Expression of a functional VEGFR-1 in tumor cells isa major determinant of anti-PlGF antibodies efficacyJenny Yaoa,1, Xiumin Wua,1, Guanglei Zhuanga, Ian M. Kasmana, Tobias Vogta, Vernon Phana, Masabumi Shibuyab,Napoleone Ferraraa,2, and Carlos Baisa,2

aGenentech, Inc., South San Francisco, CA 94080; and bDepartment of Molecular Oncology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,Tokyo 113-8519, Japan

Contributed by Napoleone Ferrara, June 7, 2011 (sent for review April 13, 2011)

PlGF, one of the ligands for VEGFR-1, has been implicated in tumorangiogenesis. However, more recent studies indicate that geneticor pharmacological inhibition of PlGF signaling does not result inreduction of microvascular density in a variety of tumor models.Here we screened 12 human tumor cell lines and identified 3 thatare growth inhibited by anti-PlGF antibodies in vivo. We found thatefficacy of anti-PlGF treatment strongly correlates with VEGFR-1expression in tumor cells, but not with antiangiogenesis. In addi-tion, PlGF induced VEGFR-1 signaling and biological responses intumor cell lines sensitive to anti-PlGF, but not in refractory tumorcell lines or in endothelial cells. Also, genetic ablation of VEGFR-1signaling in the host did not affect the efficacy of PlGF blockade.Collectively, these findings suggest that the role of PlGF in tumor-igenesis largely consists of promoting autocrine/paracrine growthof tumor cells expressing a functional VEGFR-1 rather than stimu-lation of angiogenesis.

stroma | tyrosine kinase | placental growth factor | vascular endothelialgrowth factor | angiogenesis

The VEGF signaling pathways play important roles in angio-genesis. VEGF-A binds to two tyrosine kinase receptors,

VEGFR-1 and VEGFR-2 (1). Although both receptors areexpressed in endothelial cells, VEGFR-1 is also expressed inmonocyte/macrophages, hematopoietic stem cells, and even sometumor cells (2–4). Most of the biological effects of VEGF-A aremediated by activation of VEGFR-2 (1). VEGFR-1 has a weaktyrosine kinase activity but substantially higher binding affinity forVEGF-A than VEGFR-2 (5). The biological role of VEGFR-1 ishighly complex. Although genetic data indicate that signalingdownstream of this receptor is not required for developmental an-giogenesis (6), a role for VEGFR-1 during tumor-angiogenesis hasbeen recently suggested (7–9). PlGF is a VEGFR-1 specific ligand(10) that was identified 20 years ago (11). Under pathologicalconditions, PlGF levels are increased in various cell types, includingvascular endothelial cell, smooth muscle cells, keratinocytes, he-matopoietic cells, retinal pigment epithelial cells, and many differ-ent tumor cells (12). Plgf deficient mice are born at normalMendelian ratios and donot show anyobvious vascular defects (13).PlGF overexpression enhanced tumor growth in some models (14,15), but in others, PlGFparadoxically had an inhibitory effect, likelythrough formation of VEGF/PlGF heterodimers, which down-regulate VEGFR2 signaling (16, 17).According to Fisher et al. (7), treatment with an anti-PlGF

monoclonal antibody (Mab) reducesmicrovascular density (MVD)and inhibits primary tumor growth in a variety of murine models.However, in a subsequent study, we reported that blocking PlGFdoes not result in growth inhibition in any of the tumor modelstested (12murine and 3 human tumor cell lines) (18). Importantly,the antibodies used in these studies were able to block PlGF in vivo(18) as evidenced by their ability to inhibit metastasis of B16F10cells (7, 19, 20), wound healing (13, 21), and primary tumor growthof amurine cell line overexpressingVEGFR-1.On the other hand,it has been shown that genetic ablation of plgf results in inhibitionof tumorigenesis in somemodels, but not in others (2, 8). Becauseefficacy in these models was not associated with a reduction intumor MVD, an alternative mechanism involving vascular nor-malization has been proposed (8). In addition, it has been recently

reported that an anti-human PlGFMab inhibits growth of DangGand MDA-MB-435 xenografts (8), although the mechanismremained unknown. These observations prompted us to revisit therole of PlGF in human tumor xenograft models. This issue isparticularly timely given the ongoing evaluation of anti-PlGFtherapy in clinical trials.

