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Jin Lee1, Daniel L. Karl1, Ugwuji N. Maduekwe1, Courtney Rothrock1, Sandra Ryeom3,

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ors induce new blood vessel growth primarily from host organ microvascular endothelial cells (EC), andvasculature differs significantly between the lung and liver. Vascular endothelial growth factor (VEGFGF-A) promotion of tumor angiogenesis is thought to be mediated primarily by VEGF receptor-2R-2). In this study, VEGFR-2 antibody (DC101) inhibited growth of RenCa renal cell carcinoma lungtases by 26%, whereas VEGFR-1 antibody (MF-1) had no effect. However, VEGFR-2 neutralization hadect on RenCa liver metastases, whereas VEGFR-1 neutralization decreased RenCa liver metastases byor CT26 colon carcinoma liver metastases, inhibition of both VEGFR-1 and VEGFR-2 was requireduce growth delay. VEGFR-1 or VEGFR-2 inhibition decreased tumor burden not by preventing theishment of micrometastases but rather by preventing vascularization and growth of micrometastases

and 43%, respectively. VEGF induced greater phosphorylation of VEGFR-2 in lung ECs and of-1 in liver ECs. EC proliferation, migration, and capillary tube formation in vitro were suppressed moreGFR-2 inhibition for lung EC and more by VEGFR-1 inhibition for liver EC. Collectively, our resultste that liver metastases are more reliant on VEGFR-1 than lung metastases to mediate angiogenesis

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due to differential activity of VEGFRs on liver EC versus lung EC. Thus, therapies inhibiting specific VEGFRsshould consider the targeted sites of metastatic disease. Cancer Res; 70(21); 8357–67. ©2010 AACR.

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cular endothelial growth factor (VEGF or VEGF-A) ispressed by the vast majority of solid tumors (1), andting levels of VEGF are elevated in many cancer pa-including those with colorectal and renal cell cancerhibition of VEGF can effectively suppress tumor angio-s in mouse tumor models (3), and numerous inhibitorsGF are currently in clinical use (4). VEGF exerts itsprimarily through two tyrosine kinase receptors, VEGF

FR-1; Flt-1) and VEGFR-2 (Flk-1, KDR), whichy endothelial cells (EC; ref. 3). VEGFR-2 is

vasculcell sucular)and almicroof fluiparendiffereorganmentsVEGFmetasNeu

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ns: 1Department of Surgery, Massachusetts Generalvard Medical School; 2Schepens Eye Researchrtments of Ophthalmology and Pathology, Harvardoston, Massachusetts; and 3Department of Cancerof Pennsylvania School of Medicine, Philadelphia,

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

Karl contributed equally to this work.

r Y-J. Lee: Division of Radiation Effects, Korea InstituteMedical Science, 215-4 Seoul 139-706, Korea.

uthor: Sam S. Yoon, Departments of Surgery andUniversity of Pennsylvania School of Medicine,Spruce Street, Philadelphia, PA 19104. Phone: 215--662-7476; E-mail: sam.yoon@uphs.upenn.edu.

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ed to mediate the primary downstream effects of VEGFs including increased vascular permeability, prolifera-igration, and survival (5). VEGFR-1 has generally been

ht to transmit only weak mitogenic signals (6).tumor blood vessels are derived primarily from the

vascular ECs of the host organ or tissue (7), althoughis also a contribution from bone marrow–derived ECrsors (8, 9). Significant heterogeneity exists betweenicrovascular endothelium of different organs in termscture and function (10), and ECs from different micro-ar beds have distinct gene expression patterns (11) andrface proteins (12). Morphologically, liver (microvas-sinusoidal ECs are discontinuous with fenestrationslow free passage of nutrient-rich plasma, whereas lungvascular ECs are continuous and prevent accumulationd (12). The amount of VEGF produced by surroundingchymal cells is an essential factor in these morphologicnces (13). Given that microvascular ECs from differents have such heterogeneous characteristics and require-for VEGF, it is reasonable to expect that inhibitors ofor VEGFRs may have significantly different effects ontatic tumors growing in various organ sites (14).tralizing antibodies targeting specifically VEGFR-1 orR-2 are currently being examined in clinical trials fortatic solid tumors often without consideration for theic sites of disease (15). In this study, we examined theof VEGFR-1 and VEGFR-2 inhibition in vitro on lung

ver ECs. We further examined the effects of VEGFR-1EGFR-2 inhibition on lung and liver metastases using

