
Figure 15. Fluorescence Microscopy of Effluents from MACS with Platelets Labeled
with Anti-P-Selectin PE and Tumor Cells. While performing MACS on platelet-tumor cell
complexes labeled with anti-P-selectin and MicroBeads, effluent from each step outlined in
Figure 13. The PE signal was observed only in the collection step, providing evidence that
MACS could be used to isolate platelet-tumor cell complexes with P-selectin on the platelets
as a marker. (Scale Bar: 20 pm)
4.5. Limitations of MACS of a Technique to Capture Platelet-CTC
Complexes
While experiments demonstrated the potential of MACS to isolate platelet-tumor cell
complexes, the greatest impact would come from separating a much smaller number of
complexes from whole blood. A major limitation associated with using MACS for isolating
rare cells is that the final volume is relatively large, which raises difficulties in validating
results and studying the cells further. Due to the large collection volume that would contain
captured platelet-tumor cell complexes and the small concentration of CTCs that could be
isolated, using MACS in processing samples from patients would require a large number of
slides to be scanned to confirm the presence and number of captured complexes. Further
analysis and study of the complexes could also be facilitated with alternative approaches.
Although the results from this set of experiments with MACS supported further pursuit of
testing whether platelet-CTC complexes could be captured with platelet markers, these
limitations were sufficient reason to adjust the approach in this study.
62
4.6. Interactions and Isolation of Platelet-Tumor Cell Complexes with
Flow Chamber
Using a flow chamber to isolate platelet-CTCs would provide potential benefits over MACS,
such as a much smaller capture space, which facilitates result analysis and visualization of
the interaction of the cells with the functionalized substrate.
4.6.1. Background
Flow chambers are used in a variety of experiments. Fluids generally enter and exit a flow
chamber on opposite sides. The sample introduced into the chamber, the functionalization
of the substrate over which it passes, and the flow rate are fundamental parameters that
vary according to the experiment.
General Description of Flow Chamber System
The flow chamber consists of glass slides or another type of substrate that may be
functionalized, a silicon gasket that determines the height of the chamber according to the
gasket thickness, and a distributor, which includes inlet and outlet ports and a vacuum slot.
Utilizing this latter slot, these three main components are sealed by vacuum, and a pump
introduces fluid at a controlled rate. An optical microscope can often be used to observe the
happenings inside the chamber since the bottom slide and top of the device are usually
transparent, and a computer connected to the microscope can store images and videos of
the flow experiments.
Experimental Opportunities Afforded by Either Uniform or Asymmetric Flow Cell
Functionalization Patterns
The substrate over which the sample injected into the device flows can be functionalized,
such as with various molecules, peptides, and proteins, in a uniform arrangement or in
patterns suited to the application. Visualizing cells flowing in the chamber over a uniformly
coated slide enables identification of the interaction of the cells with the functionalizing
entity. The strength of the interaction between the surface and cell determines whether the
cell adheres tightly to the surface, continues flowing unimpeded, or behaves between these
extremes, such as by rolling on the substrate.
63
A particular advantage from patterning the slide asymmetrically is potential label-free
isolation by cell sorting through rolling174. One needs to identify an appropriate pair of
ligands and surface markers, such as a receptor, that interacts in the proper range of affinity
that results in rolling. By patterning the ligand in angled lines on the slide relative to flow,
the cells can be directed to a different outlet relative to the cells of other types. One set of
ligand-receptor pairs that has been demonstrated as being capable of enabling cell rolling is
P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin. In fact, this interaction has been
quantitatively characterized in a flow chamber in the attempt to optimize cell separation
75
.
The relevance of P-selectin in interactions between platelets and tumor cells176 motivated
the testing of PSGL-1 as a potential ligand to use in isolating platelet-tumor cell complexes.
4.7. Rolling and Binding of Tumor Cells with Platelets on Functionalized
Substrates with Flow Chamber
As tumor cells have been shown to roll and adhere to endothelial cells177, which form a thin
layer on the interior surface of blood vessels, utilizing cell rolling seemed to be a natural
approach to test the interaction of platelet-CTC complexes with a ligand and attempt to
isolate them.
4.7.1. Cancer Cell Line, Whole Blood, and Formation of Platelet-Tumor Cell
Complexes
The LS180 human colorectal adenocarcinoma cell line, which was used in experiments with
MACS, was also processed through the flow chamber. Furthermore, the same procedure to
isolate platelets from whole blood and create platelet-tumor cell complexes was followed in
the flow chamber device experiments as in the MACS experiments.
4.7.2. Patterning PSGL-1 on Petri Dish for Testing with Flow Chamber
Testing whether platelet-CTC complexes could be separated by cell rolling through the
interaction between P-selectin and PSGL-1 was supported by an earlier demonstration that
the strength of affinity of this receptor-ligand pair is appropriate for cell rolling178. While
this prior proof-of-principle involved experiments with PSGL-1 expressed by the cells and
64
P-selectin patterned on the device substrate, attempting to separate platelet-CTC complexes
requires the reverse placement with PSGL-1 patterned on the substrate that is part of the
flow chamber since P-selectin is expressed on the platelets. Fortunately, PSGL-1 was
available commercially in chimeric form with the Fc region of IgG1 at the C-terminus (PSGL1 Fc). In testing interactions of platelet-tumor cell complexes with functionalized surfaces
of petri dishes, PSGL-1 Fc and Protein A were attached to the plastic through physisorption
either individually or in combination by mixing before depositing. Several regions of the
plastic were functionalized differently and observed during the flow of cells through the
flow chamber (Figure 16).
input (cells in buffer)
vacuum
output
Figure 16. Configuration of Flow Chamber on Petri Dish to Test Interaction with
PSGL-1 Fc, Protein A, and a Combination Thereof. LS180s with and without platelets
were passed through a flow chamber placed on a petri dish that was functionalized in
separate regions with PSGL-1 Fc, Protein A, and a combination thereof. A region of the petri
dish that was not functionalized with these molecules was also observed during flow to
determine the degree of non-specific interactions.
4.7.3. Patterning PSGL-1 Asymmetrically on Gold-Coated Glass Slide for
Testing with Flow Chamber
Glass slides coated with layer of gold were the substrates that PSGL-1 Fc was patterned on.
After a piranha clean of the slides, dithiobis(succinimidylpropionate), or DSP, was added to
the gold surface to allow Protein A to attach to the substrate covalently. Protein A binds to
the Fc region of the PSGL-1 chimera protein allowing for the latter's specific attachment to
the surface in a particular orientation. Finally, regions that aren't functionalized are
blocked with a serum albumin protein to avoid nonspecific binding of the cells.
65
platelet
P-selectin
PSGL-1
Fc
Protein A
I
BSA
Gold
Figure 17. Schematic Depiction of Functionalization of PSGL-1 Fc on Gold-Coated
Glass Slide. By covalently attaching Protein A to a gold surface, PSGL-1 Fc can also bind in
an oriented fashion. The intent was to make available the PSGL-1 portion of the chimera for
interaction with P-selectin displayed on activated platelets, which were expected to be
bound to tumor cells in solution.
In order to create asymmetric patterns (diagonal lines relative to the direction of fluid flow)
on the gold-coated glass slides, the pattern is first fabricated on gold by photolithography
and then etched away and cleaned by a dip in hydrofluoric acid. The slide is incubated
overnight with a low percentage of polyethylene glycol silane (PEG-silane) in toluene to
passivate the glass. The remaining steps to functionalize the gold are the same as those
described above in the preceding paragraph.
4.7.4. Instrumentation
The solution of cells was forced through the flow chambers either on the petri dish or glass
slides by using a syringe pump. To examine the interaction of the platelet-tumor cell
complexes with the functionalized surfaces substrates as the cells passed over them, images
were captured with a fluorescence microscope.
4.8. Results
4.8.1. Interaction between Platelet-Tumor Cell Complexes on Uniformly
Coated PSGL-1 Surface
Comparing single frames from videos of solutions of platelet-tumor cell complexes (Figure
18, left) and only tumor cells (Figure 18, right) passing over petri dish surfaces
66
functionalized uniformly with PSGL-1 indicate an interaction between the tumor cells and
surface very likely mediated by attached platelets. In addition, experiments with glass
slides coated with gold and uniformly patterned with PSGL-1 also demonstrated binding
and rolling of the platelet-tumor cell complexes (not presented here).
I
Figure 18. Optical Microscopy Images Showing Rolling and Attachment of Tumor Cells
Coated with Platelets and No Apparent Interaction Without Platelets. Tumor cells in
clusters of various sizes roll and attach to a PSGL-1 coated substrate (left) and appear not to
interact in the absence of platelets but with the same type of functionalized surface as
evidenced by blurred objects in the image indicating cells in motion (right). (Scale Bar: 20
Im)
4.8.2. Attempt to Separate Complexes by Rolling on Asymmetrically Patterned
Slides
Attempts to separate such complexes by directing the direction of their flow, though, with
asymmetrically patterned PSGL-1 on surfaces of gold-coated glass slides did not yield the
desired result. This is thought to be due to geometric constraints resulting from incomplete
coverage of the platelets over the surface of the tumor cells, which could preclude
consistent rolling along the edge of each stripe of the patterned PSGL-1.
4.9. Summary of Initial Two Approaches
By analyzing through fluorescence microscopy the effluent of each step in MACS,
enrichment of platelet-tumor cell complexes was evident Flow chambers with slides
67
functionalized with PSGL-1 Fc showed a platelet-dependent rolling and attachment of
tumor cells.
4.10. Significance of Results
Results from flow chamber studies supported continued testing of the general approach of
utilizing platelets to capture CTCs and guided subsequent experiments with another flowbased approach - microfluidics.
68
Chapter 4 Appendix
The following is in addition to those materials and methods described in Chapter 3
Appendix required to create samples for analysis.
Reagents
MACS Microbeads and Buffers:
Miltenyi Biotec Anti-PE MicroBeads
130-048-801
MACS Labeling Buffer: 2mM EDTA in PBS
MACS Separation Buffer: 2mM EDTA in PBS with 0.5% BSA
Antibodies
"
eBiosciences Anti-Human/Mouse CD62P (P-Selectin), (eBiosciences 17-0626) - 1:20
dilution
Santa Cruz PSEL PE (sc-101336 PE) - 1:5 dilution
Mouse IgG1 (PE) isotype antibody (abcam ab81200) - 1:10 dilution
PSGL-1 Fc
Recombinant Human PSGL-1/CD162 Fc Chimera
R&D Systems (Catalog Number: 3345-PS)
Protocols
Adding MicroBeads to Platelet-Tumor Cell Complexes (Protocol Modified from That
Provided by Supplier, Milteny Biotec)
*
*
-
Add 1 ml HBSS to the sample of platelet-tumor cell complexes that had been
incubating with antibody
Spin 1200 rpm, 5 min
Remove supernatant
Resuspend the sample in 80 ul DPBS
Add 20 gl anti-PE MicroBeads
Mix the sample well by slightly vortexing
Refrigerate at 4C, 15 min
Add 1 ml DPBS to sample
Spin at 300 g, 10 min
Remove supernatant
Add1mlDPBS
Spin at 300 g, 10 min
Remove supernatant
Resuspend in 500 ul PBS + 0.5 % BSA
69
Performing MACS with Platelet-Tumor Cell Complexes with Attached MicroBeads
(Protocol Provided by Supplier, Miltenyi Biotec)
e
*
-
e
Place column in the magnetic field of a suitable MACS Separator.