ResultsEfficacy of Anti-PlGF Antibody Treatment Correlates with VEGFR-1Expression in Tumor Cells. As a first step, we sought to identify celllines that are growth inhibited by anti-PlGF treatment. To this end,we tested the ability of the validated anti-human and mouse cross-reactive anti-PlGF mAb C9.V2 (18), hereafter referred to as anti-PlGF, to inhibit growth of CALU3, H82, U87, SW480, A549,H1299, L5180, LXFL529, H460, SKUT1b, and CAKI1 tumors (SIMaterials and Methods). Consistent with previous findings (18),mostmodels evaluated (9 of 11) did not show any growth inhibition(Fig. 1 A–D, Left, and S1 A–E). However, anti-PlGF treatmentsignificantly reduced the growth of SKUT1b (Fig. 1E, Left) andCAKi1 (Fig. 1F, Left) tumors in a dose-dependent manner. Incontrast, all tumor models tested were growth inhibited by anti-VEGF-A treatment (Fig. 1 A–F and Fig. S1 A–E, Left, red line).Together, these data suggest that anti-PlGF mAb treatment doesnot result in broad inhibition of tumor-angiogenesis and that theeffects are tumor model specific. However, PlGF is expressed inboth anti-PlGF responsive and refractory tumor models (7, 8, 18)(Fig. S2). We hypothesized that VEGFR-1 expression in tumorcells (3, 4, 22) might be a potential mechanism conferring suchmodel-specific sensitivity to anti-PlGF treatment. In agreementwith this hypothesis, we found that VEGFR-1 is expressed in theanti-PlGF sensitive cell lines CAKI1 and SKUT1b (Fig. 1 E and F,Right), but it is undetectable in anti-PlGF resistant tumor cells(Fig. 1 A–D and Fig. S1, Right). Figure 1 G and H shows thatVEGFR-1 expression was detected by flow cytometry (SIMaterialsand Methods) in the positive controls [human umbilical vein en-dothelial cells (HUVECs) and HEK293-hVEGFR-1] but not inHEK293-empty vector (negative control) cells. Next, we sought todetermine whether neutralization of PlGF might be sufficient toinhibit growth of tumors known to be dependent on VEGFR-1signaling within tumor cells. To this end, we took advantage ofDU4475, aVEGFR1-positive breast carcinoma cell line previouslyshown be sensitive to anti-hVEGFR-1 mAb treatment (4). Figure1I shows that anti-PlGF mAb treatment inhibits growth of estab-lished DU4475 orthotopic tumors. Thus, PlGF blockade can in-hibit growth of xenografts dependent on VEGFR-1 signaling and,at least among the models evaluated in this study, efficacy of anti-

Author contributions: N.F. and C.B. designed research; J.Y., X.W., G.Z., I.M.K., T.V., V.P.,and C.B. performed research; V.P. and M.S. contributed new reagents/analytic tools; J.Y.,X.W., G.Z., I.M.K., T.V., and C.B. analyzed data; and N.F. and C.B. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1J.Y. and X.W. contributed equally to this work.2To whom correspondence may be addressed. E-mail: baisc@gene.com or nf@gene.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1109029108/-/DCSupplemental.

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PlGF antibody treatment strictly correlates with VEGFR-1 ex-pression in tumor cells.

Efficacy of Anti-PlGF Mabs Is Not Mediated by Antiangiogenesis. Todetermine whether efficacy of anti-PlGF mAb treatment is me-diated by inhibition of angiogenesis, we quantified MVD (CD31positive vessels) in sections from DU4475, CAKI1, and SKUT1btumors at the end-point of the studies (SI Materials and Methods).In contrast to anti-VEGF mAb, anti-PlGF treatment did notcause a significant reduction in tumor vasculature (Fig. S3 A–C).

