differential therapeutic effects of anti vegf-a antibody...

Cancer Biology and Signal Transduction

Differential Therapeutic Effects of Anti–VEGF-A Antibody inDifferent Tumor Models: Implications for ChoosingAppropriate Tumor Models for Drug Testing

Dror Alishekevitz1, Rotem Bril1, David Loven2, Valeria Miller1, Tali Voloshin1, Svetlana Gingis-Velistki1,Ella Fremder1, Stefan J. Scherer3, and Yuval Shaked1

AbstractWe previously reported that the host response to certain chemotherapies can induce primary tumor

regrowth, angiogenesis, and even metastases in mice, but the possible impact of anti–VEGF-A therapy in

this context has not been fully explored.We, therefore, used combinations of anti–VEGF-Awith chemotherapy

on various tumor models in mice, including primary tumors, experimental lungmetastases, and spontaneous

lung metastases of 4T1-breast and CT26-colon murine cancer cell lines. Our results show that a combined

treatmentwith anti–VEGF-A and folinic acid/5-fluorouracil/oxaliplatin (FOLFOX) but notwith anti–VEGF-A

and gemcitabine/cisplatinum (Gem/CDDP) enhances the treatment outcome partly due to reduced angio-

genesis, in both primary tumors and experimental lungmetastasesmodels. However, neither treatment group

exhibited an improved treatment outcome in the spontaneous lung metastases model, nor were changes in

endothelial cell numbers found atmetastatic sites. As chemotherapy has recently been shown to induce tumor

cell invasion, we tested the invasion properties of tumor cells when exposed to plasma from FOLFOX-treated

mice or patientswith cancer.While plasma fromFOLFOX-treatedmice or patients induced invasionproperties

of tumor cells, the combination of anti–VEGF-A and FOLFOX abrogated these effects, despite the reduced

plasma VEGF-A levels detected in FOLFOX-treated mice. These results suggest that the therapeutic impact of

antiangiogenic drugs varies in different tumor models, and that anti–VEGF-A therapy can block the invasion

properties of tumor cells in response to chemotherapy. These results may implicate an additional therapeutic

role for anti–VEGF-A when combined with chemotherapy. Mol Cancer Ther; 13(1); 202–13. �2013 AACR.

IntroductionTumor angiogenesis entails endothelial cell division

from preexisting vessel capillaries and the mobilizationof circulating bone marrow–derived endothelial progen-itor cells (CEP), which home to active angiogenic sites andincorporate into tumor blood vessel walls (1). A numberof antiangiogenic-based drugs have been approved by theU.S. Food and Drug Administration (FDA) for the treat-ment of several malignancies. Bevacizumab, for example,is a humanized monoclonal antibody aimed at neutraliz-ing VEGF-A. Coadministrating bevacizumab and chemo-therapy increases overall survival and/or progression-free survival of patients with advanced metastatic

diseases, including colorectal, breast, non–small cell lung,and ovarian cancers (2–5). However, only limited clinicalevidence exists about the therapeutic impact of bevacizu-mab at the adjuvant and neoadjuvant treatment settings.Recently, it has been shown that coadministration ofbevacizumab and neoadjuvant chemotherapy significant-ly increased the pathologic complete response in patientswith triple-negative breast cancer (6, 7). In addition,emerging findings from a phase III clinical study inwhichbevacizumab was coadministered with folinic acid/5-fluorouracil/oxaliplatin (FOLFOX) to patients with colo-rectal cancer in an adjuvant setting, revealed that thistreatment combination failed to meet the predetermineddisease-free survival endpoint at 3 years (8, 9). Theseresults raise questions about the therapeutic impact ofbevacizumab and the mechanism of action of blockingVEGF-A on tumor growth, when coadministered withconventional chemotherapy.

We have recently studied the impact of antiangiogenicdrugs in combination with chemotherapy on tumorgrowth and angiogenesis. Our goalwas to revealmechan-isms that may explain how antiangiogenic drugs act aschemosensitizing agents (10). We showed that certainchemotherapydrugs, including paclitaxel and 5-flourour-acil (5-FU), induce rapid mobilization of CEPs from the

Authors' Affiliations: 1Molecular Pharmacology, Rappaport Faculty ofMedicine, Technion, Haifa; 2Department of Oncology, Ha'Emek MedicalCenter, Afula, Israel; and 3Hoffmann La Roche, Basel, Switzerland

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Yuval Shaked, Department of Molecular Phar-macology, Rappaport Faculty of Medicine, Technion–Israel Institute ofTechnology, 1 Efron St. Bat Galim, Haifa, Israel 31096. Phone: 972-4-829-5215; Fax: 972-4-855-5221; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0356

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(1) January 2014202

on June 28, 2018. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 22, 2013; DOI: 10.1158/1535-7163.MCT-13-0356