Prepare column by rinsing with appropriate amount of separation buffer (500 uL)
Apply cell suspension onto the column
Collect unlabeled cells that pass through and wash column with appropriate amount
of separation buffer (3x500 uL). Perform washing steps by adding separation buffer
three times. Only add new buffer when the column reservoir is empty. Collect total
effluent. This is the unlabeled cell fraction.
Remove column from the separator and place it on a suitable collection tube.
Pipette an appropriate amount of separation buffer onto the column. Immediately
flush out the magnetically labeled cells by firmly pushing the plunger into the
column (1mL)
Procedure to Functionalize Gold and Petri Dishes with PSLG-1
Glass Slide Coated with Gold - Uniform Coating (from Suman Bose):
*
"
*
-
Piranha clean the glass slide coated with gold (EMF)
Add DSP (10 mg / mL) [dithiobis(succinimidylpropionate)] in anhydrous DMF
(dimethylformamide) for 30 min
Wash
Incubate with Protein A (1 mg / mL) for 1 hour
Wash with PBS twice
Incubate with PSGL1-Fc (15 gg / mL in 1%BSA) for 3 hours
Immerse slide in 1% BSA
Glass Slide Coated with Gold -Asymmetric Patternson Slides (from Suman Bose):
e
e
e
e
-
Fabricate the pattern by photolithography on gold slide and then etch the gold away.
Clean by HF dip.
Treat with 2% PEG-silane in anhydrous toluene overnight (passivate glass)
Wash and treat with 1mM DPS in anhydrous DMF for 1 hr.
Put gaskets (250 um tall) and incubate with protein A (1mg / ml) for 30 min
Aspirate the Protein A, wash with buffer, fill with PSGL-1-Fc (10 or 15 gg/ml).
Incubate for 2-3 hours
Remove gasket, wash with buffer, block with 1% BSA for 1 hr
Petri Dish
e
-
Mix PSGL-1 Fc (15 g/ml) with Protein A at a ratio of at least 1:20
Incubate directly on petri dish to attach
Flowing Cells Through Flow Chamber
The solutions of cells passed through the flow chamber (Glycotec) at 75 [IL / min, which
equates to 0.5 dyn / cm 2 by using syringe pumps.
70
Analysis
Analyze MACS Samples by Fluorescence Microscopy
-
Add a small volume of the samples (typically 100 pl) to a 96-well plate (Falcon ProBind flat bottom).
TRITC and FITC filters were used to image PE and calcein-stained cells.
Brightfield images were also captured.
Analyze Cell Rolling and Attachment with Flow Chamber by Fluorescence Microscopy
Microscope: Nikon TE2000-U
Camera: Andor iXon 885
Filters: FITC, Cy3, CyS, DAPI
71
Chapter 5. Capturing CTCs with Platelets with Herringbone
Microfluidic Device
Abstract
Experiments outlined in the previous chapter supported the hypothesis that surface
markers on platelets could be used to capture platelet-tumor cell complexes, yet the results
also suggested that a technology other than MACS or a flow chamber could be critical in
enabling both rapid and sensitive analysis and capture. The progression of experiments
with the microfluidic device followed from the need initially to show the feasibility of
capturing platelet-tumor cell complexes under ideal conditions, which would demonstrate
that the strength of interaction between the antibody and platelet surface marker was
sufficiently strong to capture the complexes and also that this interaction would occur with
the necessary frequency throughout the flow of the cells through the device channels. Next,
testing samples of blood spiked with platelet-tumor cells was important to prove that the
potential interference and attachment of all the components of blood would not critically
impair the attachment yield. Finally, working with mouse models would be the most realworld demonstration of the effectiveness of this approach for capture and could even
enable further study of the spread of cancer even before transitioning to human patient
samples. These three steps are discussed in this chapter with concluding reflections and
remarks about possible future studies.
5.1. Introduction
Though similar in certain core aspects to a flow chamber, microfluidic devices provide a
much higher level of customization in physical design and functionalization, which has
enabled a rich diversity of device applications, and one of these is the capture of CTCs
through their unique or overexpressed surface markers. Especially since the report of the
CTC-chip, microfluidic design for this purpose has increased in sophistication and
effectiveness through improvements in the choice of materials to fabricate such devices, the
geometry of the channels through which the fluid flows through them, and even through
using force at a distance, such as by applying a magnetic field, that directs targets in the
solution to the substrate or a particular channel.
72
5.2. Rationale for Use of Microfluidics for CTC Capture
5.2.1. Description of Microfluidics and Applications
"Microfluidics" is a term for devices or configurations of flow that manipulate small volumes
of fluids (10-9 to 10-18 L) through channels that are on the scale of micrometers17 9 1 80. The
ability through microfluidics to manipulate fluids so precisely has naturally led to a great
diversity of applications as seen from the beginning of its development in the two original
demonstrations of microfluidics: a gas chromatograph made on a silicon wafer'81 and arrays
of ink jet printing nozzles etched in silicon' 82. The range of applications of microfluidics has
expanded significantly since its first demonstrations, and a selection of uses is outlined in
Table 4.
Table 4. Examples of Microfluidic Applications within a Selection of Fields and
Industries
Industrial applications of
combinatorial synthesis
S
Rapid chemical analyses
High throughput screening
Automotive industry
e
Mass-flow sensor' 83
Microreaction technology
*
Nanomaterial synthesis184
Printing
*
Inkjet printhead
Optics
*
*
Liquid micro-lens array1 8s
3D microfluidic microscope 186
Life sciences
e
Pharmaceuticals and biomedicine
o Drug design
o Delivery and detection
o Diagnostic devices
Medicine
o Noninvasive diagnostics and surgery
87
o Drug delivery1
S
9
5.2.2. Development of Microfluidic Devices Leading to and Including the
Capability of CTC Separation
Four great drivers of microfluidic development include the desire to use much smaller
amounts of sample and attain high sensitivity and resolution in molecular analysis, to create
73
field-deployable detectors of chemical and biological weapons for biodefense, to attain
greater throughput, sensitivity, and resolution in analytical methods for the field of
molecular biology, and the vision that the successes in silicon microelectronics could be
translated to microfluidics1 88. The foundational knowledge and technology for making
microfluidic devices - microfabrication techniques - had roots in the semiconductor
industry, which is the same field that provided a vision for the possibilities of these new
devices' 89. Interestingly, methods of producing chips to control electrons were also well
suited to create devices to precisely manipulate fluids'
90
.
For particular applications, there was a need to transition from the initial use in
microfluidics of hard materials, such as glass and silicon, to alternative fabrication methods
and materials, including soft lithography and silicone rubber' 91. One of the reasons for
moving from silicon is that it is expensive and blocks the spectrum of light that is used in
conventional optical detection methods192. Pumps and valves and other components
necessary in microanalytical systems can be built in elastomers more easily compared to
rigid materials193. Furthermore, glass and silicon are not permeable to gases, which can be
of critical importance when studying living mammalian cells194 .
Analysis with Microfluidics in Chemistry and Biology
The recognition of the strengths associated microfluidics has resulted in significant interest
in employing this technology to accelerate assays in the life sciences and chemistry and for
syntheses'
95
. There has been a long-standing vision of integrating devices designed for
different purposes, such as sample preparation, detection, and analysis, into a single system.
These lab-on-a-chip systems automate the execution of multiple protocols with one device
and performs them potentially much more quickly. For instance, a chip has been designed
and shown able to extract RNA from Gram-positive bacteria in about 10 minutes, whereas
the use of a kit for the same purpose requires an order of magnitude more time while still
necessitating an additional process to remove contaminating DNA and ultimately yielding
lower quantities of RNA196. Just this reduction in time and manual labor with microfluidics
generally has great implications, particularly in facilitating high-throughput screening
studies, yet with further potential benefits, including parallel analysis, shorter reactions,
and smaller reagent volumes, there is a firm belief that microfluidics will revolutionize
97
research techniques in biology and chemistry1 .
74
With channels and biological cells both on the scale of microns, attempting to extend the
accurate and efficient analysis of biomolecules to entire cells with microfluidics is natural in
some respects. Some challenges in molecular analysis are found also in the study of cell
samples. Separation is an essential element in analysis of biomolecules, chemicals, and
cells
19 8
, and such samples are usually very complex with various amounts of the different
components with the most important often in the smallest quantity. Also, cells even of the
same lineage must at times be studied individually due to natural variation, which requires
techniques to separate and sort cells'
99
.
Advantages of Microfluidics for CTC Enumeration and Study
The potential advantages of microfluidics over MACS, FACS, and a flow chamber as a
method to enumerate and study CTCs are significant. Fabrication and functionalization of
microfluidic devices can be much less expensive than MACS and FACS, which require
labeling antibodies, magnetic nanoparticles, and further reagents in addition to the initial
cost of $250,000 for such systems200 . Furthermore, to meet high-quality standards in
clinical diagnosis with such complex systems, the operator of the instrumentation
201
associated with MACS and FACS would need to be specialized and trained . Even for those
trained, recognizing an extraordinary low number of events by FACS may not be possible
due to inadequate sensitivity202. Built with appropriate materials, a microfluidic device is
transparent, and its relatively small area in which CTCs can be captured and detected is
visible by a simple microscope. In addition, while fluid shear stresses in microfluidic
devices can be precisely predicted and controlled, such forces in these other cell sorting
03
devices may impact cell function and change their viability2 .
Compared to a flow chamber, a microfluidic device can be more easily customized in terms
of channel geometry and even perform functions that traditionally require dedicated
instrumentation and an entire lab bench. The minimum sample volume to be processed in a
microfluidic device is also lower than a flow chamber depending on their design. Finally,
typically all channel surfaces in a microfluidic device can be functionalized with the
particular capture agent, whereas in the case of a flow chamber this is usually located only
the bottom slide.
75
The CTC-Chip Demonstrated the Effectiveness of Microfluidics to Capture CTCs
Many of the advantages in using microfluidics for cell analysis are highlighted by the
specific application of capturing CTCs from whole blood by this approach. A most
outstanding initial demonstration of the feasibility of employing microfluidic devices for
isolation of CTCs was reported in 2007, when the most advanced strategies to capture CTCs
were complex with low yield and purity 204. Designing and utilizing this "CTC-chip" was a
step forward not only in demonstrating the ability of microfluidics to process milliliters of
whole blood, which is much greater than the typical microliter amounts of simpler fluids
dealt with by microfluidics, but also in isolating epithelial CTCs with greater sensitivity and
specificity 205 .
Although optimization was still required, the CTC-chip was as straightforward in its
approach for capture as it was effective. The heart of the device consisted of an array of
78,000 microposts arranged in a pattern based on simulation results and chemically
functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies, to which
CTCs from metastatic lung, prostate, pancreatic, breast and colon cancer all adhere but to
which haematologic cells do not 2 06. The theoretical analysis to optimize the geometrical
pattern of posts utilized empirical findings, which demonstrated the range of shear stress
207
and flow rate that facilitated the greatest cell attachment . These two parameters
critically determine the cell capture efficiency as the amount of time the cell contacts the
micropost must be sufficiently long and the force between the two adequately low
2 08
.