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ifferent cancer cell lines. Surprisingly, VEGFR-1 wasto play a greater role than VEGFR-2 in liver EC prolif-

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nesCa renal carcinoma cells, CT26 mouse colon carcino-lls, SVR mouse angiosarcoma cells, and DC101 andhybridoma cells were obtained from the America Typee Collection (ATCC). MC26 mouse colon carcinomaere obtained from National Cancer Institute (NCI)r Repository. Human umbilical vein ECs (HUVEC)obtained from Lonza. Human liver sinusoidal ECsuman lung microvascular ECs were obtained fromell. All ECs were used within eight passages. Cancernes were actively passaged for <6 months from thethat they were received from ATCC or NCI Tumoritory, and the United Kingdom Coordinating Commit-Cancer Research guidelines were followed (16). Nor-ouse lung microvascular EC (m-lung EC) and mouseinusoidal EC (m-liver EC) were isolated from BALB/cas we have previously described (17). CT26 lung me-es were isolated 3 weeks following tail vein injection,T26 liver metastases were isolated 2 weeks followingplenic injection. ECs from metastases were isolatedimilar fashion.01 and MF-1 antibodies were produced from hybrid-ells using the BD CELLine 1000 system (BD Bios-s) following the manufacturer's instructions.

ro EC assaysan ECs and cancer cell lines were tested after 12 to

urs of incubation in Optimen with 1% fetal bovine(FBS) for proliferation using a colorimetric MTT assay,tion using a modified Boyden chamber, and/or capil-be formation using Matrigel, as we have previouslybed (18). Recombinant human VEGF (10 ng/mL, NCI),man VEGFR-1 antibody (AF321, 0.5 μg/mL, R&D Sys-antihuman VEGFR-2 antibody (MAB3572, 0.5 μg/mL,ystems), recombinant murine VEGF (10 ng/mL, R&D),ouse VEGFR-1 antibody (MF-1, 0.5 μg/mL), and/or anti-VEGFR-2 antibody (DC101, 0.5 μg/mL) were addedindicated. Proliferation of mouse ECs and tumor ECsssessed using a bromodeoxyuridine (BrdUrd) incor-on assay. Cells were incubated in 10 μmol/L BrdUrd) for 12 hours. The incorporated BrdUrd was stainednti-BrdUrd-FITC (1:50, BD Pharmingen), and a percent-BrdUrd positive cells were examined by fluorescence-ted cell sorting (FACS).

analysiss were fixed in 70% ethanol and incubated with mouseD31 monoclonal antibody (mAb; 1:100; Pharmingen)

our at 4°C. Cells were then incubated with goat anti-Alexa 488–conjugated secondary antibody (1:500;

immuwere i

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ular Probes) for 30 minutes at 4°C and analyzed withScan flow cytometer (Becton Dickinson).

rn blot analysisWestern blot analysis of VEGFRs, ECs in Optimem% FBS were treated with VEGF (10 ng/mL) whereted. VEGFR neutralizing antibodies were added whereted 1 hour before addition of VEGF. Cells were har-5 minutes after VEGF administration. For normal

s and metastases, tissues were snap frozen in liquiden and thawed in radioimmunoprecipitation assay buf-ntaining Complete Protease Inhibitor Cocktail (Roche)osphatase Inhibitor Cocktail (Sigma). DNA was shearedh a 21-gauge needle. Samples were sonicated for 10 toonds and then centrifuged at 4°C for 20 minutes at× g. The supernatant was collected, and protein concen-was determined by BCA Protein Assay Kit (Pierce).

rn blot analysis was performed for total and phosphor-VEGFR-1 and VEGFR-2 using the following antibodies:horylated VEGFR-1 (Tyr1213, 1:20,000, Upstate), phos-lated VEGFR-2 (Tyr1175, 1:1,000, Cell Signaling), totalR-1 (1:500, Santa Cruz), and total VEGFR-2 (1:1,000, Celling).signal transducer and activator of transcription 33) Western blot analyses, ECs were treated with VEGF,-E (10 ng/mL, Fitzgerald Industries), or placentalh factor (PlGF; 10 ng/mL, R&D Systems) for 7 minutes,sates were collected and probed for phosphorylated(1:2,000, 9145S, Cell Signaling), total STAT3 (1:1,000,