We also wished to evaluate any potential antiangiogenic effects ofPlGF Mab in short-term studies. We treated mice bearing expo-nentially growing tumors of ∼400 mm3 with anti-PlGF, anti-VEGF, or control antibodies for 48 h. CD31 IHC analyses of thesetumor tissues showed that anti-PlGF did not cause a reduction inMVD. In contrast, anti-VEGF Mab treatment induced a markedreduction in the number of CD31 positive vessels in SKUT1btumors (Fig. S3D, Upper). Furthermore, qRT-PCR analyses con-firmed that the expression of the transcripts for the pan-vascularmarkers CD31, VE-cadherin, and MCAM were significantly re-

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Fig. 1. Inhibition of tumor growth by Anti-PlGF mAb treatment is restricted to VEGFR-1 positive xenografts. (A–F, Left) Effects of anti-PlGF mAb C9.V2 onprimary growth of human tumor xenografts. (F) Dose-dependent inhibition of Caki-1 tumor growth by anti-PlGF C9.V2 Mab. (A–F, Right) Analysis of VEGFR-1expression in tumor cells. Tumor cells were incubated with biotinylated anti-VEGFR-1 mAb (blue) and or with Streptavidin-PE only as a control (red) as in-dicated. VEGFR-1 expression was analyzed by flow cytometry. Positive (pos) indicates the calculated percentage of positive cells. (G and H) Flow cytometryVEGFR-1 positive and negative controls. (G) HEK293-VEGFR-1 cells (blue) are VEGFR-1 positive and HEK293-empty vector (green) are VEGFR-1 negative. (H)Endothelial cells (HUVECs) are VEGFR-1 positive (blue). (I) Anti-PlGF inhibits growth of established DU4475 orthotopic breast carcinoma xenografts. Anti-PlGFor anti-Ragweed mAb was given at 15 mg/kg. Anti-VEGF-A mAb was given at 10 mg/kg. All antibody treatments were administrated biweekly. n = 10–15,*P < 0.05 relative to anti-ragweed treatment. Error bars represent SEM.

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duced upon VEGF blockade in SKUT1b. However, anti-PlGFtreatment did not decrease the relative mRNA expression levels inany of the vascular markers tested (Fig. S3D, Bottom).

hPlGF Induces Biological Responses in Anti-PlGF Sensitive (VEGFR-1Positive) Tumor Cells but Not in Endothelial Cells. We tested theability of anti-PlGF sensitive tumor cell lines and endothelial cellsto respond to VEGFR-1 stimulation in vitro (SI Materials andMethods). We did not observe any responses to PlGF in anti-PlGFrefractory (VEGFR-1 negative) tumor cells (Fig. S4). In contrast,anti-PlGF sensitive tumor cell lines proliferated (DU4475,SKUT1b) and migrated (CAKi1 and SKUT1b cells) in responseto hPlGF-2 (or hVEGF-A) in a dose-dependent manner (Figs. 2Aand 4D). Figure 2A also shows that anti-PlGFMab blocked PlGF-induced responses in tumor cells.We also evaluated the responsesof endothelial cells (HUVECs) to hPlGF-2 and VEGF-A. Inagreement with previous reports, HUVECs responded to VEGF-A but did not show any obvious responses to PlGF in migration(Fig. 2B,Right) and proliferation (Fig. 2B,Left) assays. It has beenpostulated that endothelial cells do not respond in vitro to exog-enous PlGF because they express high levels of endogenous PlGF(13, 23). To test this possibility, we performed PlGF knock-downin HUVECs (SI Materials and Methods). Figure 2C (Left) showsthat PlGF knock-down reduces PlGF release by more than 90%.However, HUVECs remained unresponsive to hPlGF-2 but werefully responsive to VEGF-A, bFGF, or FBS (Fig. 2C, Right).