Testing with a particular line of non-small-cell lung cancer (NSCLC) cells spiked into
phosphate buffered saline (PBS) and healthy whole blood determined that the overall
efficiency could reach greater than 60%, the ideal range of flow rate in practice, and that
there is no need to treat the blood-sample prior to flowing it through the CTC-chip, such as
centrifugation, washing, labeling, lysing, or fixation. These successful proof-of-principle
experiments were precursors to the most practical tests involving processing patient
samples, which provided powerful evidence of the CTC-chip's potential. Out of 116 patient
samples tested with the device, more than 99% of them were found to contain CTCs, and at
least five CTCs were found on the chip for each of these 115 samples.
76
Beyond enumeration, testing for the captured CTCs' expression of tumor-specific markers
2 9
was possible through immunostaining and PCR with reverse transcription . Generally, the
presence on the device of cells other than the target can hinder molecular analysis, yet the
CTC-chip yielded approximately 50% purity, which is an improvement from that of the then
210
currently available technologies by two orders of magnitude .
With the demonstrable ability to isolate CTCs and promising results for the future of being
able to test for the presence of individual transcripts associated with tumor markers, a
perhaps more immediate question of drawing meaning from data produced by the counting
211
and characterization of captured CTCs on cancer patient treatment was also addressed .
By comparing tumor volume measurements from computed tomograms (CT scans) with
CTC quantities, the number of CTCs on chip as a function of time correlated reasonably well
with response to treatment 212. Further, the high sensitivity of the CTC-chip made possible a
novel observation in this study of the same concentration of CTCs in blood samples from at
least some subjects with clinically localized prostate cancer and from those with metastatic
prostate cancer 213.
The CTC-chip was a significant advance towards reaching the potential of microfluidic
technology to discover the determining biological agents of metastases formed by spread
214
through the blood and to assist in cancer diagnosis and monitoring . Since 2007,
subsequent generations of microfluidic devices for CTC capture and analysis have
eliminated numerous limitations inherent in the CTC-chip while maintaining many of its
benefits.
5.2.3. Seeming Limitations Associated with CTC Capture with Microfluidics
Are Not Insurmountable
The CTC-chip demonstrated clearly that prevalent limitations in capturing CTCs could be
overcome through technological innovation with microfluidics. A great desire in studying
CTCs is to culture them in vitro subsequent to separation, which would be facilitated by the
ability to remove them from the chip after capture. The nature of the interaction between
an antibody and its respective antigen is both specific and nearly irreversible in normal
physiological conditions 215 . This high affinity, or tightness of binding, results in great
difficulty in releasing a target from its bound antibody while, in the case of a cell,
77
maintaining its viability 216. The main approach to allow for both capture and later release is
217
to incorporate materials, such as degradable biopolymer coatings , at the interface
between the device substrate and the antibody that can be triggered for on-demand
release 21 ,219. This broad technique is also seen in specific studies in which antibodies are
replaced with other types of adhesion molecules, such as aptamers, that are still specific and
sufficiently strong in binding to target cells yet also include built-in release mechanisms
through either the simple increase of temperature 220 or through cleavage by nucleases
added after capture 221.
Although the paratope of an antibody attaches to the epitope of its antigen like a lock and
key, that region of the antibody is only a fraction in size relative to the entire structure. As
222
such, antibodies can interact nonspecifically with other proteins . Thus, another potential
limitation to the approach of rare cell capture with microfluidics and antibodies is
biofouling 223. Specifically, leukocytes are susceptible to sticking to the surface of such a
device when processing blood, which can impair subsequent characterization and limit the
surface area useful for capture. But with careful consideration of the expression level of
genes in leukocytes relative to a specific CTC, RT-PCR has been shown capable of
meaningful mRNA expression analysis on a single breast cancer CTC surrounded by more
than 800 contaminating leukocytes 224. Thus, these and other studies support the vision of
microfluidics playing a role in helping to increase our understanding of metastasis and
ability to treat cancer through capture and analysis of CTCs.
5.3. Advantages of Herringbone Architecture for CTC Capture
At least several advantages of the herringbone-chip over the previous generation CTC-chip
have been demonstrated. The fabrication of the devices is less expensive and can be
accomplished with more readily available techniques. The transparent materials
constituting the herringbone-chip (glass and PDMS) facilitate analysis after capture of CTCs.
Finally, and perhaps even more critically, the architecture of the channels within the device
allows for trapping of not only individual CTCs but also clusters of them.
Figure 20 includes a photograph of the herringbone device employed in the studies
reported here, and Figure 34 and Figure 35 showcase a collection of images from a
microscope that demonstrates the herringbone pattern throughout the channel surfaces,
78
which results in more effective mixing of the fluid during flow. The latter two figures also
demonstrate the ability to fabricate devices with multiple identical channels per device for
increased overall surface area for interaction with the target cells.
5.4. Approach
With the foundation set by developing a protocol to create platelet-tumor cell complexes
(Chapter 3) and after testing two initial approaches for capturing them (Chapter 4), the
herringbone device began with experiments in vitro and progressed to retrieving complexes
of platelets and CTCs from the blood of mice that harbored tumors.
5.4.1. Experimental System
Herringbone Microfluidic Device
Herringbone devices were made with glass and PDMS and consisted of either just one
channel in the initial experiments or eight channels for more rapid throughput. The width
of each channel in the devices was 900 gm, and, with the 40-pm groove depths, the height of
the channel was either 40 gm (from glass slide surface to bottom of groove) or 80 pm (from
glass slide surface to top of channel without groove). Each groove was 60 pm wide.
There were two methods used to fabricate the PDMS piece in the herringbone device. The
first approach involved a silicon master mold and the second method used white plastic
molds. Creating molds that produce herringbone-grooves in the channels necessitated a
two-layer photolithography process. Standard methods were used to cure the PDMS in the
molds, plasma treat the glass and PDMS, and bond the PDMS to the glass.
Functionalization of Herringbone Device
After fabricating each herringbone device, coating their channels with the antibodies of
interest involved first attaching NeutrAvidin to the surfaces and then adding biotinylated
antibodies. The series of steps for functionalizing the channels are outlined in the schematic
of Figure 19.
79
SH
'~
3-Morcep""yI
OH
OH
OH
OH
tlhoyman
0
0
0
ON
GMSS
ii
N
Ikv
Figure 19. Functionalization Chemistry to Coat Microfluidic Channels with
Biotinylated Antibody. Silane, N-Succinimidyl 4-maleimidobutyrate, and NeutrAvidin
were added sequentially to provide a binding site for biotinylated antibodies. It should be
noted that there is not necessarily certainty about the location of biotin after conjugating it
to antibodies for attachment to NeutrAvidin, which means the site interacting with its target
may not be exposed as depicted above.
Configuration of Devices Requiring Multiple Chips
The syringe pumps used to inject solutions at a controlled rate through the microfluidic
devices had capacity for one or multiple syringes. With this ability to run sample from
multiple syringes simultaneously, the devices could be configured in parallel such that
every inlet is connected to its own syringe. Multiple chips could also be connected in series
with sample from only one syringe as input. In such a configuration, the output of one
device is connected with tubing to be the input of the next device in series. Photographs of
the syringe pumps and devices connected in series and parallel are shown in Figure 20.
80
0
a)
(4)
0
La4
a.
Figure 20. Configuration of Herringbone Devices in Series or Parallel. Sample from
one syringe flows through several devices in series in the top two photographs from an
experiment. In the bottom pair of photographs, multiple syringes are connected to the same
number of devices. Each device is approximately 2.5 cm wide and 7.5 cm long.
5.4.2. Testing Capture with Cancer Cells and Platelets in Buffer
It had been shown previously that murine 4T1 breast cancer cells are both highly metastatic
and tend to bind to platelets. In fact, past studies tested potential methods of disrupting the
interactions between platelets and 4T1s2 2s. The particular line of 4T1 cells used in these
experiments constitutively expressed a gene to produce Zoanthus green fluorescent protein
(ZsGreen), which provided a way to identify individual 4T1 cells without adding an
antibody or any further dye to them.
In testing capture of platelet-4T1 complexes in buffer with the herringbone device,
essentially the same protocol as described in Chapter 4 was used to form complexes
starting with a flask of cultured 4T1 cells and whole mouse blood with ACD as the
anticoagulant. A similar method also from Chapter 4 of verifying that platelet-tumor cell
complexes were created was employed with 4T1 cells.
81
To test the capture efficiency of various antibodies that all attach to platelet surface markers,
complexes of 4T1s and platelets or only 4T1s were suspended in equal number in a solution
with BSA and flowed through one herringbone device each that was functionalized with one
of two different types of antibodies, either anti-CD62P or anti-CD63. The CD63 molecule,
also known as LAMP-3, is expressed on activated platelets and normally associated with
intracellular vesicles membranes. With the known concentration and flow rate and an
estimate of the number of 4T1 cells input into each device, the capture efficiency for each
sample was calculated. The number of cells captured was counted manually by scanning
the device with a fluorescent microscope for objects of the known size of 4T1 with a
uniform and bright green signal.
Finally, a rough measure of the purity was determined by counting both the number of cells
with the fluorescence microscope through the FITC filter and the number of cells stained
with Hoescht as seen through the DAPI filter.
5.4.3. Testing Capture with Spiked Platelet-Cancer Cell Complexes into Blood
Blood spiked with the complexes flowed through the device to test the feasibility of
capturing them in this more relevant case. The same procedure to form 4T1-platelet
complexes in buffer was used before adding (spiking) them into blood at a known
concentration. These 4T1s also all expressed the gene for ZsGreen, which provided a signal
that greatly facilitated counting captured 4T1s and observing them in the blood on the
device.
5.4.4. Testing Capture with Samples from Mouse Models
After tumors were initiated in mice by injecting 4T1 cells into the tail vein or mammary pad,
they were allowed sufficient time to grow and be released into the blood stream from at
least the primary tumor site. At this point, blood was collected and processed on the
herringbone devices that had been functionalized with antibodies that target platelet
surface markers. An attempt was made in some cases to correlate the number of cells
captured on the device with tumor burden.
Formation of Tumors in Mouse Model and Blood Collection
82
To form tumors in mice with 4T1s, a million of the cells were injected in either the tail vein
or the mammary pad of each BALB/c mouse. Injections in the tail vein led to metastases in
the lungs. Blood was collected from the heart and stored in a tube prior to processing.
Anticoagulant (ACD or EDTA) was used to prevent clotting prior to processing on the
devices.
Processing Blood from Mice in Microfluidic Devices
Within hours of blood collection, the samples were processed with the microfluidic devices.