Cell Signaling), and β-actin (1:10,000; Abcam). EC andr cells lysates were also examined for neuropilin-11) by Western blot analysis (1:1,000, Santa Cruz).

titative reverse transcription–PCRntitative real-time PCR analysis was performed usingghtCycler Detection System (Roche Diagnostics) as pre-y described (19). Primers for mouse VEGFR-1 andR-2 were the following: VEGFR-1, (forward) 5′-CGGAA GAC AGC TCA TC-3′ and (reverse) 5′-CTT CAC

ACA GGT GTA GA-3′; VEGFR-2, (forward) 5′-GGCGT GAC AGT ATC TT-3′ and (reverse) 5′-TCT CCGGC TCA AT-3′.

nohistochemical and immunofluorescencescopy1 immunohistochemical localization and analysis ofvessel density (MVD) were performed as previously de-d (19). For VE-cadherin and CD31 immunofluorescence,ere fixed and incubated with rat VE-cadherin mAb

, R&D Systems) and mouse anti-CD31 mAb (1:100; Phar-n) overnight at 4°C. Following washing, sections wereated with goat anti-rat Alexa 594 (1:500; Moleculars) and goat antimouse Alexa 488–conjugated secondarydies (1:500; Molecular Probes) for 1 hour at room tem-re. Cell nuclei were labeled with Hoechst dye (1 μg/mL).GFR-2 and platelet/EC adhesionmolecule 1 (PECAM-1)

nofluorescence, paraffin-embedded tissue sectionsncubated with goat anti-PECAM-1 (1:100, Santa Cruz

Cancer Research

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VEGF Receptor Inhibition of Lung and Liver Metastases

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hnology) and mouse anti-VEGFR2 (1:100, Santa Cruzhnology) and then incubated with antigoat Alexa 488ntimouse Alexa 594-conjugated secondary antibodies, Molecular Probes). Subsequently, tissues were stained′,6-diamidino-2-phenylindole (0.2 μg/mL) for 3minutes.s were obtained on a Zeiss microscope and analyzedAxioVision 4.0 software (Carl Zeiss Vision).

e studiesmouse protocols were approved by the Massachusettsal Hospital Subcommittee on Research Animal Care.nerate lung and liver metastases, 0.5–1.5 × 106 cellsinjected into the tail vein or spleen. The followingice were treated with either DC101 (40 mg/kg), MF-1g/kg), a combination of DC101 and MF-1, or isotypel IgG1s (40 mg/kg) three times per week. Lungs wereted at 3 weeks, and livers were harvested at 2 weeksing tumor cell injection. Organs were weighed, fixedalin, and then photographed. To determine number

ize of metastases, lungs were harvested at 14 daysvers were harvested at 8 days, fixed in formalin, andy sectioned with 0.5 to 1 mm between sections. Twofied fields per section and five sections per organ werened, and metastases were counted by a masked obser-hotographs were taken of each counted field, and theof each metastasis was determined using SPOTced v4.6 software (Diagnostic Instruments, Inc.).

tical analysisups were compared using Instant 3.10 software (Graph-or comparisons between more than two groups, treat-groups were compared with the control group usingay ANOVA with Bonferroni adjustment for multiplerisons. P values of <0.05 were considered significant.

lts

ential effects of VEGFR-1 and VEGFR-2ition on renal cell carcinoma metastases tong versus liverspecifically assess the contributions of VEGFR-1 andR-2 activation in promoting angiogenesis in lung andetastases, we examined the effects of neutralizingdies to VEGFR-1 and/or VEGFR-2 on RenCa renal celloma liver and lung metastases in syngeneic wild-type/c mice. The RenCa cell line was chosen because itses high levels of VEGF compared with other mousecell lines (19). MF-1 and DC101 are mAbs that bind

eutralize VEGFR-1 and VEGFR-2, respectively (20, 21).ere injected via tail vein with RenCa cells to generateetastases or into the spleen to generate liver meta-