Activation of the Mitogen-Activated Protein Kinase (MAPK) Pathway IsRequired for PlGF-Induced Biological Responses in Anti-PlGF SensitiveTumor Cells. Previous studies have shown that the (MAPK) andPI3K pathways are activated in response to ligand stimulationin some cell lines overexpressing VEGFR-1 (18, 24).

To gain further insights into PlGF/VEGFR-1 signaling in tumorcells, we first performed phospho-kinase antibody array experi-ments with cell lysates fromhPlGF-2 ormock stimulatedHEK293-VEGFR-1 cells (SIMaterials andMethods). Figure 3A (Left) showsthat p42/p44 was activated by PlGF stimulation. No significantdifferences in phosphorylation of protein kinase B (PKB/AKT) orother proteins included in this array were apparent. Nearly iden-tical results were obtained when lysates from the VEGFR-1 pos-itive uterine sarcoma cell line SKUT1b were analyzed (Fig. 3A,Right). MAPK activation by PlGF was confirmed by Western blotin both SKUT1b (Fig. 3B,Left, and Fig. S5A) and CAKI1 (Fig. 3C,Left, and Fig. S5A,Right). We next usedMAPK pathway inhibitorsto investigate whether MAPK activation is required for PlGF-in-duced migration and proliferation. Figure 3 B and C (Left) showsthat the MEK inhibitor GDC-0973/XL-518 (US patent20110086837) (25) efficiently blocks PlGF-induced MAPK phos-phorylation without affecting cell viability (Fig. 3 B and C, Right,and Fig. S5C). In addition, GDC-0973 and the RAF inhibitorGDC-0879 (26) (Fig. 3 B and C, right panels), but not Rac, JNK(SP600125), or Rho inhibitors (Fig. S5B), completely suppressedPlGF-responses. However, they only slightly reduced HGF- orFBS-induced CAKi1 and SKUT1b migration and SKUT1bsurvival/proliferation (Fig. 3 B and C, Right, and Fig. 4D). In-terestingly, the dose-dependent inhibition of PlGF-inducedMAPK phosphorylation by GDC-0973 parallels the inhibition ofmigration and proliferation induced by this agent (Fig. 3 B andC).

Inhibition of PlGF/VEGFR-1 Signaling In Tumor but Not Stromal Cells Isa Major Determinant for Anti-PlGF Efficacy. To confirm the role ofVEGFR-1 in PlGF-induced responses in anti-PlGF sensitivetumor cell lines, we knocked-down VEGFR-1 in CAKI1 andSKUT1b cells using siRNA oligonucleotides (SI Materials and

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Fig. 2. Anti-PlGF tumor sensitive tumor-cell lines but not endothelial cells respond to hPlGF-2 stimulation. (A) hPlGF-2 induces dose-dependent biologicaleffects in the anti-PlGF sensitive tumor cell lines DU4475 (Left), CAKI-1 (Center), and SKUT1b (Right), and these effects are blocked by anti-PlGF mAb. (B)hPlGF-2 fails to stimulate HUVEC proliferation (Left) and migration (Right) at all doses tested. (C, Left) Quantification by ELISA of hPlGF released by PlGFknock-down (KD) or control HUVECs. (C, Right) PlGF knock-down (blue bars) and siRNA control HUVECs (green bars) remain unresponsive to hPlGF-2. Thefigure shows the average values from representative experiments. Doses of ligands are indicated in the figure. Dotted lines represent basal (control) activity.Experiments were repeated at least three times with comparable results. n = 3–5. Error bars represent SD.

11592 | www.pnas.org/cgi/doi/10.1073/pnas.1109029108 Yao et al.

