Pericytes and Endothelial Precursor Cells: Cellular ... · Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions

Pericytes and Endothelial Precursor Cells: Cellular

Interactions and Contributions to Malignancy

Rebecca G. Bagley, William Weber, Cecile Rouleau, and Beverly A. Teicher

Genzyme Corp., Framingham, Massachusetts

Abstract

Tumor vasculature is irregular, abnormal, and essential fortumor growth. Pericytes and endothelial precursor cells (EPC)contribute to the formation of blood vessels under angiogenicconditions. As primary cells in culture, pericytes and EPCshare many properties such as tube/network formation andresponse to kinase inhibitors selective for angiogenic path-ways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesionmolecule, intercellular adhesion molecule, CD105, desmin,and neural growth proteoglycan 2 was similar betweenpericytes and EPC, whereas expression of P1H12 andlymphocyte function–associated antigen-1 clearly differenti-ates the cell types. Further distinction was observed in themolecular profiles for expression of angiogenic genes.Pericytes or EPC enhanced the invasion of MDA-MB-231breast cancer cells in a coculture assay system. The s.c.coinjection of live pericytes or EPC along with MDA-MB-231cells resulted in an increased rate of tumor growth comparedwith coinjection of irradiated pericytes or EPC. Microvesseldensity analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However,immunohistochemical staining of vasculature suggested thatEPC and pericytes may stabilize or normalize vasculaturerather than initiate vasculogenesis. In addition, tumorsarising from the coinjection of EPC and cancer cells weremore likely to develop lymphatic vessels. These resultssupport the notion that pericytes and EPC contribute tomalignancy and that these cell types can be useful as cell-based models for tumor vascular development and selectionof agents that may provide therapeutic benefit. (Cancer Res2005; 65(21): 9741-50)

Introduction

The vasculature of tumors has been described as being morevein like than artery like; vascular structure exists but is abnormal.Folkman hypothesized that the switch of transformed lesions froma dormant state to malignant progression was driven by thedevelopment of vasculature; a new focus of drug discovery thusemerged towards attacking endothelial cells responsible for tumorangiogenesis (1). More recently, endothelial precursor cells (EPC)have been identified as contributors to vessel development in bothnormal physiologic processes, such as wound healing, and inpathologic settings like cancer (2). Pericytes (also known asvascular smooth muscle cells, mural cells, or myofibroblasts) wrap

around the endothelial cells. Pericytes share the same basal laminawith the endothelial cells and form an incomplete covering aroundthe abluminal surface of endothelial cells (3). Pericytes andendothelial cells communicate with each other through gapjunctions, tight junctions, soluble factors, and cell surface adhesionmolecules (4, 5).EPC can be derived from CD34+/CD133+ bone marrow

progenitor cells driven by angiogenic stimuli towards anendothelial phenotype (6–8). EPC are characterized by their abilityto form tubes and express markers typical of endothelial cells (9).EPC involvement in developing vasculature has been shown inmurine tumor models. CD34+ hematopoietic cells delivered toa model of lymphoma doubled tumor growth and were alsoidentified as contributors to vasculature in Lewis lung tumors(9, 10). Vascular endothelial growth factor (VEGF)–mobilized stemcells restored tumor angiogenesis and growth in Id-mutant mice(11). Clinically, a higher population of EPC is associated withinflammatory breast tumors versus a noninflammatory phenotype(12). Additionally, EPC have been detected in the circulationand malignant tissues of patients with multiple myeloma (13).Pericytes were first described over 100 years ago as adventitial

or Rouget cells (14) and were termed pericytes in 1923 (15).Pericytes possess a distinctive morphology comprised of anirregularly shaped plasma membrane with extended cytoplasmicprocesses. These extensions facilitate interdigitation with endo-thelial cells (16). Pericytes regulate capillary and venule bloodflow through contractile activity and can also control vascularpermeability (14, 15). In diabetic retinopathy, a degeneration ofretinal capillary pericytes results in microaneurysms (17).The role of pericytes in normal vasculature is well documented;

however, the contribution to developing tumors is just emerging.Pericytes have been identified in tumor vasculature throughimmunohistochemical staining of sections (18, 19) and couldcover 73% to 92% of endothelial sprouts in several murine tumortypes. The morphologic shape and association of the pericyteswith the endothelial cells in tumor vasculature are abnormal andthe cytoplasmic processes extend deep into tumor tissue (18). Therecruitment of pericytes to tumors may be attributed in part toplatelet-derived growth factor (PDGF) signaling. In a fibrosarcomamodel, PDGF-h was identified as an important factor in therecruitment of pericytes to tumor vessels (20). In human gliomas,PDGF-h and PDGF receptor h (PDGF-Rh) are overexpressed, andin mice, the overexpression of PDGF-h enhanced gliomaformation by attracting pericytes (21).An understanding of the role of pericytes has been gained by

histologic examination of tissues and studies of the physicalinteraction between pericytes and normal mature endothelialcells. This report begins characterization of the interactionbetween pericytes and EPC. The similarities and differencesbetween human pericytes and EPC were investigated byexamining expression of cell surface markers and behavior inin vitro assays, cocultures, and potential roles in tumor growth

