harvard-mit division of health sciences and technology hst ... · contribution of bone...

Contribution of bone marrow-derived cellsContribution of bone marrow-derived cellsduring tumor progressionduring tumor progression and treatmentand treatment

Dan G Duda, DMD, PhDDan G Duda, DMD, PhDSteele LaboratorySteele LaboratoryMassachusetts General HospitalMassachusetts General HospitalHarvard Medical SchoolHarvard Medical School

Reference:

Harvard-MIT Division of Health Sciences and Technology HST.525J: Tumor Pathophysiology and Transport Phenomena, Fall 2005 Course Director: Dr. Rakesh Jain

11.. InInttrroodduuccttiioonn: S: Stteemm cceellllss aanndd nneeoovvaassccuullaarriizzaattiioon�n22.. BBMMDDCCss iinnvvoollvveemmeenntt iinn nneeoovvaassccuullaarriizzaattiioonn dduurriinngg tumor growthtumor growth33.. BBMMDDCCss aanndd tthhee tthheerraappyy ooff ccaanncceer�r

Embryonic stem cellsEmbryonic stem cellsVasculogenesisVasculogenesis

l

Blastocyst Fetus

Inner Cell Mass

Trophob ast

In Vitro Fertilization Totipotent Cells

Figure by MIT OCW.

Blood Stem Cells

Red Blood Cells

Specialized Cells

White Blood Cells

Platelets

Other Committed Stem Cells

Pluripotent Stem Cell

Totipotent Cells

Figure by MIT OCW.

Courtesy of the National Institutes of Health.

http://www.nih.gov

Vasculogenesis in EmbryoVasculogenesis in EmbryoUndifferentiated Cells "Vascular Progenitor Cells"

E-cadherin+ E-cadherin-Flk1+

Flk1+

Flk1+ Flk1

Flk1

VEGF

VEGF

?

Hemangioblast?

PDGF-BB

Mesenchymal Stem Cells?

Other Lineage

Blood Cells EC MC

PDGF-�R+

SM-tropomyosin+

Desmin+

�SMA+Flk1

CD45+

Ter119+

VE-cadherin+

PECAM1+

CD34+

Endoglin+

Blood Vessels

Figure by MIT OCW. After Yamashita et al., 2000.

Endothelial Progenitors

Blood Cells Skeletal Muscle Haemangioblast Angioblast

Pre-venous Angioblast

Pre-arterial Angioblast

VEGF

VEGF VEGF

Endothelial Cells

Arterial Endothelial

Cells

PDGF-BB

PDGF-BB

PDGF-BB

Smooth Muscle Cells

Pericytes

Progenitors

Cells

Others?

Progenitor Expressing PDGF Receptor-�

Mesenchymal Cell Neural Crest Cell

Epicardial-derived Cell

Smooth Muscle Progenitors

After Carmeliet, 2001.

Venous

Blood Vessel

Common Vascular

Vascular Progenitor

Figure by MIT OCW.

Image removed for copyright reasons.

for Hematopoietic Stem Cells in Promoting Angiogenesis." Cell 102, no. 2 (2000): 199-209. Source: Takakura, Nobuyuki, Toshio Watanabe, Souichi Suenobu, Yoshihiro Yamada, Tetsuo Noda, Yoshiaki Ito, Masanobu Satake, and Toshio Suda. "A Role

Adult stem cells�Adult stem cellsVasculogenesis�Vasculogenesis

AdultAdult HematopoiesisHematopoiesis


Stem Cell SortingStem Cell Sorting

(CD3, CD4, CD8, CD45,(CD3, CD4, CD8, CD45, Gr-1, Mac-1, B220, and TER-119)Gr-1, Mac-1, B220, and TER-119)

BM Harvest Magnetic Selection Clone-sorting Microscopic Examination Transplantation

Figure by MIT OCW.

Hematopietic stem cells assaysHematopietic stem cells assays

Self-Renewal

Stemcell

Transient amplifying cells

Multi-lineage differentiation

WB

CXRT

Functional repopulation

A

B

C

Figure by MIT OCW.

Bone Marrow Transp

ROSA 26 (mouse transgenic for bacterial

actosidase )

mouse transgenic for

yfish green Detection of GFP (fluorescence

Detection of β-galactosidase (histochemistry)

Lethal irradiation

Tracking bone marrow progenyTracking bone marrow progeny

lant

β-gal

GFP (

jellfluorescent protein)

Male (XY) into female recipient, XX)

microscopy)

Detection of Y-chromosome (In situ hybridisation)

Figure by MIT OCW.

GFP GFP

Wt

Wt

Parabiosis

.Figure by MIT OCW

Bone Marrow hosts multipotent stem cellsBone Marrow hosts multipotent stem cells

Stem Cell Plasticity?

Haematopoietic Stem Cells

Mesenchymal Stem Cells

Endothelial Precursor Cells

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

Bronchi Alveoli Esoph Stomach Sm. Bowel Large Bowel Skin Bile Duct

M1 3.6 14.8 0 0.5 0.3 0.2 2.6 0.4 M2 2.3 10.3 0.4 0.5 0.4 0.1 2.4 0 M3 3.5 18.7 2.2 0 0 0 1.2 0 M4 2.2 10.1 2.5 0.2 0.4 0.3 1.6 2.2 M5 0 9 0.5 0.4 1.6 0 2.7 0 Mean +/- SD 2.32 +/- 1.45 12.58 +/- 4.07 1.12 +/- 1.14 0.32 +/- 0.21 0.54 +/- 0.61 0.12 +/- 0.13 2.1 +/- 0.66 0.52 +/- 0.95 Corr.* 3.74 20.30 1.81 0.52 0.87 0.19 3.39 0.84

Figure by MIT OCW.

What is theWhat is the evidence for BMDCevidence for BMDC vasculogenesisvasculogenesisin tumors?in tumors?

Putative endothelialPutative endothelial progenitor cellsprogenitor 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 cellsHuman peripheral blood mononuclear cells

CD31CD31

AcLDLAcLDL

vWFvWF

Au,Au, unpublishedunpublished 20032003Reference:

GFP-HPBMC in a collagen gel w/10T1/2GFP-HPBMC in a collagen gel w/10T1/2pericyte precursors- 3 weekspericyte precursors- 3 weeks

KoikeKoike et al.et al.,, Nature 2004Nature 2004; Au,; Au, unpublishedunpublishedReference:

BMDC rescue angiogenesis deficiency in Id1BMDC rescue angiogenesis deficiency in Id1––//––Id3Id3+/+/–– micemice


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.

GrGr++CD11bCD11b++ BMDCs directly promote tumor angiogenesisBMDCs directly promote tumor angiogenesis

�

�

�

30

20

10

0

Num

ber o

f blo

od v

esse

lspe

r fie

ld (2

00x)

MC26 plus nor. Gr+CD11b+cells

MC26 plus tu. Gr+CD1 1b+cells

*


MC26 plus tu. Gr+CD11b+cells

0

20

40

60

80 *

αSM

C +

ves

sels

/ To

tal v

esse

ls (%

)


MC26 plus tu. Gr+CD11b+cells

30

25

20

15

10

5

0

No.

of a

popt

otic

cel

ls

per f

ield

(200

x)

**

0

1

2

3

4

5

Tum

or v

olum

e (c

m3 )

MC26 MC26+Gr+CD11b+

Live volume

Necrotic volume

E

0200400600800

10001200

7 14 21Days after tumor inoculation

Tum

or v

olum

e (m

m3 )

8

6

4

2

0

**

**

VEGFR2 VE-Cad VEGFR2+

VE-Cad

No.

of G

r + C

D11

b +

Cel

ls(%

)

Nar.spleenTumor spleen

MC26 plus Gr+CD11b+MC26 plus Gr+CD11b+VEGFR2-

Figure by MIT OCW.

Figure by MIT OCW.

Figure by MIT OCW.

After Yang et al., 2004.



Endothelial Cell PrecursorsEndothelial Cell PrecursorsVasculogenesis 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 pre-existing endothelial cells

Recruitment of angioblast-like endothelial precursor cells

(Angiogenesis) (Vasculogenesis)

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 PrecursorsEndothelial Cell PrecursorsVasculogenesis 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 mobilizationBone marrow cell mobilizationfor neovascularizationfor neovascularization

VEGF

Stromal Cell NO

eNOS

MMP-9

Progenitor / Stem cell

sKitL

Figure by MIT OCW. After Duda et al., 2004.

The Weinberg hypothesisThe Weinberg hypothesis

50

100

150

200

250

300

350

400

450

500

550

600

650

0 10 20 30 40 50 60 Days

Tum

or v

olum

e (m

m3 )

n = 15

n = 15

n = 15

n = 15

n = 13

Loss of paracrine signaling

CXCR4-siRNA1,2 +SDF-1-fibro.

**

* *

Loss of endocrine signaling

CXCR4-siRNA1,2 +control GFP-fibro.

GFP-siRNA+SDF-1-fibro. Normal fibroblasts or progenitor cells

Recruitment

Invasive human breast cancer mass

Activated myofibroblasts

CXCR4 Blood vessel

EPCs

Endocrine effect

Breast carcinoma cells

Paracrine effect

SDF-1

Bone marrow Mobilization EPCs

Recruitment

A B

Figure by MIT OCW.

Figure by MIT OCW. After Peters et al., 2005.

Sample

Spindle cell sarcoma,head and neck

Hodgkin lymphoma

Mucoepidermoidcarcinoma, submandibular

Thyroid carcinoma

Osteogenic carcinoma

Mucoepidermoidcarcinoma, glossal

BMDC: bone marrow-derived cells.

Patientsex

Donorsex

Number of vesselssnalyzed

vWF+ CD45-

cellsBMDC BMDC

(percent)BMDC (percent)

normalized

F

F

F

F

F

F

M

M

M

M

M

M

58

37

36

38

95

11

215

219

192

752

293

94

1

11

7

4

3

1

0.5

5.0

3.6

0.5

1.0

1.1

1.0

12.1

7.0

1.1

4.1

4.0

Combined Immunofluorescence and FISH Data

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.