Methods). Figure 4 A and B (Left) shows that VEGFR-1 siRNAbut not control siRNA markedly decreases VEGFR-1 expressionin both cell lines. VEGFR-1 knock-down also suppressed theability of these cells to migrate in response to PlGF or VEGF-Abut did not affect their ability to respond to HGF or 10% FBS(Fig. 4 A and B, Right). Consistent with these findings, VEGFR-1depletion with a different siRNA oligonucleotide sequence(VEGFR-1 SiRNA no. 2; Fig. S6A) also specifically inhibitedVEGF- and PlGF-induced responses. We found that althoughPlGF strongly induced tyrosine phosphorylation in HEK293 cellsoverexpressing hVEGFR-1 (Fig. 4C), it barely affectedVEGFR-1phosphorylation in CAKI1 or SKUT1b (Fig. S5A). This result wasnot unexpected, because ligand-dependent tyrosine phosphory-lation of VEGFR-1 is known to be very low (or undetectable) incells endogenously expressing this receptor (27–29). To test thepotential relevance of tyrosine phosphorylation in the activationof PlGF/VEGFR-1 downstream signaling, we used the VEGFRtyrosine kinase inhibitor axitinib (30). Figure 4C shows that theMEK inhibitor GDC-0973 specifically inhibits MAPK but notVEGFR-1 phosphorylation in HEK293-VEGFR-1 cells. How-ever, axitinib inhibited both PlGF-induced phosphorylation ofVEGFR-1 and downstreamMAPKactivation in a dose-dependentmanner. Similar to anti-PlGFmAb (18) (Fig. 2A and Fig. S6C) andMEK inhibitors (Fig. 3 B and C and Fig. S6B), axitinib inhibitedhPlGF-induced signal transduction (Fig. 4C), SKUT1b cell sur-vival/proliferation (Fig. 4D) and migration of CAKI1 and SKUT1bcells (Fig. S6B). These findings indicate that VEGFR-1 expressionand phosphorylation are required for PlGF-induced biologicalresponses in anti-PlGF sensitive tumor cells.It has been postulated that anti-PlGF efficacy, in the absence of

MVD changes, is due to normalization of the vasculature asa consequence of reduced infiltration ofVEGFR-1 positive tumor-associated macrophages (TAMs) (8). To probe whether tumorgrowth inhibition by anti-PlGF indeed requires inhibition ofVEGFR-1 signaling in TAMs, hematopoietic stem cells, or otherstromal cells, we implanted SKUT1B anti-PlGF sensitive tumorcells in vegfr-1 tk −/−, rag2−/− mice (6). Because these mice expressa VEGFR-1 mutant that lacks most of its intracellular domain(including the tyrosine kinase domain), PlGF should be unable to

activate VEGFR-1 signaling in host (murine) cells. Figure 4Eshows that implantation of SKUT1b cells in vegfr-1 tk−/− does notimpair the ability of anti-PlGF to inhibit tumor growth. Similarly,Fig. S6D shows that anti-PlGF treatment has comparable effectson Caki-1 tumor growth in rag2−/− or vegfr-1 tk−/− vs. rag2−/−, vegfr-1+/+mice. These data indicate that anti-PlGF efficacy is mediatedby blockade of PlGF/hVEGFR-1 signaling in the tumor cells butnot by inhibition of VEGFR-1 signaling in host cells.

DiscussionAnti-PlGF therapy is currently being evaluated in clinical trials.Nevertheless, the significance of PlGF as a therapeutic targetremains incompletely understood.Recent studies suggest that PlGF inhibition reduces tumor

growth and angiogenesis by decreasing recruitment of macro-phages in tumor tissue (7). However, subsequent reports revealedthat inhibition of PlGF-induced signaling does not necessarilyinhibit tumor growth, nor does it correlate with pruning of tumorvessels (8). It has been also hypothesized that the efficacy of PlGFinhibition, in the absence of a significant reduction in tumorMVD, is mediated by vascular normalization following reducedTAM infiltration (8, 18). However, this hypothesis does not fullyexplain the lack of broad antitumor efficacy and the model-dependent efficacy of PlGF inhibition.AlthoughVEGFR-1 has previously been shown to be expressed