Requests for reprints: Rebecca G. Bagley, Genzyme Corp., 5 Mountain Road,Framingham, MA 01701-9322. Phone: 508-270-2455; Fax: 508-872-9080; E-mail:[email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-04-4337

www.aacrjournals.org 9741 Cancer Res 2005; 65: (21). November 1, 2005

Research Article

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using a MDA-MB-231 breast cancer xenograft tumor model. Thefindings indicate that pericytes and EPC can be useful models forantiangiogenic drug discovery, interact to form primitive vessels,and contribute to malignancy in part through the normalizationof tumor vasculature.

Materials and Methods

Cell culture. CD34+/CD133+ progenitor cells from normal bone

marrow cells were supplied as being >90% pure (Cambrex, Inc., EastRutherford, NJ). The CD34+/CD133+ cells (1-2 � 105 cells/mL) were

differentiated in Iscove’s modified Dulbecco’s medium (IMDM, Cambrex)

with 15% fetal bovine serum (FBS, Invitrogen Corp., Carlsbad, CA),

50 ng/mL VEGF165 (R&D Systems, Minneapolis, MN), 25 ng/mL basicfibroblast growth factor (bFGF, R&D Systems), and 4 units/mL heparin

(Sigma Chemical Co., St. Louis, MO) on fibronectin-coated flasks (BD

Biosciences, Franklin Lakes, NJ) to generate adherent EPC (6, 7).Adherent EPC were maintained in IMDM supplemented with 10% FBS,

without additional growth factors. Characterization of EPC has been

reported previously (8).

Pericytes from normal fetal brain (ScienCell Research Laboratories, SanDiego, CA) were cultured in pericyte medium with 2% FBS and 2 ng/mL

each of epidermal growth factor, bFGF, and insulin-like growth factor-I

(IGF-I); 5 Ag/mL insulin; 1 Ag/mL hydrocortisone; and apo-transferrin

(ScienCell Research Laboratories).Human MDA-MB-231 breast carcinoma cells (American Type Culture

Collection, ATCC, Manassas, VA) were maintained in RPMI 1640 with

10% FBS (Invitrogen). Human dermal fibroblasts (HDF, ATCC) werecultured in DMEM high glucose with 10% FBS. Human umbilical vascular

endothelial cells (HUVEC) and human microvascular endothelial cells

(HMVEC) were cultured in EGM-2 medium (Cambrex).

Reverse transcription-PCR. RNA was isolated from EPC and pericytecultures by exposure to Trizol (Invitrogen) and chloroform extraction.

Samples were applied to RNeasy miniprep spin columns (Qiagen,

Valencia, CA) and purified according to instructions. cDNA was

generated using the High-Capacity cDNA Archive Kit (Applied Biosys-tems, Foster City, CA). Real-time PCR was done with SYBR Green PCR

Master Mix with primers on an ABI Prism 7700 Sequence Detection

System (Applied Biosystems): actin, ANG-1, ANG-2, CD31, CD105,

intercellular adhesion molecule-1 (ICAM-1), lymphocyte function–accociated antigen (LFA-1), matrix metalloproteinase-1 (MMP-1), MMP-

2, MMP-9, IL-8, IGF-1, P1H12, VEGF, transforming growth factor-h3(TGF-h3), FGF, hepatocyte growth factor (HGF), VEGFR2(FLK-1) (MaximBiotech, Inc., South San Francisco, CA), PDGF-B, PDGFR-B, vascular cell

adhesion molecule-1 (VCAM-1, R&D Systems), and 18S (Applied

Biosystems).

Flow cytometry. Cells were exposed to 0.25% trypsin/1 mmol/L EDTA(Invitrogen) and washed twice in cold 0.9% PBS containing 5% FBS ( flow

cytometry buffer). Approximately 1 � 105 cells were resuspended in 50 ALof flow cytometry buffer and incubated with a primary antibody for 1 hour

on ice. Cells were washed twice and incubated with the secondaryantibody for 30 minutes on ice. Cells were washed twice and resuspended

in a final volume of 500 AL for analysis. For intracellular antigens, the cells

were first fixed and permeabilized for 20 minutes with Cytofix/Cytopermbuffer (BD PharMingen, San Diego, CA).

Three micrograms of the following primary antibodies were added: (a)

anti-neural growth proteoglycan 2 (NG2, Chemicon International,

Temecula, CA), (b) anti-asmooth muscle actin (aSMA, Zymed Laborato-ries, Inc., South San Francisco, CA), (c) anti-desmin (Chemicon), (d) anti-

P1H12 (Chemicon), (e) anti-CD11/LFA-1 (BD Biosciences, San Diego, CA),

( f) anti-CD90/Thy-1 (Chemicon), (g) anti-CD54/ICAM (BD Biosciences),

(h) anti-CD105/endoglin (BD Biosciences), (i) anti-CD106/VCAM (BDBiosciences), (j) anti-PDGF-Rh/CD140 (BD Biosciences), and (k) anti-

VEGFR2 (clone A-3, Santa Cruz Biotechnology, CA). Matched isotype

control antibodies were used (BD Biosciences, Chemicon). Two microliters

of PE-labeled secondary antibodies were used for detection (Jackson

ImmunoResearch Laboratories, Inc., West Grove, PA). Positive expressionwas determined if cells gated at z10%.