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bone marrow (11) and subsequently home to treatedtumor sites and promote angiogenesis. The administra-tion of antiangiogenic drugs 24 hours before chemother-apy resulted in the inhibition of therapy-induced CEPmobilization and subsequently enhanced treatment effi-cacy (11).Other chemotherapydrugs, such as gemcitabineand cisplatinum, which do not affect CEP levels, did notshow any added therapeutic benefitwhen combinedwithan antiangiogenic drug (11). Importantly, some of thesestudies were conducted in non–tumor-bearing mice,suggesting that antiangiogenic drugs may act as chemo-sensitizing agents by inhibiting the host systemic–rebound angiogenesis effects mediated by CEPs, leadingto improved treatment outcomes when tumors are intact(12).Counterintuitively, recent preclinical studies have sug-

gested that while antiangiogenic drugs should, in theory,act solely to inhibit the metastatic spread by decreasingangiogenesis in tumors, theymay actually promote tumorinvasion to normal adjacent tissue or even acceleratemetastasis (13). Inhibition of VEGF pathways by small-molecule antiangiogenic drugs led to increased tumor cellinvasiveness and metastatic growth in several preclinicaltumor models (14, 15). On the other hand, an anti–VEGF-A neutralizing antibody was recently shown not to pro-mote metastasis in genetically engineered mouse tumormodels (16, 17). It is suggested that various angiogenicfactors are induced by the host in response to antiangio-genic therapy (18). Furthermore, myeloid-derived sup-pressor cells (MDSC) were also found to home to tumorstreated with anti–VEGF-A therapy and contribute totumor refractoriness and subsequent regrowth (19). Over-all, these findings raise the possibility that certain cellularand molecular mechanisms may compensate for the lackof VEGF-A or its receptors by upregulating multiple"bypass" pathways in the tumor or in the host that,in turn, may promote tumor regrowth and metastasis(20–22).Similar to antiangiogenic drugs, the potential of che-

motherapy to accelerate tumor cell invasion and metas-tasis has also been investigated recently. We and othershave previously reported that chemotherapy inducesmolecular host factors that can contribute to tumor cellgrowth and tumor colonization in the lungs (11, 23–26).For example, matrix metallopeptidase 9 (MMP9) secretedfrom bone marrow–derived cells (BMDC) that colonizepaclitaxel-treated tumors may explain why mice primedwith paclitaxel chemotherapy succumb to metastasis ear-lier than mice treated with vehicle control (25). Overall,these studies suggest that the host in response to thechemotherapy generates prometastatic and protumori-genic effects thatpromote tumor regrowthandmetastasis.Therefore, drug combinations that inhibit the host’s pro-tumorigenic effects in response to chemotherapy arerequired.The aim of the current study is to investigate the effect

of combined treatments with anti–VEGF-A and chemo-therapy on breast and colon carcinomas in various

tumor models extensively used in preclinical studies,which include primary (ectopic), spontaneous lungmetastasis, and experimental lung metastasis. Usingthese models, we explored the therapeutic benefits ofan anti–VEGF-A antibody (B20) and its effects on theinvasion of tumor cells. The focus of this study is toelucidate the host effects found following the combina-tion of anti–VEGF-A and chemotherapy and its thera-peutic impact on various tumor models. The resultsshow an additional role for anti–VEGF-A other than itsantiangiogenic effect.

Materials and MethodsTumor models and cell lines

Primary ectopic tumors. 4T1 murine-breast carcino-ma cells (5 � 105) or CT26 murine-colorectal carcinomacells (2 � 106) obtained from the American Type CultureCollection (ATCC) were subcutaneously injected into theflank of 5- to 6-week-old female BALB/c mice (Harlan).Tumor size was assessed regularly with Vernier calipersusing the formula width2 � length � 0.5. When tumorsreached 200 mm3, treatment was initiated unless indicat-ed otherwise. Mice were randomly grouped before ther-apy (n ¼ 4–5 per group).

Spontaneous lungmetastases. 4T1 cells (5� 105)wereorthotopically injected into the mammary fat pad of 6-week-old female BALB/c mice. CT26 cells (2 � 106) weresubcutaneously injected into the flank of 6-week-oldfemale BALB/c mice. When tumors reached 200 to300 mm3, they were surgically removed and treatmentwas initiatedwhenmetastaseswere observed in the lungsof untreated mice (7 and 16 days following tumor resec-tion, respectively; Supplementary Fig. S1).

Experimental lung metastases. 4T1 cells (3 � 104) orCT26 cells (5� 104) were injected intravenously to the tailvein of 6-week-old female BALB/c mice. Lungmetastaticlesions were observed on day 28 following tumor cellinoculation, at which point treatment was initiated forboth tumor types (Supplementary Fig. S2).

All animal studies were approved by the Technion’s(Haifa) institutional committee. In some in vitro experi-ments, HCT116 human colon carcinoma cells (ATCC)were used. HT1080 fibrosarcoma and LM2-4 (ametastaticvariant of the MDA-MB-231) breast carcinoma cell lines,used in some experiments, were cultured in RPMI and10% fetal calf serum (FCS). All cellswere passed in culturefor no more than 4 months after being thawed fromauthentic stocks.

