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20.GEM GEM4 Summer School: Cell and Molecular Biomechanics in Medicine: Cancer
Summer 2007
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From Cell Physiology and Biology to Cellular Biomechanics:
Role of Hydrodynamics, Chemokines and Adhesion in Leukocyte-mediated Melanoma Extravasation
Cheng Dong, Ph.D.
Professor of Bioengineering &
Engineering Science and Mechanics
Department of Bioengineering &
The Huck Institutes for the Life Sciences
The Pennsylvania State University
University Park, PA 16802, USA
Courtesy of Cheng Dong. Used with permission.
USA Today (Nov. 10, 2004)
interviewed leading medical
experts about the
relationship between
inflammation and cancer:
ƒ
Doctors believe that
inflammation is involved in a
wide variety of cancers.
ƒ
Scientists say they can’t be
sure whether inflammation
produces cancer; or cancer
leads to inflammation; or the
two processes interact.
ƒ
Doctors suspect that longterm inflammation or
infection is involved in up to
20% of cancers, including
those of the esophagus,
colon, skin, stomach, liver,
bladder, breast and some
kinds of lymphoma.
Image removed due to copyright restrictions.
Scan of USA Today article.
Cancer Metastasis Cascade
Tumor in epithelium
Break through basal lamina
Invade capillary
Connective tissue
Capillary
Adhere to capillary wall
Less than 1 in 1000 cells
will survive to form metastasis
Extravasation
Proliferation
Illustration by MIT OpenCourseWare. After Alberts, et al., 1994.
BMES Conference 2001
Alberts et al., 1994
Inhibiting V600EB-Raf reduced melanoma lung metastasis in vivo
Images removed due to copyright restrictions.
A, siRNA-mediated inhibition of V600EB
Raf inhibits melanoma lung metastasis.
Left panel, 1205 Lu cell; right panel, UACC
903M cell. Number of tumors within
particular size ranges (<1500 or >1500
pixels) were quantified in a minimum of 6
fields per lung from 5 to 10 animals. Values
are means ± SEM. B, co-localization of
human melanoma cells and mouse PMNs
in vivo.
On-going Studies
Overall Hypothesis :
Tumor cells elicit a sequence of signaling events to facilitate
their transendothelial migration via immunoediting
MAPK
Melanoma
IL-8
NF-κB
PMN
Protein
secretion
Ca2+
PKC
Junction
regulation
EC
PMN : polymorphonuclear neutrophils
Working Hypothesis:
Neutrophils (PMN) facilitate melanoma adhesion leading to
increased migration under flow conditions – an important step
in tumor cell extravasation during metastasis.
ICAM-1
Melanoma
Neutrophil β2 integrins
Image removed due to copyright restrictions.
PSGL-1
L-selectin
Slex
E-selectin
Endothelium
ICAM-1
P-selectin
Flow-Migration Assay
A
Filter
Figure by MIT OpenCourseWare.
Cell
monolayer
Chemoattractant
Gasket
Images removed due to copyright restrictions.
5 cm
Inlet
B
Outlet
Modified top plate
Filter
Gasket
Gopalan et al. 1997 J. Immunol.
10 cm
Top Plate
Flow
WBC
melanoma
Cellular Monolayer
Porous membrane
Wells for
Chemoattractant
Bottom Plate
Flow
PMN Facilitates Melanoma Cell
Migration Under Flow Conditions
NHEM only - Static
C8161 only - Static
C8161 + PMN - Static
p=0.47
p=0.47
C8161 only - Dynamic
p=0.013
C8161 + PMN - Dynamic
0
100
200
300
400
500
Cells Migrated per mm 2
Dynamic: shear stress = 0.4 dyn/cm2
600
To Clarify The Signaling Events Of
Cytokine/Chemokine Induction Mediated By
PMN-melanoma Interaction
Hypothesis :
Tumor cells modulate protein secretion through
activation of transcription factor signaling in PMNs
Melanoma
MAPK
IL-8
NF-κB
PMN
Protein
secretion
PMN : polymorphonuclear neutrophils
IL-8 Secretions from PMN, Melanoma Cells and Co-cultures
1000
*
IL-8, pg/ml
800
* *
*
*
*
600
400
Images removed due to copyright restrictions.
Western blot results.
200
0
WM35
C8161
WM9
1205Lu
PMN only
Melanoma cell only
PMN + Melanoma cell transwell co-culture
PMN + Melanoma cell contact co-culture
• ELISA data
Figure by MIT OpenCourseWare.
• WM35 : low metastatic potential
• Western blots
• Increased IL-8 in co-cultures of
C8161 - PMNs
WM9 - PMNs
1205Lu - PMNs
• Increased IL-8 from PMNs (Transwell)
co-cultured with C8161, WM9, 1205Lu
• Constant IL-8 from melanoma cells
Mac-1 Expression after Melanoma-PMN
Co-culture
6
Fold Induction
Mac-1 expression on PMN
5
PMN
Co-culture
4
Transwell
3
2
1
0
Untreated
CXCR 1/2 Ab blocked
Figure by MIT OpenCourseWare.
Blocking Intercellular IL-8 Signaling Affects PMN-facilitated Melanoma Extravasation
¾ Melanoma cell extravasation
increased in response to IL-8
stimulated PMNs compared with
non-stimulated PMN groups.
¾ Melanoma extravasation
decreased significantly in the
presence of CXCR1/CXCR2
blocked PMNs or anti-IL-8
neutralizing antibody compared
with unstimulated PMNs.
Image removed due to copyright restrictions.
Graph showing results of treatment with
CXCR1/2- and IL-8-blockers.
Inhibiting V600EB-Raf Reduced Melanoma Cell Extravasation in vitro
™ B-Raf encodes a RAS-regulated kinase that mediates cell growth and malignant transformation kinase pathway activation. ™ As the most mutated gene in malignant melanomas, B-Raf has raised questions whether targeting B-Raf might effectively reduce melanoma metastasis. Graphs removed due to copyright restrictions.
¾ Inhibition of mutant V600EB-Raf greatly reduced melanoma extravasation
compared with non-inhibited melanoma cells (untransfected, nucleofected with
buffer only or scrambled siRNA) under both static and dynamic flow conditions.
Inhibiting V600EB-Raf reduced IL-8 production A, Inhibition of mutant V600EB-Raf
significantly reduced the IL-8 production
from melanoma cells (1205 Lu and
UACC 903M) cultured alone compared
with the control melanoma cells
(untransfected melanoma and
melanoma nucleofected with buffer only
or scrambled siRNA).
Graphs removed due to copyright restrictions.
B, IL-8 production from tumor
microenvironment (including both PMN
and melanoma cells) increased after
PMN co-cultured with control melanoma
cells (~1.5 fold), whereas IL-8
production either kept the same or even
reduced after PMN co-culture with
melanoma cells treated with siRNA
against mutant V600EB-Raf. Values were
normalized to the summed background
level of PMN and melanoma cell
cultured alone.
Mac-1 expression on PMN after co
culture with melanoma cells
Graphs removed due to copyright restrictions.
Mac-1 expression on PMN increased significantly after PMN co-cultured with
control melanoma cells (untransfected melanoma and melanoma nucleofected
with buffer only or scrambled siRNA). However, the co-culture between PMN and
melanoma cells treated with siRNA against V600EB-Raf did not significantly
increase Mac-1 expression on PMN.
Disruption of ICAM-1/β2 integrin binding inhibited melanoma cell extravasation
Graphs removed due to copyright restrictions.
A, ICAM-1 expression on melanoma cells (1205 Lu and UACC 903M) was
reduced after knockdown of mutant V600EB-Raf and ICAM-1 using siRNA;
B, knockdown of mutant V600EB-Raf and ICAM-1 inhibited PMN-mediated
melanoma extravasation, values are mean ± SEM;
Inhibiting V600EB-Raf disrupts NF-κB activity
Graphs removed due to copyright restrictions.
A, NF-κB activity is reduced after
inhibition of mutant V600EB-Raf in 1205
Lu (lane 10) compared with control
cases (untransfected 1205 Lu, lane 1;
1205 Lu nucleofected with scrambled
siRNA, lane 4 or buffer only, lane 7).
The complexes were supershifted by
polyclonal antibodies against p50
(lanes 2, 5, 8 and 11) and p65 (lanes 3,
6, 9 and 12). B, PDTC treatment
reduced IL-8 secretion and ICAM-1
expression on melanoma cells. 1205
Lu cells were treated with PDTC
(100µM) for 1hr. After twice washing,
1205 Lu cells were cultured using fresh
culture media. Left panel: supernatant
after 4h was collected and IL-8
secretion was detected by ELISA; right
panel: ICAM-1 expression on 1205 Lu
was examined by flow cytometry.
Mechanism of inhibiting V600EB-Raf to disrupt melanoma extravasation
β2 integrins
Melanoma
ICAM-1
IL-8
sLex
E-selectin
B-Raf
nucleus
NF-κB
IL-8
ICAM-1
PMN
EC
PMN-facilitated Melanoma Extravasation
PMN-C8161
*
= f (1/γ)
PMN
SLex
EC
E-Selection
ICAM-1
β2 Integrins
ICAM-1
C8161
PMN-EC
= f (τ)
Figure by MIT OpenCourseWare.
Parallel-Plate Flow Assay
Figures removed due to copyright restrictions.
Please see: http://www.biomedcentral.com/content/figures/1471-2172-2-9-1.jpg.
FMLP-stimulated neutrophils
were injected together with tumor
cells at a very low flow rate (0.004
ml/min) for 2 min to accumulate
cells within the flow chamber.
After the initial accumulation, a
step increase in flow rate was
applied to the cells.
BMC Immunology. 2:9, 2001.
TC-PMN Collision and Aggregation Near the Endothelium in a Shear Flow
The following parameters were
measured and found to be
influenced by shear conditions:
Image removed due to copyright restrictions.
ƒ the number of TC-PMN
collisions;
ƒ the number of TCs captured by
PMNs; and
ƒ the number of TCs adhered to
ECs as a result of TC-PMN
collision/aggregation.
Adhesion Efficiency
Number of TC adhered to EC monolayer as a result of collisions
=
Total number of collisions
Two Different Types of Cell Aggregation Are Examined:
C8161
ατ
EC
τ = µγ&
Figures by MIT OpenCourseWare.
Two different cell aggregations are examined: tumor
cell to PMN and PMN to endothelial cell.
• PMN tethering data indicates that PMN adhesion to
endothelial cells follows a more traditional hydrodynamic
relationship and is proportional to shear stress and
contact duration.
• The adhesion and migration data reveal that tumor cell
to PMN adhesion varies inversely to shear rate and is
dependant on contact duration while independent of
shear stress.
C8161 Migration per mm2
PMN
32
30
1.0 cP
28
2.0 cP
26
3.2 cP
24
22
20
18
16
40 60 80 100 120 140 160180 200 220
Shear rate (sec-1)
WM9 Adhesion Efficiency
∗
α 1/γ
Normalized tethering frequency
events/mm2/min
Tumor Cell to PMN & PMN to Endothelial Cell
350
0.7 cP
3.2 cP
7.0 cP
300
250
200
150
100
0
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
100 200 300 400 500 600
Shear rate (sec-1)
2.0 cP
1.0 cP
3.2 cP
40 60 80 100 120 140 160 180 200 220
Shear rate (sec-1)
Effects of Shear Rate and Shear Stress on PMN-Melanoma Interactions
• Experimental and
Computational Approach
– Focus on interactions between PMN and melanoma cell
– Model second step: melanoma cell colliding and adhering to a tethered PMN
on EC by capturing:
• Deformation
• Collision
• Adhesion kinetics
Side-View PIV System
Images removed due to copyright restrictions.
Velocity Profiles
• Interrogation windows:
30 x 20 pixels (W x H), 0.23 µm/pixel
Stream lines over an adhered 16-µm
bead near the microslide wall.
Velocity vectors over an adhered 16-µm
bead near the microslide wall.
Computational Fluid Dynamics and Statistic Population Balance Model
Images removed due to copyright restrictions.
Cell population studies
CFD studies
Micro-PIV experimental studies
Simulation of a melanoma cell binding to
a PMN
Cancer Immunoediting in
Leukocyte-mediated Melanoma Extravasation
collision
frequency
Flow
PMN
Shear rate vs.
Shear stress
adhesion
efficiency
tumor
cell-cell signaling,
chemokine secretion
PMN
adhesion molecule
expression
Endothelial junction regulation:
– phosphorylation state
- tumor cell extravasation model
tumor
PMN
Tumor cell
extravasation
Acknowledgement
Photographs removed due to copyright restrictions.
Shile Liang
Maggie Slattery
Dr. Andrew Henderson (BU)
Hsin H. Peng
Louis Hodgson
Dr. Jeffery Zahn (Rutgers )
Meghan Hoskins
Jun You
Dr. Gavin Robertson (PSU)
Chong-Haw Kwang Xiao X. Lei
Dr. Elise Kohn (NIH)
Sharad Gupta
Jian Cao
Dr. Lance Liotta (NIH)
Payal Khanna
Karen Perkowski
Dr. Danny Welch (UAB)
Patricia Groleau
Brad Rank
Dr. Avery August (PSU)
Tara Yunkuins
Jordan Leyton-Mange
Dr. Mike Lawrence (UVA)
Desiree Wagner
Supported by NIH and NSF grants.