In vitro tube formation. Matrigel (BD Biosciences) was added to a

48-well plate, 150 AL per well. EPC or pericytes (3 � 104) were added in

300 AL of medium: IMDM with 2% FBS for EPC and pericyte medium with

supplements and 2% FBS for pericytes. Cells were incubated for 24 hours

and stained with 8 Ag/mL calcein (Molecular Probes, Eugene, OR) for

imaging. SU5416 or SU6668 was added at final concentrations ranging

from 10 to 50 Amol/L to the cultures at the time that the cells were

seeded into the assay plates in triplicate (22). Area of tube formation was

quantified as fluorescent pixel area using SCION image software (NIH).

Results are expressed as mean F SD.Cocultures. Matrigel (BD Biosciences) was added to the wells of a 48-

well plate as before. EPC were labeled with PKH67 green dye and pericytes

were labeled with PKH27 red dye (Sigma, St. Louis, MO). EPC and pericytes

were added to Matrigel in a 1:1 ratio (1-2 � 104 cells per well). Cultures wereincubated overnight in IMDM with 2% FBS. Cells were visualized by

fluorescence using inverted-phase microscope.

MDA-MB-231 cluster assay. This model involves cancer clusters

cocultured with another cell type in suspension (23). Matrigel (BDBiosciences) was added to the wells of a 24-well plate in a volume of

300 AL. A plug of Matrigel of f1-mm diameter was removed. The hole was

filled with 1 � 106 MDA-MB-231 cells, labeled with PKH26 red dye,suspended in 1% collagen I (Cohesion Technologies, Palo Alto, CA), and

allowed to solidify. Other cells (3 � 104 cells per well) were labeled with

PKH67 green dye for distinction and added in IMDMwith 2% FBS in 300 ALfinal volume. When both EPC and pericytes were included in the samewells, a total of 3 or 6 � 104 cells per well were added. Conditioned medium

was collected from EPC and pericytes exposed to serum-free RPMI for

48 hours. The cultures were analyzed for up to 5 days. Area of MDA-MB-231

migration was quantified using SCION image software (NIH). Samples weredone in triplicate.

MDA-MB-231 tumor model. MDA-MB-231 breast carcinoma cells,

pericytes, and EPC were grown as described above. Cell suspensions were

mixed with Matrigel in a 1:1 (v/v) ratio in total volume of 300 AL containing

4 to 9 � 106 MDA-MB-231 cells with or without pericytes or EPC in a 12:1

ratio. For some implants, the pericytes or EPC (4 � 107 cells/mL) in RPMI

were exposed to g radiation (3 Gy/min) for 1 hour to a total dose of 180 Gy

(RS2000 X-ray Irradiator, Rad Source Technologies, Inc., Boca Raton, FL).

The cell suspensions in Matrigel were kept cold until implantation by s.c.

injection into the rear flank of female beige severe combined immunode-

ficient mice (C.B.-17/IcrCrl-scid-bgBR), 7 to 8 weeks of age (Charles River

Labs, Wilmington, MA). Tumors were measured twice per week and volume

was determined using the formula: (width2 � length) � 0.52. Data are

expressed as mean tumor volume F SD. Linear regression analysis was

used to determine the slopes of the growth curves. A two-way ANOVA

analysis determined significance.Immunohistochemistry. Tumors were snap-frozen in ornithine car-

bamyl transferase compound on day 29. Slides were dried for 10 minutes,

washed twice in TBS, once in TBST, and fixed in zinc/formalin buffer for 10

minutes. Slides were rinsed twice, blocked for 10 minutes, rinsed twice, andincubated with antibodies against CD31 (clone MEC13.3, BD PharMingen),

or aSMA (clone 1A4, DAKO, Carpinteria, CA) for 1 hour in a humidified

chamber. The aSMA antibody is cross-reactive for mouse and human.Slides were rinsed and incubated with CY2- and CY3-labeled secondary

antibodies (Jackson Immunochemicals, West Grove, PA). For microvessel

density quantification, four individual tumors of each group were stained

for mCD31 expression; five fields of each tumor were analyzed through a�20 objective. Data is presented as mean F SD. For analysis of lymphatic

vessels, an antibody against murine LYVE-1 was used (R&D Systems).