Drugs and their concentrationFOLFOX chemotherapy comprising folinic acid (3

mg/kg), 5-FU (5 mg/kg), and oxaliplatin (1.4 mg/kg)was administered intraperitoneally as a single bolusinjection in a 14-day cycle. GEM/CDDP chemotherapy,a combination of gemcitabine (33 mg/kg) and cisplat-inum (0.6 mg/kg), was administered intraperitoneallyas a single bolus injection in a 14-day cycle. B20, a

Differential Anti–VEGF-A Therapeutic Effects

www.aacrjournals.org Mol Cancer Ther; 13(1) January 2014 203




murine and human VEGF-A antibody (5 mg/kg; kindlyprovided by Genentech Inc.), was administered intra-peritoneally concomitantly with chemotherapy or asmonotherapy. The doses of FOLFOX and GEM/CDDPwere determined on the basis of mouse survival andbody weight loss (Supplementary Table S1) and asindicated elsewhere (27, 28). The B20 dose was used aspreviously determined (29).

Flow cytometric acquisition and analysisBlood or cell suspensions were quantitated for viable

CEPs, endothelial cells, andMDSCs, usingflowcytometryas previously described (23). Details are described inSupplementary Material (30, 31).

Quantitation and visualization of tissue hypoxia,vessel perfusion, and microvessel density

Tissue processing and immunohistochemistry wereconducted as described previously (32). Details aredescribed in Supplementary Material.

Evaluation of murine and human VEGF-A proteinlevels in the plasma

For thedetermination ofVEGF-A levels in the plasmaofmice and patients with cancer, blood was collected inEDTA tubes to prevent clotting. Plasma was obtainedafter cells were removed by centrifugation for 10 minutesat 2,000 � g and immediately stored at �20�C. Thawedplasma was applied onto a specific mouse or humanVEGF-A ELISA, respectively, according to the manufac-turer’s instructions (R&Dsystems).Resultswere analyzedin triplicates.

Evaluation of MMP9 expression using gelatinzymography

MMP9 expressionwas assessed by gelatin zymographyusing previously described method (25). Details aredescribed in Supplementary Material.

Blood samples obtained from cancer patientsPatients with cancer who underwent colorectal cancer

surgical resection (n ¼ 11) and were subsequently trea-ted with FOLFOX chemotherapy (as part of an adjuvanttreatment) at Ha’Emek Medical Center, Afula, Israel,were enrolled in the study. Bloodwas collected in EDTAtubes at baseline and 24 hours after the first cycle ofFOLFOX chemotherapy. Plasma was separated andimmediately stored at �20�C. Plasma samples wereused to assess the invasion of HCT116 cells using aBoyden chamber as described below and to determineVEGF-A levels using an ELISA. The study wasapproved by Ha’Emek Medical Center’s ethics commit-tee, and written informed consent was obtained from allthe patients before experimentation.

Invasion assayThe invasive properties of CT26 and HCT116 cells in

response to plasma of mice and humans, respectively,

were evaluated in Matrigel-coated Boyden chambers,using a previously described protocol (25). Details aredescribed in Supplementary Materials.

Quantification of the expression levels ofangiogenesis-related factors in plasma

A protein array kit (RayBio biotin label–based mouseAntibody array 1, RayBiotech, Inc.) was used to evaluatethe expression levels of angiogenesis-related factors inplasma from control or FOLFOX-treated mice in accor-dance with the manufacturer’s instruction. The resultswere analyzed using an online database for annotation,visualization and integrated discovery (DAVID, NIT)bioinformatics resources version 6.7, with categorizationfor angiogenesis. The fold change between the expressionlevels of factors in plasma of control untreated or FOL-FOX-treated mice was calculated.

Statistical analysisData are expressed as mean � SD. The statistical sig-

nificance of differences was assessed by one-wayANOVA, followed by Newman–Keuls ad hoc statisticaltest using GraphPad Prism 4 software. Differencesbetween all groups were compared with each other andwere considered significant at values below 0.05.

ResultsFOLFOX, but not GEM/CDDP, induced viable CEPmobilization, an effect that was blocked by anti–VEGF-A antibodies

In our previous studies, we evaluated systemic angio-genesis rebound by means of a substantial increase inviable CEP levels using single-agent chemotherapies(11, 12). Here, we sought to determine viable CEP levelsin mice treated with drug combinations commonly usedas first- and second-line treatments of colon and breastcarcinomas (33, 34). Levels of viable CEPs were ana-lyzed in non–tumor-bearing mice treated with the max-imum tolerated dose (MTD) of FOLFOX and GEM/CDDP. We also investigated whether coadministrationof an anti–VEGF-A antibody (B20) with these che-motherapies can affect viable CEP levels, as previouslydescribed for vascular disrupting agents (VDA) andseveral chemotherapy drugs (11, 32). For these pur-poses, blood was drawn 24 hours after the mice weretreated with FOLFOX, FOLFOXþB20, GEM/CDDP, orGEM/CDDPþB20, and viable CEP levels were analyzedby flow cytometry. As shown in Fig. 1, FOLFOX che-motherapy induced a significant increase (�4-fold) inthe mobilization of viable CEPs, whereas GEM/CDDPdid not have a significant effect on the levels of thesecells. No delayed CEP mobilization effects were docu-mented in both FOLFOX and GEM/CDDP-treated micein the first 96 hours (Supplementary Fig. S3). The B20antibody blocked FOLFOX-induced viable CEP mobili-zation, and as expected, did not affect CEP levels fol-lowing GEM/CDDP therapy.

