Harvard-MIT Division of Health Sciences and Technology
HST.525J: Tumor Pathophysiology and Transport Phenomena, Fall 2005
Course Director: Dr. Rakesh Jain
BMDCs and Cancer Therapy
Therapy
1. BMDCs for cell-based therapies
Engineered embryonic EPCs for tumor targeting
Images removed for copyright reasons.
See: Fig. 1 and 8 in Wei, et al. "Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases
after intravenous delivery." Cancer Cell 5 (2004): 477-488.
Homing kinetics after cell infusion
Cell homing after a lineage negative cell delivery, local and systemic,
in tumor bearing mice (mammary carcinoma in CW)
Images removed for copyright reasons.
90 minutes, x6.25, IVM
10 minutes, x20, MPLSM
Courtesy of Nature Publishing Group.
Source: Stroh, M., J. P. Zimmer, D. G. Duda, T. S. Levchenko, K. S. Cohen, E. B. Brown, D. T. Scadden, V. P. Torchilin, M. G. Bawendi, D. Fukumura, and R. K. Jain. "Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo." Nature Medicine 11 (2005): 678-682.
Cell cytoplasm labeling using
Quantum dots micelle and tat bioconjugate
Mammary carcinoma
in CW
100µm
2-photon image of Q-dots labeled progenitor cells (red)
Vessel enhancement by Q-dots (blue) infusion
Courtesy of Nature Publishing Group.
Source: Stroh, M., J. P. Zimmer, D. G. Duda, T. S. Levchenko, K. S. Cohen, E. B. Brown, D. T. Scadden, V. P. Torchilin, M. G. Bawendi, D. Fukumura D, R. K. Jain "Quantum
dots spectrally distinguish multiple species within the tumor milieu in vivo." Nature Medicine 11 (2005): 678-682.
2. Role of BMDCs in tumor relapse after treatment
reatment
BMDC targeting delays tumor growth
500
Images removed for copyright reasons.
See: Fig. 6 in Lyden, Rafii et al. "Impaired
recruitment of bone-marrow-derived
SURFACE AREA (mm2)
400
endothelial and hematopoietic precursor
cells blocks tumor angiogenesis and
WT (5)
WT + mF - 1 (5)
300
200
WT + DC 101 (5)
WT + mF - 1/DC
101 (5)
100
0
0
2
4
6
DAYS
8
10
12
Neutralizing the BMDCs with antibodies to both murine VEGFR1 (mF-1) and
VEGFR2 (DC101) blocks tumor angiogenesis and thus blocks tumor growth.
Figure by MIT OCW.
growth." Nature Medicine 7 (2001): 1194 ­
1201.
Tumor response to radiation is BMDC-dependent
Data
point
Asmase -/-
200
Data
point
Donor
Recipient
Asmase +/+ Asmase +/+ (10)
Asmase +/+ (9)
Asmase -/-
200
Asmase +/+ (10)
150
150
100
100
50
50
00
00
00
10
15
20
25
15Gy
00
30
10
15
20
25
Days after tumor implantation
800
20
Asmase +/+
Asmase -/-
15
10
5
0
0
4
8
10
24
Time after radiation (hours)
Tumor Volume (mm3)
per field (X400)
Apoptotic Endothelial Cells
Tumor Volume (mm3)
Recipient
Asmase +/+ Asmase +/+ (10)
250
Images removed for copyright reasons.
See: Fig. 2 in Garcia-Barros, et al. "Tumor Response to
Radiotherapy Regulated by Endothelial Cell Apoptosis."
Science 300, (May 16, 2000): 1155-1159.
Donor
Bax +/+
Bax -/­
600
400
200
0
0 10 12 14 16 18 20
Days after tumor implantation
Figure by MIT OCW.
30
Mammary tumor relapse after 25 Gy irradiation
Tie2-GFP
Tie2-GFP-BMT
Images removed for copyright reasons.
Wild-type-BMT-Tie2-GFP
BMDC infiltrate during LLC recurrence
after local irradiation using a single dose of 50Gy
Images removed for copyright reasons.
Antiangiogenic therapy by VEGFR2 blockade
for LLC tumors implanted in Actb-GFP/BMT
4
50
CD11b + CD45 + GFP +
CD31 + Sca1 + CD45 - GFP +
CD31 + Sca1 + CD45 - GFP ­
40
3
%
30
2
20
1
0
10
DC101
Figure by MIT OCW.
IgG
0
DC101
IgG
Tumor targeting using genetically engineered MSCs
Mean Lung Weight (grams)
1.2
1.0
Images removed for copyright reasons.
See: Figs. 2 and 4 in Studeny, et al. "Mesenchymal stem
0.8
0.6
cells: potential precursors for tumor stroma and targeted-
0.4
delivery vehicles for anticancer agents." JNCI 96 (2004):
0.2
1593-1603.
0
Treatment: None
Lungs: Normal
Figure by MIT OCW.
MSC-IFN-β IV
IFN-β
MSC-Gal IV
None
MDA 231
Tumors
MDA 231
Tumors
MDA 231
Tumors
MDA 231
Tumors
Reference: Andreeff and collab., JNCI 2004
3. BMDCs as surrogate markers
s
for antiangiogenic therapies
therapies
Bevacizumab:
Bevacizumab:
A blocking VEGF-specific antibody
antibody
••
Bevacizumab monotherapy significantly prolonged the time to progression in a Phase II
trial for metastatic renal cell carcinoma patients (Yang et al., New Engl J Med 2003)
••
Bevacizumab (5mg/kg) + IFL (Irinotecan-5-FU-Leucovorin)- increased the overall
survival in a Phase III trial for metastatic colorectal cancer patients (Hurwitz et al., New
Engl J Med 2004)
••
Based on these results, bevacizumab was the first antiangiogenic drug approved by the
Food and Drug Administration (February 2004)
••
Bevacizumab (5mg/kg) + FOLFOX4 (oxaliplatin, 5-fluorouracil and leucovorin) increased
overall survival in a Phase III trial for recurrent colorectal cancer patients*
••
Bevacizumab + paclitaxel and carboplatin increased progression-free survival in a
Phase III trial for lung cancer patients*
patients*
••
Bevacizumab + paclitaxel increased progression-free survival in a Phase III trial for
breast cancer patients*
patients*
*NCI Newcenter
Background
Proposed Normalization Window
Dose
Image removed for copyright reasons. Avastin (cancer therapeutic) advertisement.
Normalization Window
Excessive Pruning
Toxicity to Normal Tissue
No Effect on Vessels
Time
Figure by MIT OCW.
The unprecedented success of Avastin in the clinic was explained by its antivascular
effects and by the ability to “normalize”
normalize” tumor vasculature. The latter may improve the
delivery of cytotoxic treatments.
To date, no established, valid surrogate marker exists in the clinic to guide the
optimal dosing and scheduling of Avastin to achieve these effects
Protocol of Clinical Trial
50.4Gy EBRT / 5-FU*
Surgery
Avastin
5 or 10 mg/kg
Day
Blood/urine
-3
√
1
3
√
12 15
√
Tumor Biopsy √
√
IFP
√
√
Imaging
√
√
29 32
√
43
93
103
√
√
√
*Daily Radiation Therapy (50.4 Gy/ 28 Fx) and continuous infusion of 5-Fluorouracil (225 mg/m2/day)
Tumor Response: FDG Uptake (PET)
15
Sagittal PET scans: Patient #1-6
4
5
Reference Corrected SUV
(Rectal ROI)
1
Pt #1
2
Day 12
PrePre-Tx Day 12 PreSurgery
Surgery
Pt #5
9
Pt #6
6
3
Baseline
Day 12
Pre-Surgery
Patient
Reference Corrected SUV
(Colorectal ROI)
Pre-Tx
6
Pt #3
Pt #4
0
3
Pt #2
12
7
8
9
10
11
Pre-Treatment
Reference:
Willett et al, Nat Med 2004 (1), Willett et al, Nat Med 2004 (2), Willet et al, J Clin Oncol 2005
Day 12
Pre-Surgery
Figure by MIT OCW.
Tumor Response: Biopsy Tissue Histological Analyses
Microvascular Density
Number of vessels per field
field
After 1 BV
Before BV
80
60
40
20
0
Pt
#1
Pt
#2
Pt
#3
Pt
#4
Pt
#6
Pt
#8
Pt
#9
Pre-Tx
Day 12
16
30
20
8
10
4
0
0
1
2
3
4
2
Pt
#3
Figure by MIT OCW.
Pt
#4
Pt
#6
Pt
#8
Patient # 9
Patient # 11
6
4
Figure by MIT OCW.
Pericyte Coverage
Pre-Tx
Day 12
25
Pt
#2
Patient # 8
Patient
6
Pt
#1
After 1 BV
Before BV
40
12
Apoptosis of Epithelial Cells
8
0
20
Pt
#11
Pt
#9
Pt
#11
% α-SMA
α-SMA+ vessels
% of Apoptotic Nuclei
% of Proliferating Nuclei
Proliferation of epithelial cells
20
60
15
40
10
20
5
0
After 1 BV
Before BV
0
0 1
2
3
4
Patient
6
Patient # 8
Patient # 9
Patient # 11
Figure by MIT OCW.
Functional Tumor Vascular Parameters
after Avastin Treatment
90
10
Blood Volume
80
70
60
50
40
30
Pre-Tx
8
7
6
5
4
1
Pre-Tx
17
Day 12
3
4
5
c
16
15
14
13
Figure by MIT OCW.
12
11
10
9
Pre-Tx
6
IFP (mm Hg)
IFP During
Before B V
BV (Day12)
# 3 (5mg/kg)
16.0 ± 7.0, 5.0*
5.0
# 4 (5mg/kg)
15.0 ± 1.4, 1.0
1.0 ± 1.5, 0.9
# 5 (5mg/kg)
16.5 ± 5.0, 3.5
4.0 ± 3.7, 2.1
# 6 (5mg/kg)
12.0 ± 5.0, 3.5
6.0 ± 9.0, 4.5
# 7 (10mg/kg)
8.0 ± 2.1, 1.5
10 ± 9.2, 6.5
# 8 (10mg/kg)
15.0 ± 2.5, 1.8
5.5 ± 3.9, 2.8
# 9 (10mg/kg)
22.0 ± 20.0, 14.3
-1.5 ± 0.7, 0.5
# 10 (10mg/kg)
5.0 ± 0.6, 0.3
7.0 ± 1.5, 0.9
# 11 (10mg/kg)
29
6.0 ± 5.5, 3.2
Patient (dose)
Endoscopy:
Endoscopy:
IFP measurements
9
3
Day 12
Patient:
b
PS (ml/min per 100g tissue)
11
a
(ml per 100g tissue)
Blood Flow
CT Scan
(ml/min per 100g tissue)
100
Day 12
Solid line, p<0.05
*Mean IFP ± SD, SEM
Reference: Willett et al, Nat Med 2004, Willet et al, J Clin Oncol 2005
The Need for Surrogate Markers for Treatment
Treatment
In summary,
••
A d d itio n o f A v a s tin a s n e o a d ju v a n t t o c h e m o th e r a p y w a s d e m o n s tr a te d in th e c lin ic a l tr ia ls to
increase survival in cancer patients (for three tumor types)
••
W e h a v e s h o w n th a t A v a s tin a lo n e h a s r a p id a n d p o te n t e ffe c ts o n tu m o r v a s c u la r s tr u c tu r e a n d
function
••
In c r e a s in g e v id e n c e s u p p o r ts th e c o n c e p t th a t th e m e c h a n is m o f a c tio n o f A v a s tin is to p r u n e
abnormal vasculature and fortify and improve the function of the remaining vasculature in tumors
••
To date, no surro
rrogate marker has been established in the clinic to evaluate the anti-vascular
effects and no marker has been proposed for the normalizing effects of Avastin
••
B a s e d o n th e in c r e a s in g p r e c lin ic a l e v id e n c e th a t b lo o d m a r k e r s m a y b e u s e fu l fo r th e e v a lu a tio n o f
antiangiogenic drugs’
drugs’ efficacy, we investigated the:
i) kinetics of circulating endothelial cells (CECs) and progenitor cells (CPCs
); and
(CPCs);
and
ii) changes plasma protein
protein
in rectal cancer patients before and during Avastin treatment.
treatment.
Characterization of Peripheral Blood Cells’ Phenotype
by Four-color Flow Cytometry
Images removed for copyright reasons.
Sources: Duda, D. G., K. S. Cohen, E. di Tomaso, P. Au, R. Klein, D. Scadden, C. G. Willett, and R. K. Jain. "Differential CD146
expression on circulating versus tissue endothelial cells in cancer patients: Implications for circulating endothelial cells as biomarker
for antiangiogenic therapy." J Clin Oncol 24 (2006): 1449-53.
Kinetics of Circulating Cells in Response to Avastin
Treatment Alone in Cancer Patients by Flow Cytometry
2
0
Viable CECs
4
2
2
3
0
Base- 3 days 12 days
line
Patient:
Baseline
5 mg/kg
10 mg/kg
0.10
Percent of PC/WBC
Percent of PC/WBC
0.04
0.03
0.02
0.01
0.00
3 days 12 days
Baseline
3 days 12 days
4
6
0
CPCs
3
0.06
2
0.04
1
0.02
0
0
7
3
9
Non-viable CECs
10
0.06
11
0.04
Viable CECs
2
1
0.02
0
Baseline
CPCs
N = 5 patients
1
5
Non-viable CECs
8
0.08
0.00
5 mg/kg
10 mg/kg
2
Percent
4
6
Percent
6
Percent of CEC/WBC
Percent of CEC/WBC
Viable CECs
3 days 12 days
Figures by MIT OCW.
Baseline
Day 3
Day 12
Kinetics of plasma proteins after Avastin treatment
180
600
140
VEGF
500
100
400
80
300
60
200
Concentration (pg/ml)
bFGF
120
40
100
0
20
Pt
#1
Pt
#2
Pt
#3
Pt
#4
Pt
#6
Pt
#7
Pt
#8
Pt
#9
Pt
#10
0
Pt
#11
Pt
#2
Pt
#3
Pt
#4
Pt
#6
Pt
#7
Pt
#8
Pt
#9
Pt
#10
Pt
#11
Pt
#8
Pt
#9
Pt
#10
Pt
#11
Before BV
400
3 days after BV
sFlt1
350
12 days after BV
300
250
40
35
PLGF
30
25
200
20
150
15
100
10
50
0
Pt
#1
5
Pt
#1
Pt
#2
Pt
#3
Pt
#4
Pt
#6
Pt
#7
Pt
#8
Pt
#9
Pt
#10
Pt
#11
0
Pt
#1
Pt
#2
Pt
#3
Pt
#4
Pt
#6
Pt
#7
Figure by MIT OCW.
Effects of VEGF on Bone Marrow Precursor Cells
Cells
VEGF mobilizes precursors/stem cell for hematopoiesis and
and
vasculogenesis
vasculogenesis
VEGF is a survival factor for hematopoietic stem cells and endothelial
endothelial
cells
cells
VEGFRs are present on hematopoietic precursors/stem cells,
cells,
endothelial cells and mesenchymal stem cells
cells
Bevacizumab blockade of VEGF may affect bone marrow precursor
precursor
cells
cells
Surrogate marker candidates:
candidates:
i) kinetics of circulating endothelial cells (CECs) and progenitor/stem
progenitor/stem
cells (CPCs))
ii) changes plasma protein
protein
Characterization of Endothelial Cell Phenotype
Phenotype
in Peripheral Blood and in Tumor Tissue in Mice
Mice
Images removed for copyright reasons.
CEC kinetics in Actb-GFP/BMT mice bearing LLC
Image removed for copyright reasons.
CD31dimCD45- CECs
CD31brightCD451.2
PERCENT
PERCENT
0.6
0.4
0.2
0.9
0.6
0.3
0
0
DC101
IgG
DC101
IgG
Figure by MIT OCW.
Analysis of CD133 (Prominin 1) Expression
Expression
on Mouse Bone Marrow Cells
Cells
Images removed for copyright reasons.
Expression of CD146 (P1H12, pan-endothelial cell marker)
marker)
and CD133 (AC133, progenitor/stem cell marker) on
on
Peripheral Blood Mononuclear Cells in Cancer Patients
Patients
Rectal carcinoma patient peripheral blood
Images removed for copyright reasons.
Source: Duda, D. G., K. S. Cohen, E. di Tomaso, P. Au, R. Klein, D. Scadden, C. G. Willett, and R. K. Jain. "Differential CD146 expression on
circulating versus tissue endothelial cells in cancer patients: Implications for circulating endothelial cells as biomarker for antiangiogenic therapy."
J Clin Oncol 24 (2006): 1449-53.
What Type of Cells Is CD146 Identifying
Identifying
in Human Tissues?
Tissues?
Images removed for copyright reasons.
Source: Duda, D. G., K. S. Cohen, E. di Tomaso, P. Au, R. Klein, D. Scadden, C. G. Willett, and R. K. Jain. "Differential CD146 expression on
circulating versus tissue endothelial cells in cancer patients: Implications for circulating endothelial cells as biomarker for antiangiogenic therapy."
J Clin Oncol 24 (2006): 1449-53.
Lung carcinoma biopsy
Images removed for copyright reasons.
Source: Duda, D. G., K. S. Cohen, E. di Tomaso, P. Au, R. Klein, D. Scadden, C. G. Willett, and R. K. Jain."Differential CD146
expression on circulating versus tissue endothelial cells in cancer patients: Implications for circulating endothelial cells as biomarker
for antiangiogenic therapy." J Clin Oncol 24 (2006): 1449-53.
Glioblastoma biopsy
CD146
Images removed for copyright reasons.
1
D45
Conclusions and Future Directions
Directions
•• The study of VEGF blockade in cancer patients suggested that blood
analyses may provide useful surrogate markers for response to
bevacizumab therapy
•• VEGF withdrawal reduces early after treatment the viable (bone
marrow-derived) CECs and progenitor cells, and increases plasma
levels of VEGF and PlGF
•• The increase and then decline in non-viable CECs after bevacizumab
treatment may correlate with the anti-vascular effect
effec (at day 3) and
normalizing effect
effec (at day 12) on tumor vessels
Conclusions and Future Directions
Directions
• U n lik e tis s u e E C s , v ia b le C E C s d o n o t e x p r e s s C D 1 4 6 ; C D 1 3 3
progenitor cells are CD45+; further understanding of biology of CECs
and CPCs (markers, viability, differentiation, etc.) is warranted
• T h e c h a n g e s in p la s m a le v e ls o f o th e r fa c to r s a ffe c tin g
angiogenesis (e.g., TSP1) should also be evaluated and correlated
with circulating cell kinetics
• W e a r e e v a lu a tin g th e s e m a r k e r s a fte r V E G F - s p e c ifi c b lo c k a d e
(bevacizumab) or growth factor receptor TKI treatment in six
ongoing trials for five different cancer types
• T r a n s la tio n o n a p a tie n t- b y - p a tie n t b a s is o f th e s e m a r k e r s w ill
require more sensitive technologies as well as unitary guidelines for
CEC and CPC evaluation
Acknowledgements
Dr. Jain
Dr. Jones
Dr. Scadden
Dr. Seed
Dr. Suit
Dr. Willett
All Steele lab
National Cancer Institute, Bethesda
(PO1 to RK Jain and R21 to CG Willett)
National Cancer Foundation
(RK Jain)
Cancer Research Institute, New York
(DG Duda)
American Association for Cancer Research
& Genentech-BioOncology
(DG Duda)