in some tumor cells (2–4), the possibility that VEGFR-1 expres-sion may confer sensitivity to PlGF inhibition was not previouslyinvestigated. It is interesting to note that of the 12 murine tumormodels we recently evaluated (18), inhibition of primary tumorgrowth by anti-PlGF treatment was restricted to a cell line engi-neered to overexpress VEGFR-1. Here, we identified three un-transfected human tumor cell lines (CAKI1, SKUT1b, andDU4475) sensitive to PlGF neutralization. Remarkably, all anti-PlGF sensitive tumor cell lines identified in the present study werefound to be VEGFR-1 positive. Conversely, all anti-PlGF re-sistant cell lines were VEGFR-1 negative. These data suggest thatblockade of PlGF/VEGFR-1 signaling in tumor cells may be re-quired for anti-PlGF mAb efficacy.

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Importantly, no decreases in MVD were observed in the sensi-tive models, suggesting that efficacy is not mediated by antiangio-genesis. Consistent with these findings, in vitro experimentsindicate that anti-PlGF sensitive tumor cells lines, unlike anti-PlGFrefractory tumor cells or endothelial cells, respond to PlGF stim-ulation via VEGFR-1 signaling activation. The divergent ability ofVEGFR-1 positive tumor cells and vascular endothelial cells torespond to VEGFR-1 ligand stimulation is puzzling. However, it isconsistent with previous reports (10, 31) and also with genetic dataindicating that, at least during embryonic angiogenesis, endothelialVEGFR-1 acts mainly as a nonsignaling decoy (6, 10). Although ithas been proposed that the lack of responsiveness of endothelialcells to PlGF reflects VEGFR-1 occupation due to high levelsof endogenous PlGF, our PlGF knock-down experiment arguesagainst this possibility. Further studies are required to elucidate themechanisms underlying such cell type-dependent responses.Implantation of anti-PlGF responsive tumors in vegfr-1 tk−/−,

rag2−/− mice did not affect the efficacy of anti-PlGF treatment,indicating that VEGFR-1 signaling in stromal cells is not requiredfor the protumor effects of PlGF. Although the data presentedhere indicate that inhibition of PlGF/VEGFR-1 signaling in tu-mor cells is a major mechanism underlying anti-PlGF efficacy,

alternative mechanisms may be important in other models. In thiscontext, it is tempting to speculate that the efficacy of anti-PlGF(8) or anti-VEGFR-1 mAbs (32) in hepatocellular carcinomamodels involves, at least in part, inhibition of release of paracrinegrowth factors from sinusoidal endothelial cells (e.g., hepatocytegrowth factor or IL-6), which has been previously shown to beregulated by endothelial VEGFR-1 (33).Webelieve thatourfindingsnotonly underscore an important and

potentially clinically relevantmechanismof action of PlGFMab, butmay also help reconcile conflicting data in the literature. Indeed, therecently reported ability of an anti-human PlGFMab (8) to reduceMDA-MB-435 tumor growth very likely reflects the presence ofa previously described functional VEGFR-1 in these cells (4).It is presently unclear whether the apparent higher incidence of

anti-PlGF efficacy in human xenografts (3 of 15 models tested)compared with the lack of growth inhibition in all 12 murinetumor models truly represents a higher incidence of VEGFR-1expression/activity in human tumors. In addition, the signalingdata we present suggests that the VEGFR-1 pathway contributesto PlGF-induced effects in tumor cells mainly through MAPKactivation. Thus, VEGFR-1 expression/activity may provide a se-lective growth advantage to tumors that are highly dependent on