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Mol Cancer Ther; 13(1) January 2014 Molecular Cancer Therapeutics204




Coadministration of FOLFOX and anti–VEGF-Aantibody enhanced the treatment efficacy of theprimary tumor and experimental metastasis but didnot affect the survival of mice bearing spontaneousmetastasisTo assess whether the treatment outcome can be affect-

ed by blocking viable CEP mobilization induced by FOL-FOX, 2 tumor models, CT26 murine colon carcinoma and4T1 murine mammary carcinoma, were implanted inBALB/c mice. The cells were inoculated into mice togenerate tumor models often used for preclinical drugevaluation, namely, ectopic primary tumors, experimen-tal metastasis, or spontaneous metastasis following pri-mary tumor resection. The mice were then treated withFOLFOX, FOLFOX þ B20, GEM/CDDP, or GEM/CDDPþ B20, and tumor volumes and mouse survival (in thecases of metastasis) were monitored until the endpoint.The primary tumor growth of CT26 was suppressed inmice treatedwith FOLFOXþB20 comparedwith FOLFOXmonotherapy, whereas no differences were observed inthe tumor growth curves of CT26 and 4T1 tumor typesbetween the mice group treated with GEM/CDDP andthose treated with GEM/CDDPþ B20 (Fig. 2A andD). Ofnote, it appears that GEM/CDDP treatment efficacy inboth 4T1 breast and CT26 colon cancers was superior tothat of FOLFOX, and that anti–VEGF-A did not signifi-cantly enhance the antitumor activity of FOLFOX in 4T1tumors. In addition, mice bearing experimental lungmetastases of either CT26 or 4T1 tumors survived longerwhen treated with FOLFOX þ B20 compared with micetreated with FOLFOX monotherapy (median survival: 57vs. 44 days and 34 vs. 20 days, respectively). Again, nosignificant differences in survival curves were observedbetween the GEM/CDDP and GEM/CDDP þ B20 treat-

ment groups (Fig. 2C and F). However, in mice bearingspontaneousmetastatic disease, no differences in survivalcurves were observed for either tumor type in any of thetreatment groups (median survival: 55–57days for FOL-FOX-treated groups, 77 days for GEM/CDDP-treatedgroups in 4T1 tumors, and 85–95 days for all chemother-apy-treated groups in CT26 tumors). Yet, the mortalityrate of untreated mice bearing CT26 lung metastasis wasfaster than any of the treatment groups (Fig. 2B and E). Inaddition, no significant changes in body weight weredocumented during the therapy, excluding the possibilityof overt toxicity in surviving mice (Supplementary Fig.S4). Taken together, these results suggest that anti–VEGF-A enhance treatment efficacy of FOLFOXbut not ofGEM/CDDP chemotherapy when the tumors grown as anectopic primary tumor and as an experimental lungmetastasis but fail to do so to the same extent, in thespontaneous lung metastases tumor model.

Treatment with FOLFOX þ anti–VEGF-A increasedantiangiogenic activity in primary and experimentallung metastases but not in spontaneous lungmetastases

The induction of viable CEPs following FOLFOX che-motherapy, and a blockade of this mobilization by anti–VEGF-A, has led us to further investigate whether thisdrug combination affects angiogenesis. To test this, micebearing ectopic primary CT26 or 4T1 tumors were treatedwith FOLFOX or GEM/CDDP, with or without a singledose of the B20 antibody, to block viable CEPmobilizationand to minimally affect the local anti-angiogenic activityof the drug, as previously shown (35, 36). Tumors wereremoved at end point, and microvessel density, perfu-sion, and hypoxia were evaluated. Anti–VEGF-A therapydid not significantly affect perfusion and hypoxia in both4T1 and CT26 primary ectopic tumors, although a slight(nonsignificant) reduction in microvessel density wasobserved. Coadministration of FOLFOX þ B20 led to asignificant decrease in microvessel density and perfusionand to increased hypoxia compared with the administra-tion of FOLFOX alone in CT26 tumors, whereas only asignificant increase in hypoxia was observed in 4T1tumors from mice treated with FOLFOX þ B20 therapywhen compared with control. Importantly, no significantdifferences in antiangiogenic activity in either tumor typewere observed in mice treated with GEM/CDDP or withGEM/CDDP þ B20 (Fig. 3). In addition, we also testedlevels of MDSCs colonizing these tumors as another celltype, which is known to contribute to angiogenesis andtumor refractoriness following antiangiogenic therapy(19). The results in Supplementary Fig. S5 show thatwhilethe combination of FOLFOXþ B20 antibody significantlyand markedly suppressed the number of MDSCs colo-nizing both 4T1 and CT26 tumors, there were no signif-icant differences in the number of MDSCs in tumorstreated with GEM/CDDP with or without the B20 anti-body. These results further suggest that a VEGF-A block-ade concomitantly with FOLFOX chemotherapy inhibits

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Figure 1. FOLFOX-induced acute CEP mobilization is blocked by anti–VEGF-A therapy. Eight to 10-week-old non–tumor-bearing BALB/c micewere treated with FOLFOX or GEM/CDDP administered at the maximumtolerated dose with or without 5 mg/kg B20 antibody administered once.Blood was drawn at baseline and 24 hours after therapy. Viable CEPlevels were evaluated as described in Materials and Methods. n ¼ 4–5mice/group; data are presented as mean � SD. ��, P < 0.001.






angiogenesis in primary ectopic tumors and may alsodelay tumor refractoriness to anti–VEGF-A therapy.

