Harvard-MIT Division of Health Sciences and Technology
HST.525J: Tumor Pathophysiology and Transport Phenomena, Fall 2005
Course Director: Dr. Rakesh Jain
Contribution of bone marrow-derived cells
during tumor progression and treatment
Reference:
Dan G Duda, DMD, PhD
Steele Laboratory
Massachusetts General Hospital
Harvard Medical School
1. Introduction: Stem cells and neovascularizationn�
2. BMDCs involvement in neovascularization during
tumor growth
3. BMDCs and the therapy of cancerr�
Embryonic stem cells
Vasculogenesis
Trophoblast
Inner Cell Mass
In Vitro Fertilization
Totipotent Cells
Blastocyst
Figure by MIT OCW.
Fetus
Totipotent Cells
Pluripotent Stem Cell
Blood Stem Cells
Specialized
Cells
Red Blood
Cells
Platelets
Other Committed
Stem Cells
White Blood
Cells
Figure by MIT OCW.
Courtesy of the National Institutes of Health.
Vasculogenesis in Embryo
Undifferentiated Cells
"Vascular Progenitor Cells"
E-cadherin+
Flk1+
E-cadherin?
VEGF
Hemangioblast?
Flk1+
Flk1­
VEGF
Blood Cells
Flk1­
CD45+
Ter119+
EC
Flk1+
VE-cadherin+
PECAM1+
CD34+
Endoglin+
Mesenchymal Stem
Cells?
PDGF-BB
MC
Flk1­
�SMA+
PDGF-�R+
SM-tropomyosin+
Desmin+
Blood Vessels
Figure by MIT OCW. After Yamashita et al., 2000.
Other Lineage
Endothelial Progenitors
Blood Cells
Skeletal Muscle
Haemangioblast
Angioblast
Pre-venous
Angioblast
Pre-arterial
Angioblast
Common Vascular
Progenitors
VEGF
Venous
Endothelial
Cells
Arterial
Endothelial
Cells
VEGF
VEGF
Blood Vessel
Others?
PDGF-BB
Progenitor Expressing
PDGF Receptor-�
Mesenchymal Cell
Smooth
Muscle
Cells
Pericytes
PDGF-BB
Neural Crest
Cell
Vascular Progenitor
Cells
Epicardial-derived
Cell
Smooth Muscle Progenitors
Figure by MIT OCW. After Carmeliet, 2001.
PDGF-BB
Image removed for copyright reasons.
Source: Takakura, Nobuyuki, Toshio Watanabe, Souichi Suenobu, Yoshihiro Yamada, Tetsuo Noda, Yoshiaki Ito, Masanobu Satake, and Toshio Suda. "A Role
for Hematopoietic Stem Cells in Promoting Angiogenesis." Cell 102, no. 2 (2000): 199-209.
Adult stem cells
cells�
Vasculogenesis
Vasculogenesis�
Adult Hematopoiesis
Image removed for copyright reasons.
Stem Cell Sorting
BM Harvest
Magnetic Selection
Clone-sorting
Microscopic
Examination
.
Figure by MIT OCW
(CD3, CD4, CD8, CD45, Gr-1, Mac-1, B220, and TER-119)
Transplantation
Hematopietic stem cells assays
A Self-Renewal
Stemcell
C Functional repopulation
WBC
Transient amplifying cells
XRT
B Multi-lineage differentiation
Figure by MIT OCW.
Tracking bone marrow progeny
Lethal irradiation
ROSA 26 (mouse
transgenic
for bacterial
β-galactosidase )
Detection of
β-galactosidase
(histochemistry)
GFP (mouse
transgenic for
jellyfish green
fluorescent protein)
Detection of
GFP
(fluorescence
microscopy)
Figure by MIT OCW.
Bone Marrow Transplant
Male (XY) into female
recipient, XX)
Detection of
Y-chromosome
(In situ hybridisation)
GFP
Parabiosis
GFP
Wt
Wt
Figure by MIT OCW
.
Bone Marrow hosts multipotent stem cells
Haematopoietic Stem Cells
Mesenchymal Stem Cells
Endothelial Precursor Cells
Stem Cell Plasticity?
Images removed for copyright reasons.
Source: Krause, D., and N. Theise, et al. "Multi-Organ, Multi-Lineage Engraftment by a Single Bone Marrow-Derived Stem Cell." Cell 105, no. 3 (2001): 369-377.
Percent donor engraftment of nonhematopoietic tissues 11 months post transplant
M1
M2
M3
M4
M5
Mean +/- SD
Corr.*
Bronchi
Alveoli
Esoph
3.6
2.3
3.5
2.2
0
2.32 +/- 1.45
3.74
14.8
10.3
18.7
10.1
9
12.58 +/- 4.07
20.30
0
0.4
2.2
2.5
0.5
1.12 +/- 1.14
1.81
Stomach
Sm. Bowel
0.5
0.5
0
0.2
0.4
0.32 +/- 0.21
0.52
Figure by MIT OCW .
0.3
0.4
0
0.4
1.6
0.54 +/- 0.61
0.87
Large Bowel
0.2
0.1
0
0.3
0
0.12 +/- 0.13
0.19
Skin
2.6
2.4
1.2
1.6
2.7
2.1 +/- 0.66
3.39
Bile Duct
0.4
0
0
2.2
0
0.52 +/- 0.95
0.84
What is the evidence for BMDC vasculogenesis
in tumors?
Putative endothelial progenitor cells
Images removed for copyright reasons.
See: Fig. 1 and 2 in Asahara, Takayuki, Toyoaki Murohara, Alison Sullivan, Marcy Silver, Rien van der Zee, Li Tong, Bernhard
Witzenbichler, Gina Schatteman, and M. Isner Jeffrey. "Isolation of Putative Progenitor Endothelial Cells for Angiogenesis." Science
275 (1997): 964-966.
Human peripheral blood mononuclear cells
AcLDL
CD31
vWF
Reference: Au, unpublished 2003
GFP-HPBMC in a collagen gel w/10T1/2
pericyte precursors- 3 weeks
Reference: Koike et al., Nature 2004; Au, unpublished
+/– mice
BMDC rescue angiogenesis deficiency in Id1–/–Id3+/–
Images removed for copyright reasons.
Source: Fig. 3 and 4 from Lyden, D., et al. "Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor
cells blocks tumor angiogenesis and growth." Nature Medicine 7 (2001): 1194 - 1201.
Gr+CD11b+ BMDCs directly promote tumor angiogenesis
*
1200
1000
3
2
600
400
200
0
1
0
800
MC26
7
14
21
Days after tumor inoculation
*
MC26 plus nor
.
Gr+CD11b+cells
Live volume
*
80
60
VEGFR2 VE-Cad VEGFR2
+
VE-Cad
Nar.spleen
Tumor spleen
40
20
0
MC26 plus nor.
Gr+CD11b+cells
Necrotic volume
Figure by MIT OCW.
After Yang et al., 2004.
MC26 plus tu.
Gr+CD11b+cells
30
25
No. of apoptotic cells
per field (200x)
Figure by MIT OCW.
After Yang et al., 2004.
MC26 plus tu.
Gr+CD1 1b+cells
*
2
MC26 plus Gr+CD11b+
MC26 plus Gr+CD11b+VEGFR2-
MC26+Gr+CD11b+
0
4
0
10
**
8
6
20
αSMC + vessels / Total vessels (%)
4
No.of Gr + CD11b + Cells(%)
5
Tumor volume (mm3)
Tumor volume (cm3)
E
Number of blood vessels
per field (200x)
30
20
15
*
10
5
0
MC26 plus nor
.
Gr+CD11b+cells
�
MC26 plus tu.
Gr+CD11b+cells
Figure by MIT OCW.
After Yang et al., 2004. �
�
Endothelial Cell Precursors
Vasculogenesis in adults - Asahara T., Isner M.J., et al.
- putative endothelial progenitor cell (EPC, angioblast), CD34+
Postnatal vasculogenesis - Rafii S., et al.
- circulating endothelial progenitors (CEP), Flk-1+/AC133+
Recruitment of angioblast-like
endothelial precursor cells
(Vasculogenesis)
Recruitment of pre-existing
endothelial cells
(Angiogenesis)
Bone marrow
Tumor tissue
vascular injury
In vitro culture
Early outgrowth, low
proliferative potential
Late outgrowth, high
proliferative potential
Figure by MIT OCW. After Rafii, 2002.
Figure by MIT OCW.
�
Image removed for copyright reasons.
Source: Wade, Nicholas. "Scientists Link Tumors to Bone Marrow Cells." The New York Times, November 1, 2001: A22.�
Endothelial Cell Precursors
Vasculogenesis in adults - Asahara T., Isner M.J., et al.