The signal from ZsGreen in the 4T1s is sufficiently strong in the channel to enable manual
counting of the cells. The concentration of CTCs in each sample is unknown, so the capture
efficiency is also unknown unless the waste from the outlet is collected and analyzed. By
flowing a series of solutions through the device, the cells that are attached to the channels
can be preserved by chemical fixation and labeled by staining with nucleic acid dyes, such as
4',6-diamidino-2-phenylindole (DAPI) or a Hoechst dye, or antibodies conjugated with
fluorochromes.
Mice Tumor Burden Measurements
By imaging resected lungs to determine their relative fluorescence and by also imaging live
mice with the in vivo imaging system (IVIS), a qualitative measure of the tumor burden in
the mice was determined. The composite signal, which is expected to be a measure of
fluorescence from the tumors that consist of 4T1s, results from subtracting the
autofluorescence from the total GFP signal.
Control Experiments with Isotype Antibodies
In order to test the specificity of CTC capture, capture of anti-CD63 and anti-CD62P
antibodies was compared with their isotype equivalent. In such experiments, two devices
coated with antibodies were placed in series with the isotype antibody on the first device
and the regular antibodies on the second device. The rationale for this order was based on
the expectation that CTCs would pass through the isotype device and be captured on the
second chip.
5.5. Results
83
5.5.1. Comparison of Antibodies (Anti-CD63 and Anti-CD62) - Relative
Binding With and Without Platelets
While the anti-CD62P antibody was useful in experiments designed to determine whether
platelets were bound to tumor cells, additional antibodies were tested to optimize the
capture efficiency with the herringbone microfluidic devices. A direct comparison between
anti-CD62P and anti-CD63 antibodies showed the latter to be much more efficient in
capturing 4T1s (Figure 21). In both cases higher numbers of 4T1s were captured on chip
from solutions that contain platelets, which provides evidence that platelets are mediating
the capture at least in part. With a known flow rate and concentration of tumor cells input
through the device, an approximate efficiency was calculated.
Capture of 4T1 with and without Platelets
160
8
120
0
80
40
0
Anti-CD62P
and 4T1
Anti-CD62P
Anti-CD63
and 4T1
and 4T1
Anti-CD63
and 4T1
with platelets
with platelets
Sample
Anti-CD62P
4T1
29
9.67%
Anti-CD62P
4T1 with platelets
34
11.3%
Anti-CD63
4T1
45
15.0%
Anti-CD63
4T1 with platelets
140
46.7%
Figure 21. Comparison of Anti-CD63 and Anti-CD62 Antibodies and Relative Binding
with and without Platelets. Four samples were processed through microfluidic devices
functionalized with either anti-CD63 or anti-CD62P antibodies, and each pair of samples
contained 4T1s with and without platelets. The results from this experiment indicate
higher capture in the presence of platelets and many more cells isolated with anti-CD63
compared to anti-CD62P antibodies.
5.5.2. Capture Efficiency of 4T1-Platelet Complexes in Whole Blood With
Herringbone Device and Purity
84
To test the capture efficiency of herringbone devices, complexes of 4Tls and platelets were
spiked into blood at a known concentration and flown in parallel through two separate
devices functionalized with anti-CD62P and anti-CD63 antibodies. The capture efficiency
shown in Table 5 was calculated by manually counting the number of 4T1s on half of the
chip and dividing by the initial input, which was the product of the concentration of 4T1s
spiked into blood (5 x 104 4Tls / mL), the sample flow rate through the device (1 RL / min),
and the total time of flowing (40 min). As the data was extrapolated to provide an estimate
of the total number of captured cells, a range of estimated capture efficiencies is provided.
Table 5. Capture Efficiency of 4T1 with Anti-CD63 and Anti-CD62P
Anti-CD63
Anti-CD62P
250
15
500
30
2000
2000
12.5 - 25%
0.75 - 1.5%
The series of images in Figure 22 shows that in a particular location on the device there are
few background cells positive for Hoescht. A rough indication of the capture purity across
the entire device is provided in Table 6.
FITC
Bright Field
DAPI
Figure 22. 4T1-Platelet Complexes in Whole Blood Captured with a Herringbone
Device. Blood spiked with platelet-4T1 complexes was processed in herringbone devices,
and 4T1s (FITC) were counted along with others captured on the device labeled with
Hoescht (DAPI) to measure the purity of capture. (Scale Bar: 50 Rm)
Table 6. Rough Capture Purity of 4Ts with Anti-CD63 and Anti-CD62P Antibody
Coated Herringbone Devices.
Anti-CD63
I 1000
1 5UU
85
1 25 - 50%
Anti-CD62P
250
30
6 - 12%
5.5.3. Capture of CTCs in Blood from Mice with Tumors
Processing Blood from Mice with Tumors Formed by Injection in Mammary Pad
Blood retrieved retro-orbitally from a mouse was processed through three devices in series.
The sample flowed into the chip coated with the anti-CD63 antibody first and then the
devices functionalized with anti-CD62P and anti-EpCAM antibodies, respectively. Each of
these three chips appeared to capture 4T1 cells from blood, and images from the anti-CD63
antibody coated device are shown in Figure 23 and are produced from four channels: Cy3
(Anti-EpCAM conjugated with PE), CyS (Anti-CD45 conjugated with APC), FITC (ZsGreen
constitutively expressed in 4T1), and bright field. Whether any cells expressing CD45
should be counted as CTCs for purposes such as diagnostics is an unresolved question
generally in the field given their ambiguous identity, but the signal corresponding to
ZsGreen in these images provides compelling support that these cells originated from the
tumor formed by the injection of 4T1s.
86
Bright Field
ZsGreen
CD45
EpCAM
Figure 23. Individual Images of Devices that Captured Cells from Blood from Mouse
with Tumor Formed After Mammary Pad Injection. Blood was processed in a series of
microfluidic devices from a mouse that had been injected with 4T1 cells in the mammary
pad. The first device through which the blood flowed was functionalized with an anti-CD63
antibody, and cells positive for ZsGreen, such as shown in these images, were captured.
(Scale Bar: 50 gm)
Processing Blood from Mice with Tumors Formed by Injection in Tail Vein
Capture yields in processing blood from mice with tumors were significantly and
consistently higher in those mice injected with 4T1 cells through the tail vein compared to
in the mammary pad. In addition, larger clusters of cells, such as shown in Figure 25 and
87
Figure 26, were captured more frequently. In this particular experiment, three
microfluidic devices were each coated with a different type of antibody: anti-CD63, antiCD62P, and anti-EpCAM (Figure 24). Clusters of the greatest size and number were
separated on the first chip (anti-CD63), and fewer were on the second (anti-CD62P) and
third (anti-EpCAM), which may be due to many of the largest clusters being depleted from
flowing through the first device in series.
Input
Output
anti-CD63
anti-CD62P
anti-EpCAM
Figure 24. Order of Microfluidic Devices Each Functionalized with a Different
Antibody. Microfluidic devices were connected to the input and output and to each other in
series. The antibodies used in this experiment were anti-CD63, anti-CD62P, and antiEpCAM, and the devices coated with these antibodies were ordered respectively.
Multiple antibodies, including anti-EpCAM and anti-CD45, and Hoescht were added to gain
insight into the types of cells isolated on the devices. Noteworthy is that only a subset of
cells positive in the FITC channel (presumably for ZsGreen in the 4T1 cells), were also
positive for EpCAM, which may indicate differential degrees of epithelial-mesenchymal
transitions among the CTCs (Figure 25).
88
Hoescht (DAPI)
ZsGreen (FITC)
EpCAM (Cy3)
Bright
Field
CD45
(Cy5)
Figure 25. Individual Images of Devices with Captured Cells from Blood from Mouse
with Tumor Formed after Tail Vein Injection. Multiple images from the same location of
the device coated with anti-CD63 were recorded using different filters (in parentheses in
the label below each image) to detect the presence of antibodies (anti-EpCAM PE and antiCD45 APC) and dyes (Hoescht and ZsGreen). (Scale Bar: 50 [Lm)
89
Figure 26. Composite Images of Anti-CD63 Device that Captured Cells from Blood
from Mouse with Tumor Formed After Tail Vein Injection. Several composite images
from different locations of the device coated with anti-CD63 were captured at two different
levels of magnification (10x and 20x) after blood from a mouse that had 4T1 cancer cells
injected weeks earlier through the tail vein. In all of these images, blue represents signal
from the DAPI channel (Hoescht) and green from the FITC channel (ZsGreen), while light
blue indicates the overlap of the two signals. Red in the smaller two images on the right
side corresponds to the Cy5 channel (anti-CD45 APC), which as seen in Figure 25 was
minimal in signal for that particular image and which was not included in creating the
composite image on the left side above. (Scale Bar: 50 pim)
The capture predominantly of individual 4T1s on the downstream anti-CD62P and antiEpCAM devices is indicated in representative images shown in Figure 27.
90
Anti-CD62
Antibody
Coated
Chips
FITC (green) - Hoescht (blue) - Anti-CD45 (red)
Anti-EpCAM
Antibody
Coated
Chips
Figure 27. Composite Images of Anti-CD63 and Anti-EpCam Devices that Captured
Cells from Blood from Mouse with Tumors Formed After Tail Vein Injection. Images
from different locations on the devices coated with anti-CD62 and anti-EpCAM antibodies,
which were downstream from the anti-CD63 coated device shown in Figure 26. In all of
these images, blue represents signal from the DAPI channel (Hoescht), green from the FITC
channel (ZsGreen), and red from the anti-CD45 APC antibody (Cy5). (Scale Bar: 20 [1m)
Apparent Correlation of Capture with Tumor Burden
While platelet-4T1 complexes in buffer and spiked into blood were consistently captured on
the herringbone microfluidic devices coated with anti-CD63 antibodies, the variability in
results from experiments with blood collected from mice that had been injected with 4T1
cells motivated an investigation to determine whether the number of cells captured on the
device correlated with tumor burden of the mouse. Before sacrificing mice for the
experiments, they were imaged by IVIS, and after collecting the blood for experiments, the
lungs were resected and analyzed by capturing images through fluorescence microscopy
and observed visually at the macroscopic scale. In one experiment with three mice, the
three different methods of assessing tumor burden correlated with the capture of tumor
cells.
91
Since the cancer cell line used in these experiments fluoresces, the tumor burden was
assessed qualitatively through an imaging system that can detect fluorescence in live
animals. By removing fur, which is a source of autofluorescence, from the area of each
mouse close to the lungs, a clearer signal from tumors consisting of 4T1 cells could be
detected. In Figure 28, the autofluorescence signal (middle image) is subtracted from the
GFP signal (left image), which is from 4T1 cells and background, to yield a more accurate
measure of the signal from only the 4T1 cells in the lungs (right image). This composite
image suggests a higher tumor burden in Mouse 2 and Mouse 3 compared to Mouse 1.
GFP
Total green fluorescent signal
Tissue Autofluorescence
Gray areas in middle of mice
are regions without fur
Composite
Tissue AF subtracted from GFP
Figure 28. In Vivo Imaging System (IVIS) Pictures of Mice after Injections in the Tail
Vein to Determine Qualitatively Tumor Burden. The autofluorescence signal (middle
image) is subtracted from the GFP signal (left image) to determine the burden of tumors
consisting of 4T1 cells (right image). The regions in the chest of the mice that are associated
with no autofluorescence are areas where fur had been removed immediately prior to
imaging with IVIS. From the composite image, Mouse 2 and Mouse 3 appeared to have a
higher tumor burden than Mouse 1. In each of the three pictures, the boxes below the mice
indicate whether they are Mouse 1, Mouse 2, or Mouse 3.