Next, to assess the therapy’s impact on angiogenesis ofmetastatic tumor models, a spontaneous lung metastasesmodel and an experimental lung metastases model forboth 4T1 and CT26 tumors were treated with FOLFOX orGEM/CDDP with or without an anti–VEGF-A antibody,at the time points indicated in Materials and Methods.Three days later, themicewere sacrificed and the lungs, inwhich metastatic lesions are usually observed (see Sup-plementary Figs. S1 and S22), were removed to evaluatethe percentage of endothelial cells in the entire lungs. Theresults in Fig. 4 show that while anti–VEGF-A therapy

reduced the percentage of endothelial cells found in thelungs of mice treated with FOLFOX in the experimentallung metastases, in the spontaneous lung metastases, thepercentage of endothelial cells found in the lungs wassimilar in all treatment groups regardless ofwhether anti–VEGF-A was added to the treatment. Furthermore, thenumber of metastatic lesions in the lungs of mice bearing4T1 and CT26 experimental metastatic models was lowerin mice treated with FOLFOX þ B20 than in mice treatedwith FOLFOX monotherapy, and no significant differ-ences in the number of lung metastases were observedin mice treated with either GEM/CDDP or GEM/CDDPþB20. In addition, no significant differences in the

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Figure 2. Differential antitumor activity of anti–VEGF-A therapy in primary ectopic tumor, spontaneous lung metastases, and experimental lung metastases.Mice bearing either 4T1 (A–C) or CT26 (D–F) tumors were treated with FOLFOX, FOLFOX þ B20, GEM/CDDP, or GEM/CDDP þ B20. A and D, primaryectopic tumor: treatment was initiated when tumors reached a size of 200 mm3, and tumor volume was assessed by caliper. Data are presented asmean � SD; n ¼ 4–5 mice/group. B and E, spontaneous lung metastases: treatment was initiated on day 7 (4T1) and day 14 (CT26) following primary tumorresection, as described in Materials and Methods. Mouse survival was analyzed using the Kaplan–Meier survival curve (n ¼ 6–8 mice/group). C andF, experimental lung metastases: treatment was initiated on day 28 for both 4T1 and CT26 tumors. Mouse survival was analyzed using the Kaplan–Meiersurvival curve (n ¼ 6–8 mice/group).

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number of lesions in the lungs of mice bearing spontane-ous metastatic models of both 4T1 and CT26 wereobserved in any of the treatment groups (SupplementaryFig. S6). These results further suggest that anti–VEGF-Ainhibits angiogenesis in both primary tumors and exper-imental lungmetastases by reducing the number of endo-thelial cells in the established tumor lesions, leading toimproved therapy outcomes, yet it fails to do so in micebearing spontaneous lung metastases.

Anti–VEGF-A inhibits invasion of tumor cells inresponse toplasmafrommiceorpatients treatedwithFOLFOXRecent studies have shown that antiangiogenic therapy

canpromote tumor cell invasion and acceleratemetastasis(14, 15). We have also recently shown that several che-motherapy drugs promote a host reaction that inducestumor cell invasion and metastasis (25). We therefore

sought to evaluate the invasion properties of tumor cellsfollowing treatment with anti–VEGF-A and/or FOLFOXchemotherapy. We focused on FOLFOX-treated micebearing spontaneous lung metastases of a CT26 coloncarcinoma. Plasma samples were obtained from the mice24 hours after theywere treatedwith FOLFOXor FOLFOXþ B20. The plasma was evaluated for its potential toinduce tumor cell invasion using the modified Boydenchamber assay. The plasma from FOLFOX-treated micemarkedly induced the invasive properties of CT26 tumorcells placed at the upper Boyden chamber compared withplasma from untreated mice. However, these invasiveeffects were absent in plasma from mice treated withFOLFOX þ B20 (Fig. 5A). Similar results were obtainedwhen plasma from non–tumor-bearing mice treated withFOLFOX were used (data not shown). We should notethat the exogenous addition of escalating doses of VEGF-A to both 4T1 and CT26 did not increase the invasion

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Figure 3. VEGF-A blockade following treatment with FOLFOX but not GEM/CDDP inhibits angiogenesis in primary ectopic tumors. Eight- to 10-week-oldBALB/cmicebearing500mm3primary 4T1 (AandB) orCT26 (CandD) tumorswere treatedwithFOLFOX,FOLFOXþB20,GEM/CDDP,orGEM/CDDPþB20.At end point, tumors were removed and evaluated for microvessel density using CD31 as an endothelial cell marker (in red; bar, 80 mm; A and C),and perfusion (in blue) and hypoxia (in green; bar, 200 mm; B andD). Graphs represent a summary of the quantization (mean�SD) of microvessel density, andmean percentage (�SD) of perfusion and hypoxia per field (n > 20 fields/group). �, 0.05 > P > 0.01; ��, 0.01 > P > 0.001.






properties of these cells, and neither of these cells expressthe 3 main receptors for VEGF-A: VEGFR1, VEGFR2, andVEGFR3 (Supplementary Fig. S7).