- putative endothelial progenitor cell (EPC, angioblast), CD34+
Postnatal vasculogenesis - Rafii S., et al.
- circulating endothelial progenitors (CEP), Flk-1+/AC133+
Figure by MIT OCW. After Stoll et al., 2003.
Bone marrow cell mobilization
for neovascularization
VEGF
eNOS
Stromal Cell
Progenitor / Stem cell
NO
MMP-9
sKitL
Figure by MIT OCW. After Duda et al., 2004.
The Weinberg hypothesis
A
B
GFP-siRNA+SDF-1-fibro.
Tumor volume (mm3)
CXCR4-siRNA1,2
+SDF-1-fibro.
400
*
n = 13
*
Loss of endocrine
signaling
n = 15
Activated
myofibroblasts
n = 15
150
CXCR4-siRNA1,2
+control GFP-fibro.
100
0
10
20
30
SDF-1
EPCs
Paracrine
effect
Invasive human
breast cancer mass
n = 15
200
50
Recruitment
t
Loss of paracrine
signaling
*
450
250
EPCs
40
50
60
Days
Figure by MIT OCW.
en
itm
*
300
n
Endocrine effect
500
350
atio
Normal fibroblasts
or progenitor cells
n = 15
550
biliz
cru
600
Mo
Re
650
Bone marrow
CXCR4
Breast carcinoma cells
Blood vessel
Sample
Patient
sex
Donor
sex
Number of vessels
snalyzed
vWF+ CD45cells
BMDC
BMDC
(percent)
BMDC (percent)
normalized
Spindle cell sarcoma,
head and neck
F
M
58
215
1
0.5
1.0
Hodgkin lymphoma
F
M
37
219
11
5.0
12.1
Mucoepidermoid
carcinoma, submandibular
F
M
38
192
7
3.6
7.0
Thyroid carcinoma
F
M
95
752
4
0.5
1.1
Osteogenic carcinoma
M
F
36
293
3
1.0
4.1
Mucoepidermoid
carcinoma, glossal
M
F
11
94
1
1.1
4.0
BMDC: bone marrow-derived cells.
Combined Immunofluorescence and FISH Data
Figure by MIT OCW. After Peters et al., 2005.
Image removed for copyright reason.
Please see Fig. 1 in Peters, B. A., L. A. Diaz, K. Polyak, L. Meszler, K. Romans, E. C. Guinan, E. C. Antinx, D. Myerson, S. R. Hamilton,
B. Vogelstein, K. W. Kinzler, and C. Lengauer. "Contribution of bone marrow-derived endothelial cells to human tumor vasculature."
Nature Medicine 11 (2005): 261-262.
Tie2-expressing myeloid cells
Images removed for copyright reasons.
Source: Palma, Michele De, Mary Anna Venneri, Rossella Galli, Lucia Sergi Sergi, S. Politi Letterio, Sampaolesi Maurilio, and Luigi Naldini. "Tie2
identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte
progenitors." Cancer Cell 8 (2005): 211-226.
No detectable BMDC in tumor endothelium
Images removed for copyright reasons. See: Fig. 6 in Gothert, J. R. et al. "Genetically tagging endothelial cells in vivo: bone marrow-derived cells do not
contribute to tumor endothelium." Blood 104 (2004): 1769-1777.
De Palma et al.
Nature Medicine, June
Garcia-Barros et al.
Sciencer, May 18
Acid sphingomyelinase-/-
Tie 2p/e-GFP or PGKp
lentiviruses
BM Transplant
Tumor Transplant
MCA fibrosarcoma
B16 melanoma
TS/A mammary carcinoma
LLC
B16 melanoma
EPCs- critical for
tumor angiogenesis and growth
No EPCs
TEMs- critical for
tumor angiogenesis and growth
Images removed for copyright reasons. Source: Jain, R. K., and D. Duda.
Bone marrow derived cell contribution
to neovascularization
Figure by MIT OCW. After Jain and Duda, 2003.
Research problems
Bone marrow-derived cells infiltrate tumors and modulate
neovascularization:
-endothelial precursors (eg, Rafii, Lyden & Benezra, Nat Med 2001,
Science 2003)
- mezenchymal stromal precursors (eg, Direkze, Cancer Res 2004,
Bergers, Nat Cell Biol 2005)
-hematopoietic precursors (eg, De Palma, Nat Med 03, Cancer Cell
2005, Rafii Nat Med 01)
BM cells are mobilized, home to tumors and contribute to
neovascularization
Ionizing radiation kills cancer and normal cells, and BMDCs can
rescue tumor growth (Fuks and colab., Science 01 & 03);
Is this a mechanism of relapse?