After the collection of blood, the tumor burden was assessed qualitatively by fluorescence
microscopy of the resected lungs from each mouse. Figure 29 includes several images of the
lungs, which were the expected site of tumor formation, at a low magnification and with the
GFP filter. Of the three mice in this particular experiment, Mouse 2 and Mouse 3 showed a
significantly higher apparent tumor burden than Mouse 1.
92
Figure 29. Low Magnification Images of Resected Lungs with GFP Filter. After
collection of blood from three mice to use in microfluidic devices to attempt to capture CTCs,
the lungs from the mice were resected and imaged by fluorescence microscopy at a very low
magnification with the GFP filter to qualitatively assess tumor burden. The lungs from
Mouse 1 shown in the two images on the left side of this figure produced a lower signal than
those corresponding to Mouse 2 and Mouse 3. (Scale Bar: 2 mm)
A final indication of the degree of tumor burden came from visualizing the surface of the
resected lungs (Figure 30). The difference in color between the mice suggests that Mouse 2
and Mouse 3 (light pink and brown) suffered from higher tumor burden than Mouse 1 (light
to dark red).
Figure 30. Photograph of Resected Lungs from Mice Injected with 4T1 Cells. Pieces of
resected lung from each of three mice used in one experiment were photographed and
examined to measure qualitatively the tumor burden. These microscopic images supported
the conclusion that Mouse 2 and Mouse 3 were burdened with greater lung tumors than
Mouse 1. (Scale Bar: 10 mm - applies to the level at the bottom of well)
Whether there was capture on the herringbone microfluidic devices while processing blood
from three mice seemed to depend on their respective tumor load as assessed in the three
different ways described above. Three microfluidic devices functionalized with anti-CD63,
93
anti-CD62P, and anti-EpCAM antibodies were placed in series for processing each blood
sample. In this set of experiments, no 4T1 cells attached to the devices coated with antiCD62P or anti-EpCAM antibodies, but there was apparent binding of 4Tls from two of the
three mice on the anti-CD63 antibody-coated devices, which were positioned first in each
series of devices. In agreement with the results from tests assessing tumor burden, there
was no capture of CTCs from the blood of Mouse 1 on any of the three microfluidic devices
in series.
Processing the samples of blood from Mouse 2 and Mouse 3 through the microfluidic
devices resulted in capture of CTCs on the first device in series, respectively. Images of the
chips that processed blood from Mouse 2 showed capture both of large agglomerates of
cells, including 4T1s, and areas that contained fewer numbers of cells (Figure 31), while
images of devices associated with Mouse 3 showed only small clusters (Figure 32).
Figure 31. Captured Cells after Processing Blood from Mouse 2 on Anti-CD63
Antibody Coated Device. Both large clusters (left image and top right) and smaller
number of cells (bottom right) were captured while processing blood from Mouse 2 with an
anti-CD63 coated device. Red indicates the presence of DAPI, and green corresponds to
signal from the FITC channel, which would be provided by ZsGreen in 4T1 CTCs.
94
Figure 32. Captured Cells after Processing Blood from Mouse 3 on Anti-CD63
Antibody Coated Device. Only smaller numbers of cells were captured while processing
blood from Mouse 3 with an anti-CD63 antibody coated device. Red indicates the presence
of DAPI, and green corresponds to signal from the FITC channel, which ZsGreen in 4T1 CTCs
provides.
5.5.4. Capture of CTCs and Clusters Ranging Significantly in Size and Staining
Positively for Platelet Markers on Control and Standard Device
After processing blood from mice that had injections of 4T1 cells by tail vein, there were
cases of capturing clusters over a great range of sizes, varying from single cells to massive
agglomerates - as displayed in Figure 26. The devices were placed in series with the first
coated with the isotype antibody and the second with the regular antibody. Figure 33
includes images captured on the control device, and Figure 34 and Figure 35 are collections
of overlapping images processed by computer software for alignment after automated
acquisition by a scanning confocal microscope.
95
Figure 33. Large, Medium, and Small Clusters of CTCs Captured on the Herringbone
Microfluidic Device Coated with Isotype Antibody. Various sizes of clusters were
captured on the herringbone microfluidic device functionalized with an isotype antibody.
This chip was positioned first in series, and signals from three channels were recorded:
DAPI, FITC (ZsGreen in 4T1), and PE (Anti-CD62 antibody). Blue, green, and red correspond
to these signals, respectively. (Scale Bar: 50 [rm)
96
Figure 34. Stitched Images from Herringbone Microfluidic Device Functionalized
with Isotype Antibody. Large clusters were predominantly captured on this H B-chip
functionalized with an isotype antibody and positioned first in series before the anti-CD63
antibody-coated device. Signals from three channels were recorded: DAPI, FITC (ZsGreen in
4T1), and PE (anti-CD62 antibody). Blue, green, and red correspond to these signals,
97
respectively. Two large bubbles in two channels distort the signal of several of the clusters
(Scale Bar: 500 Vm)
Figure 35. Stitched Images from Herringbone Microfluidic Device Functionalized
with Anti-CD63 Antibody. Fewer and smaller clusters were captured on this herringbone
98
microfluidic device functionalized with anti-CD63 antibody and positioned second in series
after the isotype antibody-coated device. Signals from three channels were recorded: DAPI,
FITC (ZsGreen in 4T1), and PE (anti-CD62 antibody), which likely was not washed
completely from two channels above. Blue, green, and red correspond to these signals,
respectively. (Scale Bar: 500 gm)
5.6. Summary of Results
Experiments with the herringbone microfluidic device could be grouped into three broad
categories: (1) 4T1-platelet complexes formed and processed in buffer, (2) 4T1-platelet
complexes formed in buffer and subsequently spiked and processed in blood, and (3) blood
from mice with 4T1 tumors. Experiments from the first and second group were proof-ofprinciple and supported pursuing further testing with mouse models.
Under controlled conditions in buffer and spiked into blood, 4T1 cells were routinely
captured in experiments with herringbone microfluidic devices, yet this consistency did not
transfer after moving to mouse models. The experiments attempting to correlate tumor
burden with whether or not CTCs were captured supported the hypothesis that the lack of
capture in some experiments was due to an insufficient number of potential CTCs to capture.
The variation in tumor burden from mouse to mouse and also on the number of CTCs
released into the bloodstream after the tumor grows is, of course, a reality of working with
mouse models.
Ultimately, the experiments demonstrated that 4T1 tumor cells circulating in blood could be
captured with the herringbone microfluidic device, but questions were raised about
specificity and the minimum tumor burden necessary for capturing CTCs through this
particular approach.
5.7. Reflections and Outlook on Capturing CTCs by Means of Platelets
5.7.1. Implications of the Capture of Very Large Clusters
Given the small diameter of blood vessels in mice, the presence of a relatively large number
of CTCs in the clusters captured on the device raises a question about whether or how such
agglomerates could circulate while the mouse was alive. If the vessels could not
accommodate the passage of these large clusters, a possible explanation is that these
clusters form in the collection tube only after the mouse is sacrificed and the blood is
99
retrieved. Or these clusters originate by the attachment of a small number of cells on the
device and increase in size one or a few cells at a time during the flow of blood through the
device. If either ofthese possibilities is a cause of the presence of these massive clusters,
confirming that this phenomenon does not impact conclusions from related studies in the
past or future is critical. Alternatively, if these massive clusters do reside in mice prior to
collecting blood, attempting to correlate their presence and number with prognosis could
be worthwhile given prior studies demonstrating the greater tendency of tumor cell
clusters to form metastases compared to single tumor cells of the same total number.
5.7.2. Possible Mechanism of Capture on Control Device
Considering that many of the clusters captured on the isotype antibody device are much
larger than the height of the microfluidic channel, testing whether the complexes of
platelets and CTCs are trapped due to physical confinement is warranted. If the capture is
not due only to size, an additional consideration is that cells in the blood that could attach to
CTCs may also display Fc receptors with affinity to the Fc region of antibodies, which may
explain why CTCs were captured by both the device coated with the anti-CD63 antibody and
an isotype antibody - both of which contain the Fc region, which may be accessible to cells
given the unpredictable orientation of the biotinylated antibodies on the device. It should
also be noted that the fact that much larger clusters were observed on the isotype device
compared to the anti-CD63 antibody-coated device could be due to the depletion of these
large clusters from passing through the first chip in series and leaving none or few to be
captured on the second device.
5.7.3. Sensitivity of Capture
With variability in those experiments involving mouse models, determining the threshold
concentration of CTCs in blood required to capture any tumor cells on the device and
working to optimize the technology to reduce this threshold would be of great benefit. A
potential challenge in determining this minimum concentration for capture is the possible
difference in formation of platelet-tumor cell complexes in vivo versus in vitro. For instance,
complexes formed in solution may be of a different size and composition than those formed
in a mouse, which may impact whether the threshold determined in vitro is applicable in
experiments with animal models or even patients.
100
Chapter 5 Appendix
Reagents
Cell Staining Buffer
BioLegend (420201)
Antibodies for Capture
Anti-CD62P and Isotype:
Biotin Rat Anti-Mouse CD62P
* Clone: RB40.34
BD Biosciences (Material Number: 553743)
e
Biotin Rat IgG1 Lambda Isotype Control
* Clone: A110-1
- BD Pharmingen (Catalog Number: 553994)
Anti-CD63 and Isotype:
Biotin Goat anti-rat IgG (minimal x-reactivity) Antibody
- Clone: Poly4054
" BioLegend (405402)
Purified Anti-mouse CD63 (BioLegend)
- Clone: NVG-2
* BioLegend (143901)
Purified Rat IgG2a, K Isotype Ctrl Antibody
e
Clone: RTK2758
- BioLegend (400501)
EpCAM:
Anti-Mouse CD326 (EpCAM) Biotin
* Clone: G8.8
* eBioscience (Cat. No.: 13-5791-82)
Antibodies for Staining
Anti-Mouse CD45 APC
* Clone: 30-Fit
* eBioscience (Cat. No.: 17-0451-82)
101
Anti-Mouse CD326 (EpCAM) PE
- Clone: G8.8
- eBioscience (Cat. No.: 12-5791-81)
Anti-Human/Mouse CD62P (P-Selectin) PE
- Clone: Psel.K02.3)
- eBioscience (Cat. No.: 12-0626-82)
Nucleic acid stain
Stock solutions were diluted for use at concentration of 50 RM:
- Hoescht
" DAPI
Biological Materials and Samples
Cells
4T1 with ZsGreen - provided by Myriam Labelle from Hynes Lab at the MIT Koch Institute
Blood for Spiking Experiments and Formation of Complexes
Mouse whole blood from Bioreclamation
Mice for Animal Model Experiments
BALB/c
Equipment
Microscopes
Imaging resected lungs:
Olympus IX51 (FITC)
Imaging herringbonedevices:
e
Nikon TE2000-U (FITC, Cy3, Cy5, DAPI filters) with Andor iXon 885 camera
Nikon 1AR Ultra-Fast Spectral Scanning Confocal Microscope
IVIS
102
IVIS Spectrum-bioluminescent and fluorescent imaging system (Xenogen Corporation) at
the MIT Koch Insitute
Processing Software
o Nikon NIS Elements software
o ImageJ
Thin Glass Slides for Microfluidics and Imaging with Confocal:
Electron Microscopy Sciences
- 72192-75*, 25x75 mm, #1 (.13-.17 mm)
* Clear white borosilicate glass
Thicker Glass Slides for Microfluidics and Imaging with Nikon TE2000-U:
VWR (soda lime glass)
PDMS
Sylgard 184 Silicone Elastomer Kit, Dow Corning Corp, MI
Pumps
e
-
World Precision Instruments (WPI), SP2301W
PhD Ultra, Harvard Apparatus
PTFE tubing (Cole Parmer)
To connect devices and syringes: 100 um inside diameter
e
* For inlets, outlets, and syringe needles: 300 um inside diameter
Syringes
e
Glass (Hamilton Co)
* Disposable (BD)
Fabrication of Microfluidic Devices
Master Mold Construction:
Create on silicon wafer using SU-8 photoresist by photolithography (two-layer
photolithographic method)
White Plastic Mold:
103
Secondary polymer molds were constructed as described in the following article to facilitate
parallel device fabrication:
Salil P. Desai, Dennis M. Freeman, and Joel Voldman. "Plastic Masters - Rigid
Templates for Soft Lithography" Thirteenth International Conference on
Miniaturized Systems for Chemistry and Life Sciences. Jeju, Korea. November 1 - 5,
2009.