We next investigated whether parallel effects can befound in plasma from patients with colorectal cancertreated with FOLFOX at the adjuvant setting. After the

primary tumor was resected, plasma was collected atbaseline and 24 hours after FOLFOX therapy (n ¼ 11),and HCT116 human colon cancer cells were used to testinvasion using the Boyden chamber assay. Bevacizumab(as an anti–VEGF-A antibody) was added exogenously(5 mg/mL) to the plasma because this drug was not

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Figure 4. VEGF-A blockadefollowing treatment with FOLFOXbut not GEM/CDDP inhibitsangiogenesis in experimental lungmetastases but not in spontaneouslung metastases. BALB/c micebearing spontaneous lungmetastases (A) or experimentallung metastases (B) of 4T1 andCT26 tumors, as indicated in thefigure, were treated with FOLFOX,FOLFOX þ B20, GEM/CDDP, orGEM/CDDP þ B20. Fourteen daysafter therapy, the lungs wereremoved and evaluated formicrovessel density using CD31(in red; bar, 80 mm). Establishedmetastatic lesions in B areencircled with broken line. Inparallel, lungs were prepared assingle-cell suspensions and furtheranalyzed by flow cytometry todetermine the mean percentage(�SD) of endothelial cells(CD45�/VEGFR2þ) in thelungs. �, 0.05 > P > 0.01; ��,0.01 > P > 0.001.

Alishekevitz et al.





included in the patients’ treatment plan. Similar to mice,plasma from patients treated with FOLFOX inducedinvasion of tumor cells, an effect that was abrogated bythe exogenous addition of bevacizumab (Fig. 5B).We alsoexamined the impact of FOLFOX þ B20 therapy on micebearing LM2-4 metastatic breast carcinoma tumors andfound that FOLFOX-treated mice had a substantial num-ber of liver metastases at the endpoint compared withFOLFOX þ B20–treated mice that did not have any met-astatic lesions in the liver (Supplementary Fig. S8). Over-all, these results suggest that anti–VEGF-A blocks tumorcell invasion induced by host effects following FOLFOXtherapy.

A decrease in VEGF-A and changes in MMP9expression in the plasma of FOLFOX-treated miceTo determine whether neutralization of VEGF-A in the

plasma of treated mice or patients with cancer mayaccount for the inhibition of tumor cell invasion, ELISAsfor murine and human VEGF-A were conducted on plas-ma obtained frommice or patients with cancer at baselineand 24 hours after treatment with FOLFOX. The resultsin Fig. 6A and B show that circulating plasma VEGF-Alevels of FOLFOX-treated mice were significantly lowerthan those of control mice, and plasma VEGF-A levels ofpatients with cancer did not significantly change between

baseline and following FOLFOX chemotherapy. In addi-tion, plasma fromFOLFOX-treated or untreatedmicewasalso applied on protein array to assess the changes in thelevels of additional pro-angiogenic factors besides VEGF-A.UsingDAVIDanalysis for angiogenic factors,we founda marked increase in several angiogenic factors, amongthose are factors associated with VEGF and VEGF recep-tor pathways (Supplementary Table S2). Taken together,these results indicate that although low levels of VEGF-Adetected by ELISA following FOLFOX therapy did notcorrelate with the increased tumor cell invasion proper-ties, other pro-angiogenic factors were upregulated in theplasma of FOLFOX-treated mice and anti–VEGF-A wasable to block therapy-induced tumor cell invasion.

As our previous study showed that MMP9 secreted byBMDCs may account for the invasive properties of tumorcells following chemotherapy (25), we sought to deter-mine the levels of MMP9 in the plasma of FOLFOX- orFOLFOX þ B20–treated mice. Plasma from treated micewas applied onto gel zymography and levels of MMP9were evaluated. The results in Fig. 6C show that whileFOLFOX induced MMP9 activity in the plasma of treatedmice, FOLFOX þ B20 antibody markedly reduced itsactivity. MMP9 activity was not different betweenGEM/CDDP and GEM/CDDP þ B20–treated groups. Inaddition, no significant differences in the level of MMP9

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Figure 5. Plasma from mice and patients with cancer treated with FOLFOX induces tumor cell invasion, an effect that is blocked by anti–VEGF-A.A, eight to 10-week-oldmice bearing CT26 spontaneous lungmetastases were treated with FOLFOX or FOLFOXþB20. Blood was drawn 24 hours later andapplied onto a Boyden chamber to evaluate CT26 tumor cell invasion. B, plasma from patients with colon cancer who underwent tumor resection (n ¼ 11)was obtained at baseline and 24 hours after treatment with FOLFOX. The plasma was applied onto a Boyden chamber to evaluate HCT116 tumor cellinvasion in the presence or absence of 5 mg/mL bevacizumab. Graphs represent the mean � SD of number of cells counted per field (n > 10 fields/group).�, 0.05 > P > 0.01; ��, 0.01 > P > 0.001.






were observed in primary tumors of both 4T1 and CT26removed at endpoint in any of the treatment groups (datanot shown). These results suggest that anti–VEGF-A mayinhibit the activity of MMP9 induced by the host in theplasma of FOLFOX-treated mice but may not affect thelevels of MMP9 in the treated tumor site.