Endogenous peroxidase was blocked and sections were then exposed to

10% rabbit serum. Slides were incubated with anti-mLYVE-1 at 7.5 Ag/mLfor 60 minutes at room temperature. A secondary biotinylated antibody

was added at 1.5 ng/AL for 30 minutes at room temperature followed by

avidin-biotin complex method peroxidase, 3,3V-diaminobenzidine fordetection, and counterstained with hematoxylin. Sections were washed

between steps. Slides were scanned at �20 magnification.

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Cancer Res 2005; 65: (21). November 1, 2005 9742 www.aacrjournals.org



Results

The expression of several cell surface markers characteristic ofpericytes in tissues has been confirmed by flow cytometry inpericytes maintained as a primary culture (24–28). The expressionof characteristic cell surface endothelial markers on EPC has beendescribed previously (8); the EPC are CD45 negative but expressCD31, VEGFR2, and VE-cadherin. Several cell surface proteins areexpressed similarly by pericytes and EPC, although each cell typeis maintained in culture under different conditions (Fig. 1A).The profiles for pericyte and EPC expression of CD54/ICAM,desmin, aSMA, and CD106/VCAM were nearly identical. NG2,VEGFR2, and PDGF-Rh were strongly expressed on both EPC andpericytes, whereas CD90/Thy-1 was more highly expressed by EPC.VEGFR2 expression has previously been detected in rat brainpericytes and in myofibroblasts associated with human endome-trial cancer (29, 30). EPC and pericytes also produce fibronectinthat is secreted to generate the basal lamina that is a componentof vasculature.Differences in cell surface protein expression between EPC and

pericytes were notable for markers associated with endothelialcells such as CD105/endoglin and P1H12. CD105, a TGF-hreceptor, was detectable both on EPC and pericytes but was morestrongly expressed by EPC (Fig. 1A). P1H12 and LFA-1, which arestrongly expressed by EPC, were weakly expressed by pericytesat the protein level.Expression of multiple angiogenic genes by EPC and pericytes

were determined by reverse transcription-PCR (RT-PCR) andnormalized to 18S RNA levels (Fig. 1B). Genes expressed by bothEPC and pericytes include FGF, IGF-I, MMP-2, PDGF, TGF-h3, andVEGF. IL-8 and MMP-1 expression was minor in EPC butsignificantly up-regulated in pericytes by comparison. Only VEGFwas expressed at higher levels in EPC than pericytes, whereasANG-2 was expressed by pericytes and not EPC. MMP-9 expressionwas low in both cell types, and neither EPC nor pericytes expressedANG-1 or HGF. This data indicates that similarities exist betweenEPC and pericytes in the secretion of angiogenic factors, but differ-ential expression of some genes signifies the distinction betweenthese two primary cell lines. Additional markers commonly asso-ciated with vasculature, such as CD31, CD105, VEGFR2, and P1H12,were also confirmed by RT-PCR (Fig. 1C).Tube formation is an event fundamental to vasculogenesis and

growth factors from malignant cells can stimulate the process. Inculture, EPC tube formation is dependent upon cell density andserum concentration and is explored here with pericytes. EPC andpericytes were each plated onto Matrigel at optimal cell densitiesin their respective culture medium. Pericytes actively formedtubes/networks (Fig. 2A). The pericytes formed shorter networksof tubes in a web-like pattern with some symmetry that covereda large area of the well. The tubes formed by EPC seemed longer,more linear, and to have less symmetry. The pericyte tubesremained stable for several days, whereas tubes formed by EPCdegenerated after 24 hours (data not shown).Both pericytes and EPC express the receptors for PDGF and

VEGF and therefore may be sensitive to inhibitors of PDGF andVEGF signaling (Fig. 1A and C). SU6668 is a tyrosine kinaseinhibitor known to block the function of PDGF-R (19, 22). Theeffect of SU6668 on the tube formation by EPC and pericytes wasevaluated. The model of analysis measuring area was selected as itquantifies the length of the cells as they elongate to form capillary-like networks. By comparison, a single round cell would cover less

area than a network of cells that connect with one another.Exposure to SU6668 inhibited tube formation by both EPC andpericytes in a concentration-dependent manner (Fig. 2A). TheSU6668 IC50 values for decrease in tube area was f25 Amol/L forEPC and 2 Amol/L for pericytes suggesting that pericytes are moresensitive to inhibitors of PDGF-R than EPC (Fig. 2B). SU5416,a small-molecule tyrosine kinase inhibitor with selectivity forVEGFR2, also reduced the tube areas formed by EPC and pericytesin a concentration-dependent manner with IC50 values of 35 and25 Amol/L, respectively (31). These tube formation assay resultssupport the value of incorporating pericytes and EPC into cell-based assays as models of tumor vasculature.Although EPC or pericytes when plated at optimal cell density