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Fig. 4. Inhibition of PlGF/VEGFR-1 signaling intumor but not stromal cells is a major de-terminant for anti-PlGF efficacy. (A and B, Left)VEGFR-1 siRNA but not control siRNA reducesVEGFR-1 expression by FACS. (A and B, Right)Effects of VEGFR-1 knock-down on migration ofCaki-1 and SKUT1b cells in response to PlGF,VEGF, HGF, or 10% FBS. (C, Upper) Effects ofaxitinib (VEGFR inhibitor) and GDC-0973 onhPlGF-2-induced phosphorylation of VEGFR-1and p42/p44 in HEK293-VEGFR-1 cells. (C,Lower) Effects of axinitinib and GDC-0973 onhPlGF-2-induced phosphorylation of p42/p44 inHEK-293-CAKI-1 and SKUT1b cells. (D) Effects ofaxinitinib and GDC-0973 on hPlGF-2-inducedproliferation/survival of SKUT1b cells. Dottedlines represent basal levels of migration orproliferation. Experiments were repeated atleast three times with comparable results. n =3–5. Error bars represent SD. (E) Effects of anti-PlGF, anti-VEGF-A, or anti-ragweed mAb on thegrowth of tumors implanted in vegfr-1 tk−/−,rag2−/− mice. Antibodies were administered asindicated in Fig. 1 and in Materials and Meth-ods. n = 10, relative to anti-ragweed treatment.Error bars represent SEM.

11594 | www.pnas.org/cgi/doi/10.1073/pnas.1109029108 Yao et al.

Ras/Raf/MAPK signaling. In this context it is interesting thatVEGFR-1 signaling within tumor cells previously has been shownto modulate growth and survival of several Ras/MAPK pathway-driven mouse tumor models and cell lines (3, 34). Growing evi-dence also supports a possible role for VEGFR-1 signaling incertain human cancers. In vitro studies suggested a role forVEGFR-1 signaling in survival of colorectal and pancreatic cancercell lines during epithelial to mesenchymal transition (22, 35–37).Also, VEGFR-1 signaling is required for growth of patient-derivedmalignant melanoma-initiating human cells in mice (38), and anti-hVEGFR-1 mAb treatment increases the survival of mice injectedwith acute lymphoblastic leukemia cells (39) and also inhibits tu-mor growth of VEGFR-1 positive breast carcinoma and mela-noma xenografts (4). Furthermore, expression of VEGFR-1 intumor cells has been observed in human biopsies (3, 40, 41). Fi-nally, mutations in VEGFR-1 have been found in human cancers,including ∼10% of melanomas (42).In conclusion, we show that, among the models we tested, effi-

cacy of anti-PlGF mAb treatment is limited to VEGFR-1expressing tumors, because it requires inhibition of PlGF/VEGFR-1 signaling within tumor cells. These findings may berelevant in the context of ongoing clinical evaluation of anti-PlGF(43), anti-VEGFR-1 (44) Mabs, VEGF-Trap (45), and other

VEGFR inhibitor therapies. It is tempting to speculate thatVEGFR-1 expression/activity may be a biomarker to selectpatients and indications likely to benefit from anti-PlGF therapies.

Materials and MethodsAnimals and Cell Lines. Female Beige nude and BALB/c nude mice wereobtained from Charles River. RAG2−/− mice were from Jackson Laboratories.flt1 tk−/− mice were generated as described (6). flt-1 tk, rag-2 double ko micewere generated by crossing Flt-1 tk −/− with with rag2−/− mice.

Tumor cell lines were obtained from theATCC. Tumor cells weremaintainedin RPMI-1640 containing 10% FBS (Sigma, Sigma-Aldrich), penicillin (100 units/mL), streptomycin (100 μg/mL), and L-glutamine (2 mmol/L). Hek293 cells werecultured in DMEM supplemented with 10% FBS (Sigma, Sigma-Aldrich), L-glu-tamine (2 mmol/L), and puromycin (1 μg/mL). Primary HUVEC were purchasedfrom Lonza and maintained in EGM-2 medium (Lonza). Only low-passageHUVECs were used in our experiments. All cells were cultured at 37 °C in a hu-midified incubator containing 5% CO2. Hek293-hVEGFR-1 and HEK293-controlcell lines were generated by transfection followed by puromycin selection.

ACKNOWLEDGMENTS. We thank the Genentech animal facility and theprotein purification and antibody technology groups. We also thankH. Koeppen for histopathological analysis and L. Gilmour, R. Neupane, andC.P. Poon from the FACS lab for excellent support.

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