DiscussionOne of the current problems facing antiagiogenic drug

therapy is the lack of marked and significant antitumoractivity in several randomized phase III clinical studiesfor several malignancies despite the remarkable thera-peutic benefits such drugs showed in preclinical models.These discrepancies have recently been accounted forby showing that the animal models used did not appro-priately mimic the clinical scenario (37). A growing num-ber of preclinical studies have shown antiangiogenicdrug efficacy by using preclinical models such as GEMMsor patient-derived xenografts. However, these modelsrequire high maintenance and need large numbers ofmice for each treatment groupmainly because of the highvariation and timing of the tumor take (37, 38). Onequestion raised is whether the differences in treatmentoutcomeof antiangiogenic drugs in the various preclinicalmodels stem from a spectrum of tumor models tested,including primary ectopic tumors, small or large tumors,established metastases, and micrometastasis amongothers. Clinically, anti–VEGF-A therapy, that is, bevaci-

zumab, has been tested and was found to be beneficial inseveral advanced metastatic malignancies (2–4). It hasalso shown promising benefits in limited numbers ofneoadjuvant treatment setting trials, that is, before pri-mary tumor resection (6, 7). However, in the adjuvantsetting, after tumor resection, bevacizumab did not showany significant benefit at least not in the treatment ofcolorectal cancer (9). In this study, coadministration ofanti–VEGF-A and chemotherapy improved the treatmentoutcomes of primary ectopic tumors and experimentallung metastases with established colon and breast carci-noma tumor lesions. However, no survival benefit wasseen in mice bearing spontaneous lung metastasis inwhich only a microscopic disease of either tumor typewas observed in the lungs. Guerin and colleagues haverecently shown that the antitumor activity of antiangio-genic drugs is effective in the treatment of primary tumorsbut was either ineffective or showed modest therapeuticbenefit in a postsurgical advanced metastatic diseasemodel (39). In addition, a recent study by Rovida andcolleagues showed that the combination of various che-motherapy drugs, including doxorubicin, topotecan, andgemcitabine but not paclitaxel and cisplatin, inhibitedthe prometastatic effects of sunitinib (40). Importantly, asopposed to our study, which focuses on the impact of thetherapy on the host leading to metastasis spread usingvarious tumor models and tumor stages, in Rovida andcolleagues study, the authors suggested that increasedtumor hypoxia following antiangiogenic therapy is

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Figure 6. Plasma VEGF-A andMMP9 expression levels inFOLFOX- or FOLFOX þ B20–treated mice or patients withFOLFOX-treated cancer. A, VEGF-A levels (mean � SD) in plasmaobtained from non–tumor-bearingmice (n ¼ 5/group) 24 hours afterthey were treated with FOLFOX orFOLFOXþ B20 was applied onto amurine-VEGF-A ELISA. B, VEGF-Alevels (mean � SD) detected byspecific human-VEGF-A ELISA inplasma obtained frompatientswithcolon cancer (n ¼ 11) whounderwent tumor resection andwere subsequently treated withFOLFOX. C, MMP9 levels (mean) inplasma obtained from mice, 24hours after treatment with B20,FOLFOX, FOLFOX þ B20, GEM/CDDP, orGEM/CDDPþB20,wereevaluated using gel zymographyand densitometry. Theexperiments were carried out intriplicates. �, 0.05 > P > 0.01.

Alishekevitz et al.





minimized when chemotherapy is added at the neo-adjuvant treatment setting (40). Overall, the tumor modelused for drug testing, and the impact of the therapy onthe host and on the tumor may be critical for the assess-ment of the therapeutic benefit of any particular anti-cancer drug.In our study, we used different tumor models to eluci-

date the antiangiogenic effects of anti–VEGF-A therapywhen coadministered with chemotherapy. We found thatthe endothelial cell number or microvessel density andperfusion were significantly decreased, whereas hypoxiawas significantly increased in primary tumors or estab-lished tumor lesions in the lungs of mice treated withFOLFOXþ B20when comparedwith FOLFOXalone. Suchantiangiogenic benefits were absent in mice treated withGEM/CDDP in either tumor type.These results emphasize2 important points. First they suggest that, like in otherstudies, the efficacy of antiangiogenic therapydoesdependnot only on the type of tumor being treated but also on thetype of chemotherapy that the anti–VEGF-A agent is coad-ministered with (11, 12). Second our results emphasize,once again, the use of a reproducible cancermodel for drugtesting, a problem raised in recent reports (37, 38).The host’s response to several chemotherapy drugs