Source: Palma, Michele De, Mary Anna Venneri, Rossella Galli, Lucia Sergi Sergi, S. Politi Letterio, Sampaolesi Maurilio, and Luigi Naldini. "Tie2

Tie2-expressing myeloid cellsTie2-expressing myeloid cells


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 endotheliumNo 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.

Garcia-Barros et al. De Palma et al. Sciencer, May 18 Nature Medicine, June

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 No EPCs tumor angiogenesis and growth TEMs- critical for

tumor angiogenesis and growth

Images removed for copyright reasons. Source: Jain, R. K., and D. Duda.

i ll i il i i

Bone marrow der ved ce contr but on to neovascu ar zat on

Figure by MIT OCW. After Jain and Duda, 2003.

li lls i l l

l i i- lial ( i

i ) - l l ( , Di

ll Biol ) - i i ( l Na ed ll

i Na ed )

ll ili il i i

I izi i i ill l ll( l Science );

I i i f l

Research prob ems Bone marrow-der ved ce nfi trate tumors and modu ate neovascu ar zat on: endothe precursors eg, Rafi , Lyden & Benezra, Nat Med 2001,

Sc ence 2003mezenchyma stroma precursors eg rekze, Cancer Res 2004,

Bergers, Nat Ce 2005hematopo et c precursors eg, De Pa ma, t M 03, Cancer Ce

2005, Rafi t M 01

BM ce s are mob zed, home to tumors and contr bute to neovascu ar zat on

on ng rad at on k s cancer and norma ce s, and BMDCs can rescue tumor growth Fuks and co ab., 01 & 03s th s a mechan sm o re apse?

Courtesy of RK Jain, et al. Used with permission.

Courtesy of RK Jain, et al. Used with permission.

Figure by MIT OCW.

Image courtesty of NIH.

What is theWhat is the evidence for BMDCevidence for BMDC incorporation asincorporation asPerivascular cellsPerivascular cells in tumors?in tumors?

Smooth muscle precursor cells in bloodSmooth muscle precursor cells in blood


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 diameterQuantification of number and diameterof EPC-derived vesselsof EPC-derived vessels

Percent GFP+ functional vessels

Tumor type Implantation si te

Actb-GFP/BMT (C57BL6)

1

Tie2-GFP/BMT

(FVB)2

Lung adenocarcinoma Subcutaneous < 1%4 4.5 ± 1.6

4

Lung adenocarcinoma metastasis

Lung3 < 1%

5 10.6 ± 3.6

5

Melanoma Subcutaneous < 1%4 N/A

6

Mammary carcinoma Subcutaneous/

mammary fat pad N/A 1.3 ± 0.1

7

Mammary carcinoma metastasis

Brain N/A 58.4 ± 8.47

Spontaneous mammary carcinoma

– N/A 20.78

Spontaneous fibrosarcoma – N/A 3.28

Spontaneous hemangiosarcoma

– N/A < 1%8

Spontaneous squamous cell carcinoma

– N/A 5.38

What is theWhat is the evidence for BMDCevidence for BMDC incorporation asincorporation asPerivascular cellsPerivascular cells in tumors?in tumors?

Smooth muscle precursor cells in bloodSmooth muscle precursor cells in blood


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,TG1-1 in CW (VEGF-GFP/FVB mouse, 2 weeks, rhorhodextrandextran perfusion)perfusion)

500500µµmm

100100µµmm 5050µµmm

Fukumura et al., Cell 1998; Duda et al. CancerFukumura et al., Cell 1998; Duda et al. Cancer ResRes 20042004Reference:

ααSMASMA staining in mammary carcinoma tissuestaining in mammary carcinoma tissuein VEGF-GFP mousein VEGF-GFP mouse

Tong et al., PhD Thesis 2005Tong et al., PhD Thesis 2005Reference:

Image removed due to copyright reasons.


Bone marrow-derived stromal cells in mouse insulinoma after BMT

Perivascular cells originate from Sca1Perivascular cells originate from Sca1++ hematopoietic cellshematopoietic cells


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 Circulating VEGF-GFP+ cells in orthotopicorthotopicmammary carcinoma-bearingmammary carcinoma-bearing immunodeficient immunodeficient micemice

TumorTumor ControlControl

100

100

101

101 102 103 104

102

103

104

VEGF-GFP

CD

45Pe

rCP

051404.Rag2.005

100

100

101

101 102 103 104

102

103

104

VEGF-GFP

CD

45Pe

rCP

051404.Rag2.002

R2 R3

Figure by MIT OCW.

+

+

=

=

Multiple stem cells

Cell fusion

Pluripotency

A

B

C

D

Trans de-/re-differentiation

Figure by MIT OCW.

Stem Cell- entity or functionStem Cell- entity or function


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.

MAPC,MAPC, VerfaillieVerfaillie, Nature 2001, Nature 2001


TUMOR

E-selectin

VEGF

BMCMCBMCs

MPC HMPC

F

ECVEGF PIGF SDF1

Bone Marrow

Radiation

Blood Vessel

Figure by MIT OCW.

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