PDMS processing with mold and after curing:
e
*
e
Mix silicone elastomer polydimethoxysilane (PDMS) (Sylgard 184, Dow Corning
Corp, MI) with curing agent at 1:10 ratio
Degas the mixture with vacuum chamber
Pour onto master mold
Degas to remove trapped air again with vacuume chamber
Cure at 60*C
After curing, remove the PDMS from mold, place in 80*C oven for at least several hours,
clean the surfaces of the PDMS with by applying removing clear tape, and punch holes for
inlet and outlet ports to connect with tubing to the syringe and waste collection tube.
Bonding of PDMS with glass slide
-
Plasma treat up to two glass slides and two PDMS pieces simultaneously for 45
seconds at the highest RF setting (Herric Instruments)
Immediately press the PDMS to the glass slide to bond and remove trapped air
bubbles
Functionalize Devices
Solutionsfordevicefunctionalization
"
e
-
Ethanol
Deionized water
DPBS
Silane (100 VL / mL EtOH
GMBS (1 mM in EtOH)
Neutravidin (1 mg / mL)
Ab (at least 20 [tg / mL in 1% BSA solution or cell staining buffer)
Procedureforfunctionalizing microfluidic devices
*
Fill device with silane solution
Incubate 30 - 45 min
Wash with 50 [tL EtOH
Add 50 pL GMBS solution
104
e
e
"
-
Incubate 30 min
Wash with 50 iL EtOH
Wash with 50 uL DPBS
Wash with 30 - 50 [iL Neutravidin solution
Incubate overnight or longer than 2 hours in humidified environment
Wash with 50 uL BSA or cell staining buffer
Add 50 uL antibody solution
Incubate for 2 hours and afterward:
o Wash with BSA when attaching anti-EpCAM antibody and anti-P-selectin
antibody or
o Wash with BSA or cell staining buffer for anti-CD63 antibody and then:
- Add antibody to the device
- Incubate for 2 hours
- Wash with BSA
Protocols
Add Platelet-Tumor Cells into Blood for Testing Capture
o
o
o
o
o
Determine concentration of tumor cells through microscopy
Dilute solution of platelets and tumor cells in buffer to desired concentration
Centrifuge at 1200 rpm, 5 min
Remove supernatant
Add appropriate volume of blood to reach desired concentration of spiked cells
Form Tumors in Mice for Experiments Involving Animal Models
Inject 4T1 cells before confluency under the fat pad or into tail vein (1
x
106 cells)
Collecting Blood from Mice with Tumors
From heart:
e
Sacrifice mice by asphyxiation after approximately ten days
Collect blood immediately from heart and store in ACD or EDTA anticoagulant prior
to processing on chip the same day
For collection retro-orbitally, the animal did not need to be sacrificed, but less blood was
retrieved
Process Samples on Herringbone Device
Infuse sample at a rate of 1 pL / min (single channel device) or 8 [tL / min (eight channel
device) through devices either in series or parallel
Washing, Fixing, and Staining Cells on Device
*
Wash with cell staining buffer
105
-
Fix cells with 5% formaldehyde in DPBS
Flow in staining solution with antibodies (10 ig / mL) and nucleic acid stain (50
IM)
Incubate staining solution
Wash with cell staining buffer
Details of experiment to determine efficiency:
Flow 4T1 with and without PriorComplexing through HerringboneDevice to Compare
Efficiency of Various Antibodies
-
Prepare solutions of 4T1s with and without platelets at a concentration of 1 x 104
cells / mL in 1% BSA
Flow at 1 L / min for 30 min
Count green cells in chamber after flowing 30 min
Estimate of number of cells flowed in after 30 min based on concentration and flow
rate
Analysis
Imaging mice with IVIS
Remove fur by shaving chest and using Nair
Acquire images of GFP and tissue autofluorescence
Use software to substract signal
106
Chapter 6. Imaging Bacteria Treated with an Antimicrobial Peptide by
High-Speed Atomic Force Microscopy
Abstract
New views granted by advances in instrumentation into processes hitherto never seen
provide phenomenal insights and questions for continued investigation. By examining the
response of the entire surface of several bacterial cells simultaneously responding to the
action of an antimicrobial peptide, unanticipated variability in the kinetics was observed,
which, in connection with data on the degree of corrugation over time, led to the proposal
that the killing of bacteria occurs in two phases.
6.1. Introduction
The increase in the rate of image acquisition while using the atomic force microscope (AFM)
has provided insights otherwise impossible at the nanoscale and at timescales of seconds.
Processes of biomolecules have been examined by high-speed AFM, but this work had not
been extended to entire cells. The growing urgency to understand bacteria for human
health given the increasing prevalence of strains resistant to antibiotics motivates studying
behavior of this pathogen with the high-speed AFM. With renewed interest in antimicrobial
peptides as agents to treat human disease, furthering understanding of their mechanisms of
action could lead to increased effectiveness in combating bacterial infections. With this in
mind, the kinetics of CM15, an antimicrobial peptide (AmP), acting on E. coli was examined.
6.2. Atomic Force Microscopy (AFM)
The authors of the seminal paper on AFM conceived of the instrument as measuring
"ultrasmall forces on particles as small as single atoms" by the scanning tunneling
226
microscope (STM) measuring movement of a very small cantilever . Thus, just five years
after the STM was developed and in the year that the creators of the STM won a Nobel Prize
for that invention, 1986, the first AFM was demonstrated. Conceptually straightforward in
operation yet quite sophisticated in implementation, atomic force microscopy (AFM) has
enabled study of a broad range of samples at such an incredibly small scale that new
insights into phenomena have been generated through visualizations at the ultra smallscale generated by its use. Technological advancements have eliminated some of the
107
instrument's limitations and, thus, removed barriers to examining phenomena at scales of
time and length previously impossible.
6.2.1. AFM Operating Principles
While AFM is commonly used to measure a small region of a sample's physical topography,
it can be utilized with special instrumentation to study, for instance, magnetization patterns
on a surface 227 or the mechanical properties of particular proteins by unfolding them
individually 228. This is the rare type of microscope that produces an image of a sample by
"feeling" it, which is accomplished by rastering a sharp tip across the substrate surface
while recording tiny deflections of the cantilever at every location.
6.2.2. AFM Components and Functions
The highly sophisticated components of an AFM that in symphony sense and record the
topography of samples necessitate such great precision due to requirements of measuring
at the nanoscale. Originally, electron tunneling was the basis for measuring cantilever
deflection, and with this approach atomic resolution was achieved. Yet, contaminants and
additional interactions can interfere with tunneling. Thus, optical interferometry,
capacitance methods, and laser beam deflection have all been utilized as methods to detect
the distance of the cantilever from the sample. Among these four techniques, measuring the
tip position with a laser and photodiode is very popular (Figure 36).
108
Photodiode
Laser
Sa
& Tip
eCantilever
PZT Scanner
Figure 36. Schematic of AFM Components. Typically an AFM measures the nanoscale
topography of a surface by rastering a tip over it. The tip and cantilever respond to changes
in the properties of the sample, and deflections are measured by a photodiode's
determination of the location of a laser spot that is reflected of the tip. Feedback electronics
direct the scanner according to real-time changes in the tip deflection. 229
The AFM probe is a minute and sharp tip located on a cantilever typically made from silicon
or silicon nitride 230 that deflects like a spring in response to surface properties while
scanning the sample and which reflects the laser onto the array of detectors. With this laser
position information, a computer and further set of controller electronics serve as the main
components of a feedback system to acquire, display, and analyze data and control a
piezoelectric scanner that can rapidly change the position of the sample relative to the tip in
three dimensions.
Modes of Operation
An AFM can operate in various modes that have different strengths and disadvantages,
which influence the choice of mode to employ for a particular sample. While the tip is
scanning a sample in contact mode, it constantly touches the surface. This interaction may
be too harsh when examining biological specimens, which can require gentler approaches.
109
In contrast to contact mode, in both non-contact mode and tapping mode, the cantilever on
which the tip is located is oscillated near or at its resonant frequency. Two major
distinctions between these latter two modes include the amplitude of oscillation and the
nature of the interaction with the sample. In non-contact mode, the cantilever oscillates
usually fewer than 10 nm and as small as a few picometers. To overcome difficulties
associated with the tip sticking to surface due to the formation of a liquid meniscus, tapping
mode was developed. In this mode, the cantilever's amplitude of oscillation is typically at
least an order of magnitude greater (100 to 200 nm) than in non-contact mode.
While operating in non-contact mode, long-range forces, such as van der Waals forces,
acting on the cantilever decrease its resonant frequency. A feedback loop adjusts the
average tip-to-sample distance in order to maintain the original oscillation amplitude or
frequency. By compiling the distance measurements at every point, a topographic image of
the sample is created. While operating in either non-contact or tapping mode results in less
damage done to both the sample and tip, in tapping mode, the tip does in fact make
intermittent repulsive contact with the sample surface when it reaches the low point of
oscillation, yet the contact force is as low as 0.1 nN 231. A significant advantage associated
with tapping mode is that the larger amplitude of oscillation prevents the tip from being
captured by the fluid at the surface, which can form from capillary condensation in ambient
conditions 232.
That the oscillation of the cantilever in tapping mode or non-contact mode changes in
response to interacting with the substrate provides additional methods of gaining insight
into both the topography of the sample and also into material properties at every point.
Most straightforward is the height signal, which, as described above, is recorded by
measuring the response needed to establish a constant oscillation amplitude after this
decreases due to forces at the surface interacting with the tip.