has recently been investigated in various studies(11, 25, 26, 41–43). The first study reported that adminis-tration of cytotoxic-like vascular disrupting agents tonon–tumor-bearing mice induced a rapid CEP mobiliza-tion (32). On the basis of this study, other studies alsoreported that various cytotoxic drugs may induce themobilization of different types of BMDCs in non–tumor-bearing mice, suggesting that these effects aresolely related to the host and not to the tumor. Welfordand colleagues have recently shown that as opposed toCEPs, Tie2-expressingmacrophageswere found to highlycolonize in tumors treated with a vascular disruptingagent (44). DeNardo and colleagues have also reportedthat in response to chemotherapy, tumor immunecells home to the tumor microenvironment, especiallymacrophages, which highly infiltrate mammary adeno-carcinomas (42). In a previous study, we reported that thechemotherapy drug, paclitaxel, which induces a rapidmobilization of CEPs, administered in combination withan antiangiogenic drug therapy had an enhanced treat-ment outcome and the progression of the establishedtumors decreased (11). In the current study, we show thatsimilarly to paclitaxel, FOLFOX chemotherapy, a drugcombination used to clinically treat colon cancer amongother malignancies (45), induces CEP mobilization. Con-sequently, the concomitant administration of anti–VEGF-A and FOLFOX resulted in a significant inhibition of CEPmobilization, as previously documented for paclitaxel(11). Importantly, when the same experiments were car-ried out in mice treated with GEM/CDDP, in which CEPmobilization was not induced, the addition of B20 to thechemotherapy did not result in an additional therapeuticbenefit. Overall, our results suggest that anti–VEGF-Ablocks systemic angiogenesis, which can be induced in

response to certain chemotherapies and therefore canimprove therapy outcome.

An intriguing question arising from this study is thatwhile treatment with FOLFOX led to reduced levels ofVEGF-A in the plasma of mice and, to some extent, in theplasma of patients with cancer, anti–VEGF-A therapycould still inhibit host effects promoting tumor cell inva-sion. Although we did not address these counterintuitiveresults by carryingout possible experiments,we speculatethat the reduced levels of plasma VEGF-A detected bycommercially available ELISA kits do not necessarilydetect all VEGF-A isoforms, which in fact could still beactive. Under such circumstances, anti–VEGF-A therapy(B20) could block the undetectable isoforms of VEGF-Aand therefore inhibit metastasis-promoting host effectsdespite the low VEGF-A levels detected. This speculationcoincides with several studies showing that VEGF-Alevels in patients with cancer do not correlate with theantiangiogenic treatment efficacy and do not provide apredictive biomarker for the treatment success of anti-angiogenic drugs (46, 47). Furthermore, new ELISA kitsfor VEGF-A, which are not commercially available andtherefore,werenot available tous for this study, candetectthe low-molecular-weight isoforms of VEGF-A. Usingsuch ELISA kits, it has been shown that the low-molec-ular-weight VEGF-A levels correlate with the efficacy ofanti–VEGF-A therapy (S.J. Scherer, unpublished observa-tions). It is therefore plausible that FOLFOX chemother-apy upregulates undetected low-molecular-weightVEGF-A isoforms, which are then neutralized by anti–VEGF-A therapy, thus blocking the invasive properties oftumor cells. Overall, it would be of interest to furtherdetermine the levels of low-molecular-weight VEGF-Aisoforms in the plasma of mice and patients with cancerfollowing cytotoxic drug therapy.

In summary, this study suggests that anti–VEGF-A, incombination with chemotherapy, may have additionaltherapeutic activities besides antiangiogenesis that arerelated to blocking host protumorigenic and prometa-static effects that exist in response to certain chemother-apy drugs and can explain why antiangiogenic therapy inthis contextmay improve therapy outcome, depending onthe preclinical tumor models used.

Disclosure of Potential Conflicts of InterestY. Shaked has received commercial research grant from and is a

consultant/advisory board member of Hoffmann La Roche. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design:D. Alishekevitz, D. Loven, S.J. Scherer, Y. ShakedDevelopment of methodology: D. Alishekevitz, S. Gingis-VelitskiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Alishekevitz, D. Loven, V. Miller, E. FremderAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): D. Alishekevitz, E. Fremder, S.J. Scherer,Y. ShakedWriting, review, and/or revision of themanuscript:D.Loven, S.J. Scherer,Y. ShakedAdministrative, technical, ormaterial support (i.e., reportingororganizingdata, constructing databases): R. Bril, T. Voloshin, S. Gingis-VelitskiStudy supervision: Y. Shaked






AcknowledgmentsThe B20 antibody was kindly provided by Genentech Inc.

Grant SupportThis work was supported by research grants from the European

Research Commission under the FP7 program (260633), the Israel ScienceFoundation (565/09), and a sponsored research agreementwithHoffmannLa Roche to Y. Shaked.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 6, 2013; revised October 14, 2013; accepted October 15,2013; published OnlineFirst October 22, 2013.

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2014;13:202-213. Published OnlineFirst October 22, 2013.Mol Cancer Ther Dror Alishekevitz, Rotem Bril, David Loven, et al. Tumor Models for Drug TestingDifferent Tumor Models: Implications for Choosing Appropriate

VEGF-A Antibody in−Differential Therapeutic Effects of Anti

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