can form tubes/networks, at suboptimal concentrations, thesestructures do not form. However, when lower cell numbers of EPCand pericytes are cocultured, interactions between the two celltypes occur and the formation of tubes/networks proceeds.Structures resembling primitive vessels form involving both celltypes, EPC (green) and pericytes (red ; Fig. 3A). The structuresformed present as multiple lumens that seem to consist of EPCstudded with pericytes. Although EPC and pericytes were equal innumber, the lumens that developed seemed derived mainly fromEPC. The cords and rings comprised of EPC are dotted withpericytes and the connections between cell groups seem mainlypericytes. Because the pericytes frequently seem to connect groupsof EPC through extensions, this suggests the possibility thatpericytes are activated first and may drive vessel extension(Fig. 3B). Changing the ratio of EPC to pericytes from 1:1 to 1:2or 2:1 did not generate significantly different results. EPC andpericytes can interact closely to form cords composed of one layerof each cell type (Fig. 3C). EPC seem to migrate along the alignedpericytes to form multicellular vessels. Exposure of EPC andpericyte cocultures to SU6668 resulted in inhibition of theformation of primitive vessels and apparent disruption ofinteractions between EPC and pericytes, such that the cells seemdisassociated and the cultures lack any form of organization(Fig. 3D).The behavior of EPC and pericytes in the presence of MDA-

MB-231 breast cancer cells was investigated. The MDA-MB-231clusters were prepared as 1- to 2-mm plugs. In the presence EPCor pericytes, the migration of MDA-MB-231 cells into theMatrigel was enhanced (Fig. 4A). This effect was not observedwith conditioned medium collected from EPC or pericytes (datanot shown). The pixel area covered by the cancer cells wasquantified (Fig. 4B). A comparison was made with HUVEC,HMVEC, and HDF, but these cells did not generate similar resultsindicating that not all cells possess the ability to enhance theoutgrowth and migration of MDA-MB-231 cancer clusters. Thearea covered by cancer cells migrating into the Matrigel wasthrice greater in the presence of EPC or pericytes on day3 versus MDA-MB-231 cells alone. Differences between pericytesand EPC arose when pericytes formed stable tubes for severaldays, whereas the EPC did not. By comparison, the EPC formedmultiple clusters within the wells. Overall, pixel area covered bythe migrating cells was similar in MDA-MB-231 cocultures withEPC or pericytes. However, the inclusion of both EPC andpericytes in the same well did not have an additive or synergisticeffect.The tyrosine kinase inhibitor SU6668 resulted in the inhibition

of EPC and pericyte tube formation (Fig. 2A and B). To determineif SU6668 could inhibit the migration of MDA-MB-231 clusters in

Pericytes and Endothelial Precursor Cells




Figure 1. A, flow cytometry profiles ofEPC and pericytes for expression of cellsurface molecular markers characteristicfor pericytes or endothelial cells. Solidprofiles represent matched IgG isotypecontrols. Several markers were expressedby both pericytes and EPC includingCD105/endoglin, fibronectin, SMA,CD54/ICAM, NG2, desmin, PDGF-R, andThy-1. Cell surface molecules expressedby EPC but not detected at significantlevels in pericytes were P1H12 and LFA-1.B, molecular expression of angiogenicgenes by EPC and pericytes as detectedby RT-PCR. MMP-2 was highly expressedby both EPC and pericytes, whereasneither cell expressed ANG-1 or HGF.EPC had greater expression of VEGF,whereas pericytes had higher levels ofTGFh3, MMP-1, and IL8. C, expressionof markers detected by flow cytometrywas confirmed by RT-PCR.

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the presence of EPC or pericytes, cocultures were exposed to10 Amol/L SU6668. Migration was inhibited by 36% in the wellswith EPC and 20% in wells with pericytes. Cocultures of all threecell types were also inhibited somewhat by SU6668, by 10% to25% depending upon the final number of EPC or pericytes in thewells (data not shown).The coculture of EPC or pericytes with MDA-MB-231 breast

cancer cells indicated that EPC and pericytes can increasemigration of malignant cells in vitro . To determine whether asimilar effect could be observed in vivo , MDA-MB-231 breastcancer cells were coinjected s.c. with pericytes or EPC in a 12:1ratio. The tumors that arose from the implantation of cellmixtures had a greater rate of growth versus those that arose fromMDA-MB-231 cells alone. A two-way ANOVA analysis indicatedthat the differences were significant (P < 0.0001). Slopes of thegrowth curves were determined by linear regression analysis. Theslope for the tumor growth curve for the EPC/MDA-MB-231

mixture was 2.6-fold greater than for the slope for the MDA-MB-231 cells alone and the slope for the tumor growth curve for thepericyte/MDA-MB-231 cell mixture was 2.5-fold greater than thatfor the tumor cells alone (Fig. 5A). Thus, the incorporation of EPCor pericytes into the initial implant provided a growth advantageto tumors.The coinjection of irradiated cells with cancer cells has been

a technique employed to enhance the establishment of s.c.tumors (32). Therefore, the effects of coinjection of viable versusradiation-killed pericytes or EPC on tumor growth wereexamined (Fig. 5B and C). The addition of viable pericytes toimplantation of MDA-MB-231 cells significantly increased theslope of the tumor growth curve 3-fold (P < 0.001) versus cancercells alone, whereas addition of irradiated pericytes to the tumorimplantation mixture increased the slope of the tumor growthcurve 1.8-fold versus MDA-MB-231 cells alone. Similarly, additionof viable EPC to implantation of MDA-MB-231 cells significantly