. Neutralization of VEGFR-2 with DC101 over 21 dayscantly inhibited RenCa lung metastases (26% reductionan organ weight), whereas neutralization of VEGFR-1F-1 had minimal effect (Fig. 1A and B). In contrast,de of VEGFR-1 with MF1 over 14 days inhibited RenCa

etastases (31% reduction in mean organ weight),

as blockade of VEGFR-2 with DC101 had minimalresponinhibi

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Neutralization of both VEGFR-1 and VEGFR-2 with anation of MF-1 and DC101 did not lead to additionaltion of either lung or liver metastases. These experi-were repeated two more times with similar results.ause VEGF signaling is known to play a role in both theishment of metastatic lesions as well as the growth oftases, we examined the effects of VEGFR-1 and VEGFR-ition on the number and size of metastases in the lungver. Mice with RenCa lung metastases were treatedeutralizing antibodies, but lungs were harvested ats rather than at 21 days, and the number and size oftases were examined in serial sections. DC101 treat-led to an appreciable decrease in the size of lungtases (Fig. 1C) but had no effect on the mean numberg metastases (Supplementary Fig. S1A and B). MiceenCa liver metastases treated with MF-1 (sacrificedays instead of 14 days) had a significant decrease inze of liver metastases with no change in the numberr metastases. Analysis of MVD using CD31 immuno-hemistry or PECAM immunoflurescence of lung andetastases, respectively, revealed that decreases in size

tastases due to MF-1 or DC101 treatment was accom-by decreases in MVD (Fig. 1D).

ential activation of VEGFR-1 and VEGFR-2 inand liver ECsdetermine if variations in VEGFR-1 and VEGFR-2 levelsferent organ environments could explain differencesponse to VEGFR inhibitors, we examined levels ofR-1 and VEGFR-2 in normal mouse lung and liver byuantitative reverse transcription–PCR (RT-PCR) andrn blot analysis. VEGFR-1 levels were moderately high-lung compared with liver, and VEGFR-2 levels werely equivalent in lung compared with liver (Supplemen-ig. S2A and B). Thus, different levels of VEGFR-1 andR-2 in lung and liver did not account for the distinctof VEGFR inhibition on lung and liver metastases.better assess VEGFR signaling in lung and liver ECs,xt analyzed VEGFRs in lung ECs and liver ECs. Cells were analyzed by Western blot analysis for totalhosphorylated VEGFR-1 and VEGFR-2 before and afterdition of VEGF. In lung EC, VEGF treatment led tohorylation of both VEGFR-1 and VEGFR-2 (Fig. 2A).ver, in liver EC, the addition of VEGF resulted in sig-t phosphorylation of VEGFR-1 and only minimal phos-lation of VEGFR-2. Phosphorylation of VEGFR-1 andR-2 were inhibited by neutralizing antibodies to theseors. Quantification of VEGFR phosphorylation follow-EGF treatment showed greater VEGFR-2 activationg EC and greater VEGFR-1 activation in liver ECB).directly examine the effects of VEGFR-1 and VEGFR-2ing on EC function, we analyzed lung EC and liver ECee different endothelial activation assays: proliferation,tion, and tube formation (Fig. 2C). Proliferation asred by both lung EC and liver EC was increased in

se to VEGF stimulation. Proliferation of lung EC wasted to a greater degree by neutralizing antibody to

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1. Differential effects of VEGFR-1 and VEGFR-2 inhibition on lung and liver metastases. A, RenCa renal carcinoma lung and liver metastasestreated with VEGFR antibodies MF-1 (αVEGFR-1), DC101 (αVEGFR-2), and/or control IgG (n = 6 mice per group). B, mean weight of lung ands percentage of control. C, mean size of RenCa metastases. D, MVD as percentage of control. Bars, SD. *, P < 0.05, compared with IgG

group.

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VEGFversusEC wathanP < 0.EC miAnti-Vlung Eshowe10%, Pfactorer inhanti-VEC (16anti-VeffectmatioTo c

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R-2 than by neutralizing antibody to VEGFR-1 (42%22%, P < 0.01). In contrast, proliferation of livers inhibited more by VEGFR-1 neutralizing antibodyby VEGFR-2 neutralizing antibody (49% versus 9%,001). The same pattern of responses was seen whengration was analyzed in a modified Boyden chamber.EGFR-2 antibody had a greater inhibitory effect forC (30% versus 12%, P < 0.01), and anti-VEGFR-1d greater inhibition of liver EC migration (39% versus< 0.05). Finally, for capillary tube formation on growth–reduced Matrigel, anti-VEGFR-2 antibody had a great-ibitory effect on lung EC (27% versus 11%, P < 0.01) andEGFR-1 antibody had a greater inhibitory effect on liver% versus 7%, P < 0.001). Combination therapy with bothEGFR-1 and anti-VEGFR-2 antibodies had an additivein inhibiting EC proliferation, migration, and tube for-n in lung EC but had no additive effect in liver EC.onfirm the differential effects of VEGFR-1 and VEGFR-ition on m-lung EC and m-liver EC, we isolated theseom normal mouse lung and mouse liver. FACS analysis31 before and after the second round of isolationd significantly increased CD31 expression (Supplemen-ig. S3A). EC purity was also assessed by double labeling-cadherin and CD31, and the vast majority of cellssed both EC markers (Supplementary Fig. S3B). VEGF

addition of VEGF alone given a value of 100%. Bars, SD. P values are in

ent of m-lung EC and m-liver EC led to differential ac-n of VEGFR-1 and VEGFR-2 similar to that seen in

MF-1ment

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n liver and lung ECs. VEGF treatment in m-lung ECmore phosphorylation of VEGFR-2 than VEGFR-1,as VEGF treatment of m-liver EC led to more phosphor-of VEGFR-1 than VEGFR-2 (Fig. 3A). Similarly, inhibi-f m-lung EC proliferation in vitro was greatest withR-2 neutralization, whereas inhibition of m-liver EC pro-ion was greatest with VEGFR-1 neutralization (Fig. 3B).

s of VEGFR-1 and VEGFR-2 inhibition onectal carcinoma lung and liver metastasesensure that the differential effects of VEGFR-1 andR-2 inhibition against liver and lung metastases were aon of the host organ environment and not specific to thecell line, we performed similar in vivometastasis experi-using CT26 colon carcinoma cells. For lung metastases,EGFR-2 therapy with DC101 significantly attenuated theh of lung metastases (23% reduction in mean weight),ti-VEGFR-1 therapy had minimal effect (Fig. 4A ande addition of MF-1 to DC101 decreased the mean weight. In contrast to lung metastases, MF-1 or DC101 aloneeffect on liver metastases and only the combination of

and DC101 inhibited liver metastases (mean weight re-n, 25%). These experiments were repeated two morewith similar results. MVD was decreased up to 55% inetastases treated with DC101 or a combination of

2. VEGFR-1 and VEGFR-2 expression in human lung and liver ECs and in vitro EC assays. A, Western blot analysis for total and phosphorylated-1 and VEGFR-2 in lung ECs and liver EC lysates. VEGF, neutralizing antibody to VEGFR-1 (αVEGFR-1), and neutralizing antibody to VEGFR-2R-2) were added where indicated. β-Actin blot serves as loading control. B, quantification of VEGFR-1 and VEGFR-2 phosphorylation relativeVEGFR-1 and VEGFR-2 expression. C, EC proliferation (using MTT assay), migration (using modified Boyden chamber), and capillary tube formation

and DC101 (Fig. 4C and D). For liver metastases, treat-with MF-1 and DC101 reduced MVD by 43%.

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R-1 and VEGFR-2 activation in cancer cellse cancer cells have been reported to express VEGFRs,hibition of these receptors can have cancer cell auton-effects (22). To ensure that the results observed in ourstudies were due to effects on ECs and not on the can-lls, we examined the effect of VEGFR-1 and VEGFR-2ion on CT26 and RenCa in vitro. Western blot analysised VEGFR-1 expression, but not VEGFR-2 expression, byand RenCa cells (Fig. 5A). These results were confirmedantitative RT-PCR (Supplementary Fig. S2C). VEGFent led to VEGFR-1 phosphorylation on both cell typesB) but had no effect on CT26 or RenCa proliferationC). VEGF led to a significant increase in CT26 migration;crease was small when compared with migrationd 10% serum, and this increase was not inhibited byEGFR-1 antibody. VEGF had no effect on RenCa migra-ig. 5D). To examine other possible VEGFRs that mayte CT26 migration toward VEGF, we examined levels ofin CT26 (and RenCa) cells and found NRP-1 to be pres-

Bars, SD. *, P < 0.05, compared with VEGF group.

t at low levels compared with HUVEC or SVR mousearcoma cells (Supplementary Fig. S2C and D).

was inpleme

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and VEGFRs in ECs isolated from metastaseshe CT26 lung and liver metastasis models, inhibitionGFRs transiently blocked the growth of metastases,en with continuous treatment, metastases eventuallyto lethal sizes (data not shown). We next comparedpression of VEGFRs in CT26 lung metastases com-with normal mouse lung and found that VEGFR-1remained similar whereas VEGFR-2 levels were sig-tly lower in lung metastases compared with normalSupplementary Fig. S4A). To specifically investigater versus normal ECs, ECs were next isolated fromlung metastases and normal mouse lung (Supplemen-ig. S4B). Mouse lung EC grew in vitro as elongated,e-shaped cells, whereas EC isolated from lung metasta-peared more rounded (Supplementary Fig. S4C). West-ot analysis showed that VEGFR-1 levels were similar ino EC types, but that VEGFR-2 levels were significantlyd in lung metastasis EC compared with normal lungig. 6A). Furthermore, VEGF treatment of normal lungd to VEGFR-2 phosphorylation, whereas VEGFR-2horylation was not detectable in lung metastasis EC.al lung ECs showed significant proliferation in responseF treatment (as assessed by BrdUrd incorporation) thathibited by VEGFR-2 neutralizing antibody (Fig. 6B).trast, ECs derived from lung metastases showed a min-ncrease in proliferation in response to VEGF and mini-sponse to VEGFR-2 inhibition.g Western blot analysis, CT26 liver metastases exam-ere found to have lower levels of VEGFR-2 comparedormal liver, whereas levels of VEGFR-1 were equivalentnot shown). A decrease in VEGFR-2 expression in theature of liver metastases compared with the vascula-f normal liver was confirmed using coimmunofluores-for VEGFR-2 and PECAM-1 (Supplementary Fig. S4D).olated from CT26 liver metastases were compared withisolated from normal liver. VEGFR-1 expression wasr in both EC types (Fig. 6C). After treatment with, VEGFR-1 was less phosphorylated in liver metastasesmpared with normal liver EC. As shown previously,l liver ECs showed increased proliferation in responseGF, and this proliferation was significantly reduced byEGFR-1 antibody (Fig. 6D). In contrast, the prolifera-f liver metastasis ECs was only modestly stimulatedGF, and this proliferation was minimally inhibited byEGFR-1 antibody.

ssion

ying dependence on VEGFR-1 versus VEGFR-2 duringigenesis in different host organ environments has notell characterized. This study was initiated following anected finding in a mouse model with a targeted dele-f the calcineurin inhibitor Dscr1, which causes a defectFR-2 signaling and delayed growth of flank xenograftshen experimental lung and liver metastases were

ated in these mice, the growth of lung metastases

3. VEGFRs on isolated mouse lung and liver ECs. A, Western blotfor total andphosphorylatedVEGFR-1 andVEGFR-2.RecombinantVEGF (mVEGF) was added where indicated. β-Actin blot serves ascontrol. B, EC proliferation (using BrdUrd incorporation). VEGF,-1, and/or αVEGFR-2 were added where indicated. All data are

hibited but the growth of liver metastases was not (Sup-ntary Fig. S5). We thus hypothesized that VEGFR-1

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4. Effect of VEGFR-1 and VEGFR-2 neutralization on CT26 colon carcinoma lung and liver metastases. A, CT26 lung and liver metastaseswith VEGFR antibodies MF-1, DC101, or control IgG (n = 6 mice per group). B, mean weight of lung and livers as percentage of control. C, mean

f lung and liver metastases as percentage of control. Bars, SD. *, P < 0.05, compared with IgG control group. D, representative images followingmmunohistochemistry of lung metastases from designated treatment groups.