Courtesy of RK Jain, et al. Used with permission.
Figure by MIT OCW.
Courtesy of RK Jain, et al. Used with permission.
Image courtesty of NIH.
What is the evidence for BMDC incorporation as
Perivascular cells in tumors?
Smooth muscle precursor cells in blood
Images removed for copyright reasons.
Source: Fig. 4 and 6 in Simper, D., P. G. Stalboerger, C. J. Panetta, S. Wang, and N. M. Caplice. "Smooth muscle progenitor cells in
human blood." Circulation 106 (2002): 1199-1204.
Quantification of number and diameter
of EPC-derived vessels
+
Percent GFP functional vessels
Tumor type
Implantation si t e
Lung adenocarcinoma
Subcutaneous
Lung adenocarcinoma
metastasis
Lung
Melanoma
3
Subcutaneous
Actb-GFP/BMT
1
(C57BL6)
Tie2-GFP/BMT
2
(FVB)
4
< 1%
4
4.5 ± 1.6
< 1%
5
10.6 ± 3.6
< 1%
4
N/A
5
6
Mammary carcinoma
Subcutaneous/
mammary fat pad
N/A
1.3 ± 0.1
Mammary carcinoma
metastasis
Brain
N/A
58.4 ± 8.4
Spontaneous mammary
carcinoma
–
N/A
20.7
Spontaneous fibrosarcoma
–
N/A
3.2
Spontaneous
hemangiosarcoma
–
N/A
< 1%
Spontaneous squamous cell
carcinoma
–
N/A
5.3
7
7
8
8
8
8
What is the evidence for BMDC incorporation as
Perivascular cells in tumors?
Smooth muscle precursor cells in blood
Images removed for copyright reasons.
Source: Fig. 4 and 6 in Simper D., P. G. Stalboerger, C. J. Panetta, S. Wang, and N. M. Caplice. "Smooth muscle progenitor cells in human blood."
Circulation 106 (2002): 1199-1204.
TG1-1 in CW (VEGF-GFP/FVB mouse, 2 weeks, rho
dextran perfusion)
500µ
500µm
100µ
100µm
50µ
50µm
Reference: Fukumura et al., Cell 1998; Duda et al. Cancer Res 2004
αSMA staining in mammary carcinoma tissue
in VEGF-GFP mouse
Image removed due to copyright reasons.
Reference: Tong et al., PhD Thesis 2005
Image removed for copyright reasons.
Bone marrow-derived stromal cells
in mouse insulinoma after BMT
Perivascular cells originate from Sca1+ hematopoietic cells
Image removed for copyright reasons.
See Fig. 4 in Song, Steven, J. Ewald Andrew, William Stallcup, Zena Werb, and Gabriele Bergers. "PDGFRß+ perivascular progenitor
cells in tumours regulate pericyte differentiation and vascular survival." Nature Cell Biology 7 (2005): 870-879.
Circulating VEGF-GFP+ cells in orthotopic
mammary carcinoma-bearing immunodeficient mice
051404.Rag2.005
104
CD45PerCP
CD45PerCP
103
R2
102
101
100
051404.Rag2.002
104
103
R3
102
101
100
101
102
103
104
100
100
VEGF-GFP
101
102
103
VEGF-GFP
Figure by MIT OCW.
Tumor
Control
104
Multiple stem cells
A
=
C
+
B
Cell fusion
+
=
Trans de-/re-differentiation
D
Pluripotency
Figure by MIT OCW.
Images removed for copyright reasons.
See: Figs. 1, 4, 6 and 7 in Blau, H. M., T. R. Brazelton, and J. M. Weimann. "The Evolving Concept of a Stem Cell: Entity or
Function?" Cell 105 (2001): 829-841.
Stem Cell- entity or function
MAPC, Verfaillie, Nature 2001
Images removed for copyright reasons.
TUMOR
MPC
HMPC
F
EC
VEGF
PIGF
SDF1
BMCs
MC
BMC
Bone Marrow
Blood Vessel
Radiation
Figure by MIT OCW.
E-selectin
VEGF