Figure 2. A, tube formation assay imagesof pericytes and EPC cultures onMatrigel. Pericytes form short, intricatetube networks while EPC form more linear,asymmetric tube networks. WhenSU6668 and SU5416 (10-50 Amol/L,24 hours) were added to the cultures,tube formation was inhibited in aconcentration-dependent manner. B,concentration response curves forinhibition of pericytes and EPC tubeformation upon exposure to SU5416 orSU6668 for 24 hours. IC50 concentrationsfor SU5416 exposure were 25 and35 Amol/L for pericytes and EPC,respectively. IC50 concentrations forSU6668 exposure were 2 and 25 Amol/L forpericytes and EPC, respectively.





increased tumor growth compared with controls or irradiatedEPC (P < 0.0001). The slope of the tumor growth curve forviable EPC increased 2.2-fold versus cancer cells alone, whereasaddition of irradiated EPC to MDA-MB-231 cell implantationincreased the slope of the tumor growth curve 1.2-fold. Thus,irradiated EPC or pericytes enhance tumor growth slightly,although there were no significant differences when comparedwith control in contrast to viable EPC or pericytes.The tumors that arose from MDA-MB-231 cells mixed with EPC

or pericytes were further evaluated for vessel morphology andmicrovessel density (Fig. 6). Endothelium was identified withantibodies against CD31 (red) and perivascular cells with anantibody against aSMA (green). Although the antibodies cannotspecifically identify the human pericytes and EPC, the pattern ofstaining indicated a change in morphology of the vasculature.In tumors consisting of MDA-MB-231 cells alone (Fig. 6A), therewas little association between CD31- and aSMA-positive cells. Intumors arising from the coinjection with EPC (Fig. 6B), or withpericytes (Fig. 6C), the blood vessels seem stabilized and betterorganized with distinct lumens and direct contact betweenendothelium and pericytes. No significant differences in micro-vessel density were noted between tumors (Fig. 6D) suggestingthat EPC or pericytes may enhance tumor growth by stabilizingor normalizing the vasculature, rendering the vessels morecapable of supplying oxygen and nutrients to the tumors. Thelack of increase in microvessel density suggests that EPC orpericytes are less likely to initiate vasculogenesis, the creation ofnew blood vessels, and more likely to promote angiogenesis, theextension of existing blood vessels. Further quantitative analysisof the tumors indicated that the inclusion of EPC promoted thedevelopment of vessels with a lymphatic component (P < 0.05;Fig. 6E). This effect was observed to a lesser degree with

pericytes. In humans, expression of the antigen LYVE-1 isassociated with a poor prognosis in breast cancer (33). Theincorporation of EPC or pericytes into tumors rendered theMDA-MB-231 model more aggressive thus indicating that EPCand pericytes can enhance malignancy, in part, through thenormalization of blood vessels.

Discussion

The focus of therapeutics against tumor vasculature has beenmature endothelial cells. Recent findings expanded that focus toinclude circulating endothelial cells and EPC mobilized from thebone marrow. Although the existence of pericytes and inter-actions between pericytes and endothelial cells as functionalcomponents of vasculature have been well documented, theimportance of pericytes to the function and survival ofendothelial cells is only now being appreciated. The distinctionbetween pericytes and other cells types in the vasculature oftissues is often made through immunohistochemical staining fordesmin. Other markers expressed by pericytes include NG2,aSMA, and VCAM (24–28). The expression of some markers canvary depending upon the tissue in which the pericytes reside. Forexample, aSMA is detected on pericytes from pre- and post-capillary microvascular segments, but those from midcapillariesdo not (34).Pericytes and EPC share many properties such as the ability to

form tubes and express specific molecular markers. CD90 is ofparticular interest, because its expression is associated with asubset of CD34+ stem cells that are predictive of successfulhematopoietic engraftments evaluated in cancer patients under-going peripheral blood stem cell transplantations (35). Theexpression of CD90 on EPC and pericytes may be indicative of

Figure 3. Tube formation images ofpericytes and EPC cocultures on Matrigel.EPC were labeled with PKH67 green dyeand pericytes were labeled with PKH27 reddye. Mixed cultures (1:1) of each cell type(1-1.5 � 104) were incubated overnighton Matrigel in basal medium with 2% FBS.A, pericytes (red) extend and form tubesto connect groups of EPC (green ). B, EPCand pericytes could be found directlyassociating with one another to form alinear structure comprised of both celltypes. C, EPC form lumens and ring-likestructures on Matrigel in the presence ofpericytes. Pericytes reorganize to stud theEPC as they form vessels in culture. D,organization and formation of primitivevessels in culture are inhibited by SU6668.