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signaliliver mVEGFRalonewhererequirmore,and livof VEVEGFinducefromthat dVEGFVEGFRSev

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ng may play a significant role in the vascularization ofetastases. Using neutralizing antibodies specific for-1 and VEGFR-2, we found that blockade of VEGFR-1

was sufficient to delay growth of RenCa liver metastases,as blockade of both VEGFR-1 and VEGFR-2 wased to delay growth of CT26 liver metastases. Further-in vitro analyses of ECs from human and murine lungsers revealed that liver ECs have more phosphorylationGFR-1 than VEGFR-2 in response to VEGF and thatR-1 inhibition was more effective in blocking VEGF-d liver EC functions. Finally, analysis of ECs isolatedmetastases compared with normal tissues suggestedownregulation of VEGFRs and/or independence from-mediated proliferation may account for resistance toinhibitors.

eral studies have examined organ-specific differencesGFR-1 and VEGFR-2 signaling in liver and lung dev-ent and regeneration. It has been reported that acti-of VEGFR-1 in liver ECs led to the production of

s that protected the liver parenchyma from injury and

ed liver regeneration (23). In another study, VEGFR-2 (29), a

hout addition of mVEGF (10 ng/mL). β-Actin blots serve as loading controls. C, pro, migration of CT26 and RenCa cells toward mVEGF or 10% FBS. Anti-VEGFR-1

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following partial hepatectomy; VEGFR-1 inhibitionot examined (24). Using a transgenic mouse expressingerase reporter gene under the control of the VEGFR-2ter, the highest level of VEGFR-2 activity was foundlung (25). Blockade of VEGFR-2 signaling in the peri-period disrupted lung development in mice, whereasR-1 blockade had no effect (26). Our observation ofnces in the efficacy of VEGFR inhibitors for lung andetastases are consistent with these reported organ-

ic differences.erous VEGF pathway inhibitors are currently in clin-se for patients with metastatic disease from solids. Some of these therapies, such as bevacizumab (anEGF antibody), are effective as single agents againstvascular metastases, such as those from renal cell

r (27). Bevacizumab is also effective when combinedhemotherapy for metastases for less vascular tumors,s colorectal cancer (28). The majority of the effectsacizumab against metastases have previously beenht of as a result of the inhibition of VEGFR-2 signaling

nd specific VEGFR-2 inhibitors are currently in clinical

tion had only a minor effect on liver regeneration in trials (30). However, preclinical testing of new antiangiogenic

5. VEGFR expressions by cancer cells. A, Western blot analysis for VEGFR-1 and VEGFR-2 in RenCa and CT26 cell lysates. SVR is a transformedEC line, and this cell lysate serves as a positive control. B, Western blot analysis for phosphorylated VEGFR-1 in RenCa and CT26 cells with

liferation of CT26 and RenCa cells in response to mVEGF or 10%antibody (MF-1, 0.5 μg/mL) was added where indicated.

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020. © 2010 American Association for Cancer

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does not often include examination of metastases inle different organ environments, and this is the firstto examine the effects of VEGFR-1 and VEGFR-2 inhi-against ECs from different host organs and againsttases in different host organs.h CT26 and RenCa cells express VEGFR-1, and VEGFromote CT26 migration in vivo. However, the disparateof VEGFR-1 inhibition in the liver and lung are unlike-to the effects of VEGFR-1 inhibition on cancer cellsit had no effect on CT26 migration. Moreover, the dif-ial effect of VEGFR-1 inhibition against metastases iner and lung environments was also observed for RenCawhose proliferation or migration is unaffected byR-1 inhibition. CT26 and RenCa liver metastases re-ed differently to VEGFR inhibition; RenCa cells re-ed to neutralization of only VEGFR-1 whereas CT26equired blockage of both VEGFR-1 and VEGFR-2. Theely higher levels of VEGF secreted by RenCa may selectGF-dependent EC growth, which may sensitize thesetases to VEGFR inhibition to a greater extent thanmetastases.re are a variety of mechanisms by which tumors may

e angiogenesis inhibition, including upregulation ofative angiogenic factors and pathways, development