The phase signal is a somewhat less intuitive means of measuring sample properties in that
it derives from the "delay" in oscillation of the tip or, more technically, the difference in
phase between the tip and the driver of its oscillation. The phase of oscillation can change
when the tip is near or in contact with the surface, and the magnitude of this phase
110
disruption is dependent on numerous factors that all affect the degree of energy dissipation
due to the tip interacting with the sample. Such factors include viscoelasticity, adhesion,
and contact area, the last of which depends on the topography of the sample. As such,
determining what factors contribute to the phase signal at any given point is complex, yet it
is still a common technique to characterize composition of surfaces.
A third signal produced in non-contact or tapping mode is the amplitude error, which
corresponds to changes in surface height. The feedback circuit is responsible for
maintaining the tip's oscillation amplitude at a setpoint value by appropriately controlling
the height of the cantilever relative to the sample surface. As there is a delay between the
change of the tip's amplitude and the subsequent adjustment in scanning height, there is
potentially a discrepancy between the amplitude setpoint and the actual amplitude of
oscillation at a given moment, and this difference is the amplitude error signal.
6.2.3. Development of the AFM to Image Soft Materials
The capability of the AFM was first demonstrated in contact mode on inorganic materials,
and methods and additional modes for imaging were developed to measure sample
properties beyond height and gently scan fragile materials, such as films and biological
samples, which may also be soft and only bound weakly to the underlying substrate.
6.2.4. Limitations
A high-quality image taken by a conventional AFM often requires scanning for 1 to 100 min,
which is considered in practice to be a slow speed that has limited the productivity and also
the increased use of this instrument 233. At least several explanations have been offered for
why advancements in the image acquisition rate have not been rapid and widely
implemented. In the mid-1990s, since all the components were matched well in terms of
performance, an increase in scan speed would require replacing most of the system's
components 234. Adding to the dilemma is the hesitance of AFM manufactures to build
instruments capable of using small cantilevers, which would increase performance, and
major-manufactures of cantilevers reciprocal reluctance to invest in producing smaller
23 5
cantilevers due to the lack of AFMs built to utilize them .
111
6.2.5. High-speed AFM
Fundamentally, the motivation to advance the image acquisition speed of the AFM to videorate is to understand aspects of phenomena previously unexamined in such fields as
chemistry, biology, and materials science. Examples include
"
surface diffusion
"
phase transition
*
self-assembly
*
film growth and etching
"
biomineralization
"
biomolecular motors
236
:
Technological developments in the past decades have raised the instrument's imaging
capability to the point of being able to visualize entire cells with nanometer resolution and
on the scale of seconds 237. Attaining higher scanning speeds has necessitated
improvements in major AFM components, including cantilevers, deflection measurement,
scanners, and controllers 238.
Contemporary Work with High-Speed AFM
As an indication of the magnitude of progress in increasing the image acquisition rate while
maintaining quality, in 2006, an AFM zoom series was produced in 0.56 s with an initial
image width of 2 gm down to a final width of 470 nm. The sample studied was rat tail
collagen, and the images, which would have required about fifteen minutes of conventional
239
AFM scanning, show a characteristic 67-nm banding pattern .
Two studies conducted with high-speed AFM reported within the previous several years
include investigations of walking myosin V and an adenosine triphosphate (ATP)-drive
motor, F1. In the former, further details from high-resolution movies led to a fuller
understanding of myosin V's motor mechanism
240
. In the second study, the high-speed AFM
enabled examination of the potential core feature of F1 that is responsible for torque
generation, which is not possible with conventional single-molecule optical microscopy
24 1
since that technique requires connecting a probe to the protein's rotary shaft .
112
Next Stage: Studying Cellular Processes
There are several essential requirements to study cellular processes with the AFM
heretofore unmet. The scan size must be sufficiently large to capture at least one cell, and
the acquisition speed must be very rapid while maintaining imaging forces on the cells that
are not so low as to lose important sample information but also not so high that damage the
cells. Prototype AFM components and smaller probes were two aspects of the enabling
technology in the present work reported here. The micro-fabricated cantilevers were about
a thousand times smaller than conventional cantilevers, had a higher resonance frequency,
and a lower quality factor and spring constant. A prototype optics head was utilized that
was compatible with such small probes (Figure 37).
Schematic
of optics
design
quadrant
tapping
P
-
piemr
cantikever
sca.nner
a
u""
MultiM
V
Objective lens
Flow-through pump
l-fluc
ideell
thud
c
Fabricated
prototype head
CAD representation of
prototype head
Figure 37. Representations of Prototype Head Design and Photographs of Fabricated
Head with MultiMode V. The version of this optics head is based on a design by Hansma,
et al. and is described in detail in U.S. patent 6,871,527. In sum, several novel features of
this prototype head enable the AFM to operate with a smaller focused spot of light that
previously was impossible.
While the task of increasing the performance level of the AFM to the required level is one
that has been fulfilled through decades of effort of numerous scientists and engineers of
113
various disciplines, the success of imaging live cells with AFM depends also on their
adhesion to the underlying substrate that is sufficiently strong so as to prevent their release
during the multiple scans in solution. The method of attaching the cells must also not affect
their health and functioning in a way that interferes with the phenomenon under
investigation. In the particular case of imaging microbes, the contact area between the cell
and support is quite small due to their tendency to maintain their shape and not spread
over the surface, which, without suitable attachment chemistries, results in cell detachment
particularly after interacting forcefully with the scanning
tip242.
6.3. Antimicrobial Peptides (AmPs)
While humans first postulated the germ theory of disease only about 250 years ago 24 3 ,
organisms, including us, have internally been aware much longer about the dangers
pathogens pose and have developed defenses to combat their intrusion. The formal study,
though, of innate antimicrobials has been ongoing since at least the early 20th century. In
the 192 Os, Alexander Fleming was first to discover a soluble antimicrobial substance made
244
by humans, lysozyme, which is a protein 130 amino acids in length and considered by
245
some to be the first report of a peptide that has antimicrobial activity . Several years later,
he also found the power of penicillin, a group of antibiotics produced by a particular fungus.
Beyond these two specific antimicrobials created by certain organisms, there is a whole
category of broad-spectrum antibiotics made by all classes of life - antimicrobial peptides
(AmPs).
6.3.1. Significance
Antimicrobial peptides produced by organisms constitute their oldest defense system
246
against pathogens, including bacteria, viruses, fungi, and protozoa . The true origin of
AmP research is thought by some to be in studies conducted in the 1950s and 1960S247.
While perhaps the first description of an animal AmP came as late as 1962248, as of the end
of 2011, there had been 1,200 types of AmPs isolated, and all organisms appear to use AmPs
as part of their host defense 249. Interestingly, despite their ancient history and prevalence,
there is a relative lack of microbial resistance development to them, which, in combination
with the observed resistance of bacteria to existing antibiotics, drives interest in studying
AmPs towards employing them clinically 25 .
114
It should be noted, too, that AmPs influence processes beyond just the direct destruction of
invaders, including cytokine release, cell proliferation, angiogenesis, wound healing,
25 1
chemotaxis, immune induction, and protease-antiprotease balance .
6.3.2. Modes of Action
The modes by which AmPs act on cells to eliminate them are broadly categorized into either
25 2
membrane or non-membrane acting . Within the category of the membrane acting mode,
there are several proposed models explaining how AmPs form transmembrane pores to
25 3
permeabilize the membrane and thereby cause cell death . Although all AmPs acting on
bacteria must interact with the cytoplasmic membrane in the process of damaging them, it
25 4
is not the case that cell membrane permeabilization is the only AmP mode of action .
Essential intracellular processes, such as nucleic acid synthesis, protein synthesis,
enzymatic activity, and cell wall synthesis, are targeted by some AmPs that, while
255
translocating into cells, do not also cause membrane permeabilization . Further, it is
postulated that the mode of action of a particular peptide might depend on the type of
bacteria it targets and its membrane's physical properties and the concentration of
peptide 25 6. The minimum inhibitory concentration (MIC) is the lowest concentration that
inhibits bacterial growth.
6.3.3. Size, Structure, and Composition of AmPs
2
The size of AmPs is considered to range from 10 - 100 amino acids
7,2
s8 .
Specifically
regarding those AmPs characterized by antibacterial activity, three features essential to the
successful completion of the first step of interacting with the bacterial membranes include
25 9
net positive charge, hydrophobicity, and flexibility . The positive overall charge increases
the AmP's interaction with anionic lipids, while the hydrophobicity is necessary for
insertion into the membrane 260. The mixture of hydrophilic and hydrophobic amino acids
261
in the AmP results in its amphipathicity, which is also characteristic of cell membranes .
Finally, structural flexibility allows the AmP to switch conformations from its soluble form
262
to its membrane-interacting conformation .
115
Four structural classes generally used to categorize cationic antimicrobial peptides are ahelical, P-sheet, loop, or extended structures, yet this simplified scheme of structures does
not accommodate many AmPs 2 63 . For instance, many AmPs produced by bacteria have two
different structural domains 264.
6.3.4. Frontier of Learning about AmPs
Synthetic membranes or vesicles have been analyzed spectroscopically to learn about pore
sizes and structures created by AmPs 2 65 . While the morphological changes caused by AmPs
on the surface of cells has been visualized previously by electron microscopy and AFM,
266
these effects had only been observed at the endpoint . The early kinetics of AmP activity
on membranes of live cells at the nanometer scale and on the order of seconds had not been
reported in the literature 267.
6.4. Experimental Details
6.4.1. Preparation of Bacteria and Attachment on Slide for Imaging and
Interaction with AmP
The protocol to attach bacteria to the glass slide for imaging with the AFM and inverted
microscope needed to avoid significantly influencing cell behavior while still providing a
sufficiently strong attachment of the bacteria in solution during imaging so as not to detach
from the substrate.
Growth and Washing of Bacteria Prior to Attachment
A culture of E. coli bacteria was prepared overnight, diluted the following day, and grown
for several hours prior to imaging. The initial culture was produced from a single colony.
To remove material that could interfere with bacterial attachment, the bacteria that had
been diluted from the overnight culture and then grown for three hours were spun down
and washed with Millipore.
Preparing Glass Coverslips
A coating of poly-L-lysine on the glass slides increased the strength of binding of the
bacteria to the substrates. First, the glass was cleaned in acid. The slides were rinsed with
116
Millipore water before then immersing them in a solution of poly-L-lysine hydrobromide.
After this short incubation, the glass slides were dried vertically at least overnight and used
within a week.
Incubating Bacteria on Slides for Attachment
A small drop of bacteria in Millipore water was transferred to a coverslip coated with polyL-lysine. After 30 min of incubation, unbound cells were washed off with Millipore water.
6.4.2. Description and Handling of CM15
Characteristics of CM15
The kinetics of CM15 acting on E. coli was examined. This AmP is a linear peptide consisting
of the following sequence of amino acids: KWKLFKKIGAVLKVL268. It is a synthetic hybrid
composed of seven residues of cecropin A and eight residues of a bee venom peptide,
mellitin 269. Pores of 2.2 - 3.8 nm have previously been observed as result of CM15's
bactericidal activity, which involves first binding to the cell membrane and then folding into
an alpha-helix 270 .
Synthesis, Storage, and Dilution of CM1S
The AmP, CM15, was produced by solid-phase peptide synthesis methods, desalted for
purification, and lyophilized for shipment. The peptide was then resuspended with
Millipore water, stored in this solution, and diluted further in Millipore water for particular
experiments.
6.4.3. Imaging with High-Speed AFM and Fluid Cell
The AFM operated with an open fluid cell to enable fluid exchange. This ability to inject a
solution of CM15 after the sample of bacteria was initially imaged was important in order to
determine the starting condition of the surface of the bacteria. The resulting concentration
of CM15 after introducing it into the flow cell was five times the MIC, which is 50 mg mL-'.
Images were acquired typically every 13 s in tapping mode. At a resolution of 1024 x 256
pixels, this requires an imaging speed of 20 lines per second. The recorded signals included
height, amplitude, and phase for both trace and retrace.
117
6.4.4. Correlating Bacterial Surface Corrugation with Cell Death
To analyze samples by AFM and fluorescence microscopy concurrently, an AFM was
mounted on an inverted microscope. The former captured images in tapping mode, and
fluorescence images were produced with an oil immersion objective. To determine whether
each cell was alive or dead, a mixture of two nucleic acid stains with different cell
penetration capabilities dependent on cell membrane permeability was introduced to the
sample of bacteria during the experiment. The green dye can enter healthy cells, while the
red dye passes only through membrane-compromised cells.
6.4.5. Bulk Cell Killing Assay
The purpose of the assay on bulk cell death is to verify conclusions of kinetics drawn from
experiments with the AFM that were on a very small population of bacteria. Bacteria for
these studies were grown according to the same protocol as that used to produce bacteria
for experiments with the high-speed AFM. After overnight growth, a bacteria culture was
diluted and grown three hours, and the resulting cells were washed and resuspended in
Millipore water to a concentration of 2
x
106 cells mL-1. After adding the CM15 peptide to
yield a concentration (20 mg mL-1) that was equivalent to AFM experiments when
comparing the ratio of peptides to cells. By plating aliquots from this solution of cells and
CM15 at different time points, colonies were formed overnight, which indicated the number
of viable cells at each respective moment of collection.
6.4.6. Comparison with Control Peptides
In addition to imaging the bacteria for a minimum of 10 min to be sure that they were firmly
attached and not altered by the presence of poly-L-lysine on the glass coverslip, a control
peptide, 2K1, which has no known antimicrobial activity at the concentrations tested herein
and which is highly positively charged with the sequence, (GK) 6AS(GK) 6 , was introduced in
separate studies to help confirm that the cell corrugation after the addition of CM15 was
due specifically to its activity on the bacteria. Similarly, the effects on E. coli of a
conventional antibiotic, ampicillin, were examined by AFM for comparison with CM15.
6.5. Analysis
6.5.1. Phase Data as Choice for Evaluating Bacterial Surface Changes
118
Among the signals recorded in tapping mode during imaging, the phase data was chosen for
evaluation of the changes in surface smoothness because of increased contrast Materials
properties and topographical changes do both influence the phase signal, but since the
phase data appeared to be scan direction dependent to a degree, the information from the
phase signal in this case is thought to be influenced primarily by topography.
6.5.2. Quantifying CM15 Kinetics on E. Coli
To quantify the kinetics of CM15 acting on the bacteria, the axial root mean square (RMS)
variation of the bacteria surface was calculated. At least twenty line sections with phase
data over each bacterial surface were extracted as raw data to determine the RMS variation.
Subtracting a third-degree polynomial line fit from each line section yields higher spatial
frequencies, which are the basis of the RMS calculation. The value of RMS variation was
calculated at multiple time points as a measure of the change in bacterial surface
corrugation as CM15 acted on the cells.
6.6. Results
6.6.1. Addition of CM15 Changes E. Coli from Smooth to Corrugated with
Variation of Onset of Roughening
Observed with the high-speed AFM, not only do the surfaces of bacteria become
pronouncedly corrugated on the order of minutes, the time from the introduction of CM15
into the flow cell until the onset of roughening is variable even between directly adjacent
cells (Figure 38).
119
Figure 38. Change in Bacterial Cell Surface from Smooth to Corrugated After Addition
of CM15. This series of images from the high-speed AFM demonstrates that the onset of cell
surface corrugation after introducing CM15 (t = 0 s) varies from cell to cell. The difference
in time between capturing each image is 13 s, and the images are from the phase data (Scale
Bar: 1 pm).
6.6.2. Effects on E. Coli from Control Peptide and Conventional Antibiotic
Compared to CM1S
The addition of 2K1 produced minimal changes in the surfaces of E. coli as examined by
AFM over the course of at least 43 minutes. As shown in Figure 39, the cell surfaces
appeared to remain smooth according to both whole cell images and comparison of crosssections of the bacterium produced from phase data.
120
|After 1 hr
IAddition of 2K1
I Addition of 2K1
I After 43 min
2s-
S 202 Is-
1'
C 10-
j
-s
Cross-sections
from phase data
over bacteria
before 2KI
-
1 -10
Z
1il
-
before and after
after 2K1
addition of 2K1
_,__
0
0.2
0.4
0.6
Distance (urn)
0.8
1
Figure 39. No Significant Cell Surface Corrugation after Introduction of Control
Peptide. The addition of 2K1 (A and C), which is the positively charged control peptide
with no known antimicrobial activity at this concentration, did not result in significant
change of surface variations after at least 43 min (B and D) as supported by comparison of
cross-sections before and after addition of 2K1 of normalized phase (bottom).
Bacteria treated with ampicillin demonstrated a similar consistency in surface smoothness.
After 112 min, small changes in the surface appeared albeit much less significant than that
observed seconds after injection of CM15.
6.6.3. Connection between Corrugation and Cell Death
Through a dual imaging approach and by employing a stain that helps measure cell viability,
a correspondence was found between corrugation of the cell surface and each bacterium's
survival (Figure 40).
121
AFM tapping-mode images
Fluorescence images
Bacterium with smooth
b
surface is aliv (a
U
n
0
W
40
CO
S
are tyically dead (e)
.E
C
Figure 40. Images from AFM and Fluorescence Microscopy Captured at the Same
Location Prior to and After Adding CM1S. Comparing tapping-mode images (A and C)
and fluorescence images (B3 and D) at the same location shows that corrugated bacteria are
typically dead (red) and bacteria with smooth surfaces are alive (green). The surfaces and
health of the cells before addition of CM15 (A and B) were more often smooth and viable
than 30 min after (C and D). These AFM images were recorded at a scan rate of 0.5 Hz and a
resolution of 512 x 256 pixels. (Scale Bar: 5 pm)
6.6.4. Quantification of Cell Surface Corrugation Before and After Addition of
CM15
Quantification of the RMS variation of the bacterial cell surface as a function of time
revealed that, while the time before onset of corrugation on the cell surface is variable, the
time required from the onset of change to reach maximum roughness is markedly more
consistent from cell to cell. As indicated in Figure 41, in one experiment, the range of time
before the beginning of roughening was from 40 s (bacterium 7) to at least 240 s (bacterium
2) with a mean of 155 s (standard deviation: 89 s). In this same experiment, half of the
damage to the bacteria happened on average within 52 s (standard deviation: 16 s).
122
LO
00
Bact 2
0
zBact I
00
Bact 9
Cu
0
-105
315
210
10s
Time after AMP addition s)
Average bacterial
roughness before and
after addition of CM15
E
250
200
~150
0
1100
0
-105
0
.1 210
1
Cross-sectional data
extracted from phase
images (bacterium 1)
E
Figure 41. Quantification of Bacterial Roughness Prior to and after Addition of CM15.
The series of AFM images (left) shows bacteria at 105 s intervals after injection of CM15.
Images were recorded every 21 s (fifth image shown) at a resolution of 1024 x 256 and a
scan rate of 12.2 lines s-1. From each phase image in the time progression, cross-sectional
data was extracted as shown for bacterium 1 (bottom right). The average bacterial
roughness as a function of time before and after addition of CM15 is presented in the top
right with numbers corresponding to labels in the time series of AFM images.
123
6.6.5. Assay on Bulk Cell Killing with CM15 and E. Coli
Fit reasonably well with a single exponential, the data from the bulk cell killing assay
indicate a killing rate half-time of CM15 is 4.6 min (Figure 42), which correlates well with
the measurements by AFM on single cells.
Ln
.E 1.2-
o1.0
0 0.8
c 0.6U
E
o
z
0.40.20-
60
50
40
30
20
Time after addition of AMP (min)
Figure 42. Bulk Cell Killing Assay with CM15 and E. Coli. Behavior interpolated through
the first 5 min of CM15 antimicrobial activity in bulk correlates well with measurements on
single cells with the AFM
0
10
6.7. Discussion
The combination of the variability in the onset of cell surface corrugation and the
consistency in time required for CM15 to complete damage introduces a question about this
AmP's mechanism of action. In eukaryotic cell biology, apoptotic death, which involves
permeabilization of the mitochondrial outer membrane, follows an incubation and
execution phase2 71 . Although there are differences between the overall mechanism
involved in this process and that of bacterial cell death induced by AmPs, both include a
critical component of pore formation initiated by a protein or peptide. This suggests that
CM15 acts according to two phases: incubation and execution. Further understanding
about these phases could be necessary in learning how bacterial resistance to AmPs is
developed.
These developments provided by the high-speed AFM and the technique described is
generally applicable to study other cell types, such as yeast or mammalian cells and even to
eukaryotic cell organelles. In sum, these measurements demonstrate the enormous
potential of high-speed AFM imaging for cellular biology.
124
Chapter 6 Appendix
For details on methods and materials other than those involved in attaching bacteria to the
substrate prior to imaging, please see the original publication:
Fantner, G.E., Barbero, R.J., Gray, D.S. & Belcher, A.M. "Kinetics of antimicrobial
peptide activity measured on individual bacterial cells using high-speed atomic
force microscopy." Nature Nanotechnology 5, 280-285 (2010).
Biological Material
E. coli (ATCC 25922)
Reagent
Poly-L-lysine hydrobromide (Sigma part number P1524)
Protocols
Growth and Preparationof Bacteria Priorto Attaching on Slide
e
*
e
Grow E. coli overnight from a single colony in LB growth medium at 37*C.
Dilute the culture of bacteria 1:100 in fresh LB medium and grown for 3 hours the
following day
Centrifuge the cells
Wash three times with Millipore water
Glass Slide Preparationto IncreaseAdherence
e
*
*
*
*
Boil round glass coverslips in 2.5 M HCl solution for 10 min to clean the glass
Rinse the coverslips with Millipore water
Immerse the coverslips for 10 min in a pH 8.0 solution of 0.05 mg ml-1 poly-L-lysine
hydrobromide and 10 mM Tris.
Dry vertically overnight at room temperature and covered
Store at room temperature use within one week
Incubation of Bacteria on Coverslip
*
*
Deposit concentrated bacteria in Millipore water on coverslips
Incubate 30 min
Wash three times with 1 ml Millipore water.
125
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