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their angiogenic potential and capacity for proliferation. Thesimilarities in cell surface proteins between EPC and pericytessuggest the possibility that these two cell types could arise froma common progenitor cell.The full pluripotency of CD133+ stem cells remains to be

characterized, and it is possible that these cells may be driven to adifferent phenotype under an alternative set of stimulatoryconditions. CD105/endoglin expression has also been observed ina small subset of CD34+CD133+ hematopoietic stem cells (36),markers that are present in the progenitor cells used here to derivethe EPC. CD34+ is recognized as a hematopoietic stem cell marker,and the detection of a lymphocytic marker, LFA-1, on EPC but notHMVEC or HUVEC (data not shown) maintains a link betweenEPC and hematopoiesis. LFA-1 is expressed selectively onmonocytes and macrophages (37), thereby linking the EPC withcells derived from a hematopoietic precursor. By comparison,pericytes are believed to be of mesenchymal origin, althoughconclusive evidence is lacking (34).The multistage nature of differentiation from stem cells to

mature endothelial cells results in a spectrum of EPC with varyingexpression of molecular markers depending upon stage ofdifferentiation. The expression of aSMA on the surface of EPCderived from CD34+ stem cells brings into question the exclusivityof aSMA as a marker of mesenchymal lineage cells or CD34 of as amarker of hematopoietic lineage cells. The detection of aSMA on

endothelial cells has been observed previously in endothelial cellsisolated from porcine and murine capillaries. The expression ofaSMA arose when heparin was removed from cells in culture,thereby driving the endothelial cells toward a less maturephenotype (38). The plasticity of endothelial cells and EPC inculture indicates that these cells respond phenotypically to varyingstimuli in the environment.Most antiangiogenic strategies have been directed toward

mature endothelial cells; however, the angiogenesis process drivenby EPC may also be a useful target. For example, delivery ofendostatin has shown effects toward EPC in models, wherebyendostatin inhibited EPC differentiation and mobilization (39, 40).The incorporation of human EPC or pericytes into murine tumorsadvances the use of traditional models, where previously, thevasculature was solely murine in origin. The development ofhuman-mouse chimeras of vasculature within tumors is a moreappropriate setting that better mimics the conditions presented inthe clinic and, as such, may be more informative and predictable inthe evaluation of novel therapies.Circulating EPC (CEPC) or endothelial cells may be useful

surrogate markers for angiogenesis and for antiangiogenictherapy response. In a human lymphoma model, a strongcorrelation was found between CEPC and tumor volume. VEGFserum levels paralleled the increase in CEPC (41). In breastcancer and lymphoma patients, CEPC were increased by 5-fold.

Figure 4. A, MDA-MB-231 human breastcancer cell invasion coculture assayimages. MDA-MB-231 human breastcancer cells embedded in collagen wereinserted into Matrigel in a 24-well plate.EPC or pericytes were added as asingle-cell suspension to some of the wells.The sprouting or outgrowth of the cancercells from their origin was visually greaterin the presence of EPC or pericytes on day5. Top, individual wells; magnification, �1.Pericytes formed tube networks thatwere stable on day 5 and extended acrossthe area of cancer cell migration zone.HUVEC did not increase migration ofcancer cells. B, Matrigel invasion ofMDA-MB-231 cells was quantified usingSCION Image Software. Coculture witheither EPC and pericytes enhanced theinvasion of MDA-MB-231 cells comparedwith MDA-MB-231 cells alone (control ) onday 3. HMVEC, HUVEC, and HDF did nothave the same effect. Columns, mean;bars, FSD.





CEPC numbers were similar between controls and lymphomapatients in remission and in breast cancer patients followingsurgery (42). In a similar study, peripheral blood was collectedfrom breast cancer patients with infiltrating carcinoma, ductalcarcinoma in situ , and from normal controls. mRNA analysis

identified CEPC elevation in patients with infiltrating carcinomas(43). Angiogenic profiles were assessed from blood samples of 82cancer patients presenting a variety of cancers (lymphomas,leukemias, breast and hepatocellular carcinomas, ovarian, neuro-endocrine, and lung). Compared with healthy volunteers, mRNAexpression indicated that VE-cadherin was increased in patientswith tumors (44). Circulating endothelial cells and EPC may be avaluable indicator of patients who may benefit most fromantiangiogenic therapy.EPC and pericytes here were found to contribute to the