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ablished/mature tumor vasculature, and co-option oforing normal vasculature (31). It has been shown thatng VEGFR-2 in a transgenic model of spontaneouseatic islet tumors led to an increase in hypoxia andulation not only of VEGF but also of the basic fibroblasth factor, angiopoietin 1 and other angiogenic factorse previously found that the profile of proangiogenic

s upregulated in response to overexpression of endog-angiogenesis inhibitors varied among different tumor(19). In this study, we further find that ECs in lungver metastases exhibit reduced expression of VEGFRsr adopt mechanisms for VEGF-independent growth.ould postulate that the bulk of the inhibitory effect ofR-1 or VEGFR-2 antibody on metastases occurs in thevascularization of microscopic metastases from nor-ver or lung ECs and that macroscopic metastases andECs become dependent on non-VEGF pathways toangiogenesis.re are several possible explanations why VEGFR-1a more prominent role in liver EC function and livertases, whereas VEGFR-2 plays a more prominent roleg EC function and lung metastases. For example, the

6. Tumor ECs withdownregulation andndependent growth.tern blots for total-1, total VEGFR-2, andorylated VEGFR-2 beforeer mVEGF treatment forEC, CT26 lung metastasisng metastasis EC), andells. β-Actin blot servesing control. B, proliferationng EC and lung metastasiseasured by BrdUrd

ration. C, Western blots forGFR-1 and phosphorylated-1 for m-liver ECs andver metastasis ECs (liversis EC) before andatment with mVEGF.feration of m-liver EC andtastasis EC as measuredrd incorporation. Murine

10 ng/mL), αVEGFR-10.5 μg/mL), and αVEGFR-2, 0.5 μg/mL) were addedndicated. All data arented as a percentageaddition of mVEGF alonevalue of 100%. Bars, SD.

ction of VEGFR-1 and VEGFR-2 in activating intracel-ignaling pathways such as STAT3 (33) may vary in liver

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EC veand thactivaVEGF-lung Eused emetasexperiof VEGmetasinhibiteases.to accprimaficult tof boexpresdifferVEGFRtion oECs infor pr

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rsus lung EC. Using the VEGFR-1–specific ligand PlGFe VEGFR-2 specific ligand VEGF-E, we found that PlGFted STAT3 more in liver EC than in lung EC and thatE (compared with VEGF-A) activated STAT3 more inC than in liver EC (Supplementary Fig. S6). Our studiesxperimental metastasis models and not spontaneoustasis models. The primary rationale for the use ofmental metastasis models was to examine the effectsFR-1 and VEGFR-2 inhibition on established micro-

tatic disease. The vast majority of patients receivingors of VEGF signaling have established metastatic dis-The use of spontaneous metastasis models would needount for the effects of VEGFR inhibition on both thery tumor and developing metastases, and it may be dif-o separate these two effects. In addition, the traffickingne marrow–derived cells (BMDC), some of whichs VEGFRs, into the tumor microenvironment, maybetween lung and liver metastases, and effects on-1 inhibition on BMDCs may contribute to the inhibi-f liver metastases (34). However, our analysis of liver

vitro identifies VEGFR-1 as an important receptor Rece

:639–48.nway EM, Carmeliet P. The diversity of endothelial cells: a chal-ge for therapeutic angiogenesis. Genome Biol 2004;5:207.

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s, the activity of VEGFR-1 and VEGFR-2 neutralizingdies against metastases varies based on host organnment. Knowing that there is significant heteroge-among ECs in various microvascular beds, it is logicalhe effects of VEGFR inhibition against metastases ins locations may differ. As specific VEGFR-targeteds move forward into clinical trials for the treatmentossibly prevention of tumors and metastases, differ-in the activity and efficacy of these agents in variousenvironments should be considered.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

Support

grants 5 K12 CA 87723-03 and 1 R21 CA117129-01 (S.S. Yoon). P.A.e is a Research to Prevent Blindness Senior Scientific Investigator.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 04/01/2010; revised 08/02/2010; accepted 08/22/2010; published

oliferation, migration, and tube formation. OnlineFirst 10/26/2010.

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2010;70:8357-8367. Cancer Res   Yoon-Jin Lee, Daniel L. Karl, Ugwuji N. Maduekwe, et al.   Tumor Metastases Based on Host Organ EnvironmentDifferential Effects of VEGFR-1 and VEGFR-2 Inhibition on

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