malignancy of human breast cancer cells using both in vitro andin vivo examples of a tumor microenvironment. However, thedegree of contribution by EPC and pericytes to vasculature mayvary with tumor type, and the role of EPC, in particular, remainssomewhat controversial. In a murine model, orthotopic ands.c. GL261 glioma tumors were evaluated for engraftment of bonemarrow–derived endothelial cells. Little engraftment was ob-served, even in highly vascularized tumors overexpressing VEGF(45). In a transgenic model created to trace the origin of tumorendothelium, results indicated that endothelium of Lewis lung orB6RV2 lymphoma tumors did not originate from bone marrowcells (46). By contrast, coinjection of murine endothelial cellswith human epidermoid cancer cells in vivo increased tumor sizeand vascularity (47). Pericyte coverage can vary from tissue totissue and among tumors. A comparison of human glioblastomas,renal cell, colon, breast, lung, and prostate carcinomas showedthat breast and colon tumors having significantly greater pericyterecruitment than gliomas or renal cell carcinomas (48). Theexpansion of tumor vasculature may be enhanced by interactionsbetween EPC and pericytes as coculture experiments show theability of these two cell types to form primitive vessels. Inaddition, molecular analysis revealed the expression of matrixmetalloproteinases, particularly by pericytes, which may assist indriving angiogenic reactions by breaking down structuralcomponents of the basement membrane thereby facilitating themigration of endothelial cells and pericytes.Like platelets, endothelial cells are a source of PDGFh (49).

EPC, as precursors to mature endothelial cells, secrete PDGFh(Fig. 1B) contributing to the structural network of EPC andpericytes in early stages of vascular development. PDGFh isessential for normal vascular development; thus, genetic ablationof PDGFh in mice resulted in impaired recruitment of pericytes toblood vessels (50). Pericytes were validated as a therapeutic targetusing kinase inhibitors selective for PDGF-Rh. Pancreatic tumorsin Rip1Tag2 mice regressed in part by destabilization ofinteractions between pericytes and endothelial cells followingtreatment with Gleevec or SU6668 (19, 22). Inhibiting VEGF/PDGFsignaling in gliomal cells produced tumor regression by interferingwith pericyte-mediated endothelial cell survival mechanisms (51).The pericytes and EPC presented here were sensitive to kinaseinhibitors selective for PDGF or VEGF pathways; thus, thecontribution these cells make to vascular development can bedestabilized to therapeutic advantage.Development of the first generation of antiangiogenic drugs

used mature, fully differentiated endothelial cells, such as HMVECand HUVEC, in the discovery process; yet, these agents met limitedclinical success. The incorporation of pericytes and EPC as modelsof tumor vasculature into drug development schemes maygenerate more fruitful results. Previous serial analysis of geneexpression analysis of EPC, HMVEC, and endothelial cells isolatedfrom several tumor types indicated that EPC expressed more of the

Figure 5. A, MDA-MB-231 human breast cancer cells were implanted s.c. intothe flank of female beige severe combined immunodeficient mice either aloneor with human EPC or pericytes. MDA-MB-231 cells (9 � 106) were mixed withEPC or pericytes (7.5 � 105) in Matrigel/RPMI 1640 (1:1) before injection. Thepresence EPC or pericytes increased the initial rate of tumor growth. Points,mean (n = 5); bars, FSD. Two-way ANOVA analysis indicated differences weresignificant (P < 0.0001). B-C, MDA-MB-231 tumor growth after s.c. coinjectionof MDA-MB-231 cancer cells (4 � 106) alone or with live or irradiated pericytes orEPC (3.3 � 105). The rate of tumor growth was greater following coinjectionwith live pericytes or EPC than with killed pericytes or EPC versus tumor growthrate with tumor cells alone. Points, mean (n = 5); bars, FSD. Two-wayANOVA analysis indicated differences were significant when EPC or pericyteswere viable compared with control or irradiated cells (P < 0.0001).

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genes selectively up-regulated by tumor endothelium than HMVEC(11). Pericytes and EPC are important participants among themany cell types that give rise to progressing malignant disease.Targeting pericytes and EPC may lead to more effective therapiesfor cancer and increase our understanding of tumor development.

Acknowledgments

Received 12/5/2004; revised 7/8/2005; accepted 8/22/2005.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Figure 6. Vasculature of tumors was analyzed by immunohistochemistry with antibodies against CD31 (red) and aSMA (green ). A, blood vessels in control tumors aredisorganized with little association between endothelium and perivascular cells. B, blood vessel of a tumor arising from the coinjection of MDA-MB-231 cells and EPC;direct contact is evident between CD31+ and aSMA+ cells. C, coinjection of cancer cells and pericytes generated vasculature that is organized with stable lumenformation. D, microvessel density analysis of MDA-MB-231 tumors with or without EPC or pericytes. Tumors were stained for mCD31 expression and vessel densitywas determined over five fields per sample (n = 4). There was no difference in microvessel density when EPC or pericytes were coinjected with cancer cells. E,lymphatic vessels were identified with an antibody against murine LYVE-1. The degree of lymphatic vessels that developed was increased by the incorporation of EPC(P < 0.05, Student’s t test). The effect of pericytes on lymphatic development was not as significant. Inset, immunohistochemical staining for LYVE-1 in a tumorgenerated from the coinjection of EPC and MDA-MB-231 cancer cells. Bar, 100 Am.

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2005;65:9741-9750. Cancer Res Rebecca G. Bagley, William Weber, Cecile Rouleau, et al. Interactions and Contributions to MalignancyPericytes and Endothelial Precursor Cells: Cellular

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Pericytes and Endothelial Precursor Cells: Cellular ... · Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions