Anti-VEGF agents confer survival advantages totumor-bearing mice by improving cancer-associatedsystemic syndromeYuan Xuea, Piotr Religaa, Renhai Caoa, Anker Jon Hansenb, Franco Lucchinic, Bernt Jonesd, Yan Wue, Zhenping Zhue,Bronislaw Pytowskie, Yuxiang Liangf, Weide Zhongf, Paolo Vezzonig,h, Björn Rozelli, and Yihai Caoa,1

aDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden; bBiopharmaceuticals Research, Novo Nordisk A/S, DK-2760Malov, Copenhagen, Denmark; cCentro Ricerche Biotecnologiche and Istituto di Microbiologia, Universita’ Cattolica del Sacro Cuore, 26100 Cremona, Italy;dDepartment of Clinical Sciences, Faculty of Veterinary Medicine and Animal Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden; eImCloneSystems Incorporated, 108 Varick Street, New York, NY 10014; fDepartment of Urology, First Hospital of Guangzhou, People�s Republic of China; gIstituto diTecnologie Biomediche, Consiglio Nazionale delle Ricerche, 20090 Segrate, Italy; iIstituto Clinico Humanitas IRCCS, via Manzoni 56, Rozzano, Italy; andhDepartment of Laboratory Medicine, Karolinska University Hospital, Huddinge, Karolinska Institute, SE141 86 Stockholm, Sweden

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved September 25, 2008 (received for review August 12, 2008)

The underlying mechanism by which anti-VEGF agents prolong cancerpatient survival is poorly understood. We show that in a mouse tumormodel, VEGF systemically impairs functions of multiple organs includ-ing those in the hematopoietic and endocrine systems, leading toearly death. Anti-VEGF antibody, bevacizumab, and anti-VEGF recep-tor 2 (VEGFR-2), but not anti-VEGFR-1, reversed VEGF-induced cancer-associated systemic syndrome (CASS) and prevented death in tumor-bearing mice. Surprisingly, VEGFR2 blockage improved survival byrescuing mice from CASS without significantly compromising tumorgrowth, suggesting that ‘‘off-tumor’’ VEGF targets are more sensitivethan the tumor vasculature to anti-VEGF drugs. Similarly, VEGF-inducedCASS occurred in a spontaneous breast cancer mouse model overex-pressing neu. Clinically, VEGF expression and CASS severity positivelycorrelated in various human cancers. These findings define novel ther-apeutic targetsofanti-VEGFagentsandprovidemechanistic insights intothe action of this new class of clinically available anti-VEGF cancer drugs.

angiogenesis � antiangiogenic therapy � cancer syndrome �tumor growth � VEGF

Although various anti-VEGF agents delivered as monotherapydisplay significant anti-cancer effects in different experimentaltumor models, their therapeutic efficacies in clinical settings havebeen often evaluated as adjuvant therapies to chemotherapeuticagents (1, 2). In contrast to mouse tumor studies in which tumormasses are monitored, the clinically therapeutic benefits are mainlydetermined based on prolonged survival time of cancer patients (1,2). Intriguingly, anti-angiogenic drugs approved on the basis of asurrogate marker of tumor size do not always reduce mortality (3).

The underlying mechanisms by which VEGF antagonists confersurvival advantages to cancer patients have not been fully eluci-dated. In combinatorial therapy regimens, anti-VEGF agents mightmodulate the efficacy of chemotherapeutic agents by normalizationof tumor blood vessels (4). Most preclinical and clinical studies ofanti-VEGF agents have focused on tumor vasculature or tumorgrowth, and little is known about the systemic effects of thesetherapeutic agents in the body. Most cancer patients at the ad-vanced stage of disease encounter cancer-associated systemic syn-drome (CASS), which significantly impairs the quality of life andshortens lifespan. Clinical manifestation of CASS includes a broadspectrum of symptoms including defective hematopoiesis, endo-crine system, ascites, GI track disorders, muscular and adiposeatrophy, and functional impairment of liver, spleen, and kidney (5).Here, we report that tumor-produced VEGF had extensivelydestructive effects on multiple organs/tissues in mice and that ananti-VEGF receptor 2 (VEGFR-2) agent significantly prolongedmouse life time by improving CASS. A similar correlation betweenVEGF expression and CASS has also been detected in patients withvarious cancers.

ResultsTumor-Derived VEGF Induced CASS in Immuno-Competent and -Defi-cient Mice. Tumor-derived VEGF induces CASS affecting multipletissues and organs in both immunocompetent and immunodeficientmice. See supporting information (SI) Text and Figs. S1–S4 fordetailed results.

To define the threshold level at which VEGF induced CASS,different ratios of vector- and VEGF-transfected tumor cells weremixed to create a series of in vivo tumors expressing different levelsof VEGF in the in vivo tumors. At a serum concentration of VEGFof 1.2 ng/ml, CASS was clearly manifested in liver, spleen, bonemarrow (BM) and adrenal gland (Fig. 1B, Fig. S5). In contrast, 0.8ng/ml of serum VEGF did not result in any obvious CASS pheno-types, indicating that approximately 1 ng/ml of serum VEGF wasthe threshold level required to cause CASS in this particularxenograft tumor model. Similar results were seen in mice bearinganother VEGF-overexpressing tumor type, Lewis lung carcinomatumors (Fig. S6). These findings show that the tumor-producedVEGF affects multiple healthy organs in mice.

Vascular Phenotypes. Immunohistochemical analysis of xenografttumor models using anti-CD31 antibody showed that blood vesselsin the liver, spleen, BM, and adrenal cortex of VEGF tumor-bearingmice appeared as primitive and dilated sinusoidal vascular struc-tures, which consisted of disorganized, tortuous, and intercon-nected vascular plexuses (Fig. 1A). Quantification analysis showedthat although the vessel density in the spleen was remarkablyincreased, the total vessel density in the liver was significantlydecreased (Fig. 1 C and D). In addition to the cortex, the adrenalmedulla developed a high density of vascular plexuses (Fig. 1A).Consistent with structural alterations of the adrenal cortex, theserum corticosterone level in VEGF tumor-bearing mice wasconsequentially reduced (Fig. 1E). The reduction of corticosteronelevels was reminiscent of hypoadrenocorticism found in Addison’sdisease (6).

Hepatic Necrosis, Apoptosis Endothelial Cells (ECs) in the SinusoidalBlood Vessels. The pathological hepatic changes induced by thetumor-produced VEGF led to regional necrosis in the liver tissue

Author contributions: Y.X., P.R., R.C., and Y.C. designed research; Y.X., P.R., R.C., F.L., Y.L.,W.Z., and B.R. performed research; Y.X., P.R., R.C., A.J.H., F.L., B.J., Y.W., Z.Z., B.P., Y.L., W.Z.,P.V., B.R., and Y.C. analyzed data; A.J.H., F.L., B.J., Y.W., Z.Z., B.P., P.V., and B.R. contributednew reagents/analytic tools; and Y.X. and Y.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: yihai.cao@ki.se.

This article contains supporting information online at www.pnas.org/cgi/content/full/0807967105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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(Fig. S7A). Ki67, a proliferating marker, staining showed that ECsin the hepatic sinusoidal blood vessels were actively proliferating(Fig. S7B). There was an approximately sixfold increase in prolif-erating ECs in the liver of VEGF tumor-bearing mice as comparedwith the control group (Fig. S7D). TUNEL staining showed anapproximately sixfold increase in hepatocyte death in the VEGFgroup compared with the vector control (Fig. S7 B and E). Thesefindings demonstrate that VEGF-induced hyperproliferation ofECs in the sinusoidal blood vessels leads to an elevated apoptoticrate accompanied by liver necrosis.

The increased death rate of hepatocytes might trigger an inflam-matory response by infiltration of high numbers of macrophages.F4/80 staining showed a �3-fold increase of macrophages in hepatictissues and a 2-fold increase in spleen (Fig. S7 B, C, and H).

Impairment of Liver Function. VEGF-induced hepatic tissue damageresulted from the high rate of hepatocyte apoptosis and necrosis;the expansion of sinusoidal blood vessels also suggested impairmentof liver function. Indeed, measurement of serum transaminasesshowed that levels of alanine transaminase (ALT) and asparatetransaminase (AST) were already considerably elevated at day 14after tumor implantation in VEGF tumor-bearing mice (Table S1).In contrast, serum levels of other parameters reflecting hepaticfunction such as cholesterol and albumin remained unchangedduring the entire 14-day period, probably due to the highly com-pensatory capacity of the remaining hepatocytes (Table S1).

Severe Anemia in VEGF Tumor-Bearing Mice. Depletion of hemato-poietic cells from BM suggested an anemic phenotype in VEGFtumor-bearing mice. Gross examination of these mice revealed asevere anemic phenotype, which manifested as considerable pale-ness of several hairless regions of the mouse body, including thepaws, mouth, nose, and genitals (Fig. S1C). Hematological analysisof the peripheral blood showed a significant decrease in hematocritin both immunocompetent and immunoincompetent mice at day 14after VEGF tumor implantation (Table S2). The level of hemo-globin and the number of erythrocytes in the peripheral blood weresignificantly decreased (Table S2). These results showed thatVEGF tumor-bearing mice suffered from a severe anemia. Inaddition, the total number of white blood cells was also significantlydecreased, suggesting defective myelogenesis (Table S3). Takentogether, depletion of BM hematopoietic cells and decreasednumbers of red blood cells and white blood cells demonstrate thattumor-produced VEGF results in severe anemia in mice.

Extramedullary Hematopoiesis and Mobilization of BM Cells. Hepa-tomegaly and splenomegaly, as well as infiltration of hematopoieticcells, suggested that in VEGF-expressing tumor-bearing mice ex-hibited active extramedullary hematopoiesis occurred in theseorgans. Immunohistochemical analysis with a specific anti-erythroblast antibody (Ter119) demonstrated a high density oferythroblasts and reticulocytes in the liver and spleen tissues ofVEGF tumor-bearing mice as compared with those of control mice.These erythroblasts formed clusters, which appeared as hemato-poietic islets (Fig. S7 B, C, F, and G). BM transplantation ofsyngeneic EGFP� cells to irradiated recipient mice showed signif-icant mobilization of GFP� BM-derived cells to the liver and spleentissues (Fig. S8 A and B). These findings demonstrate that activeextramedullary hematopoiesis occurs in livers and spleens of mice withVEGF-expressing tumors, which mobilized BM cells to these sites.

Consistent with extramedullary hematopoiesis, plasma levels oferythropoietin (EPO) were significantly elevated in VEGF tumor-bearing mice (Fig. S8C). Surprisingly, high levels of EPO wereunable to initiate active BM hematopoiesis, suggesting a defectiveresponse of BM hematopoietic cells to EPO. Although a significantdecrease in circulating soluble VEGFR-2 was detected, levels ofsoluble VEGFR-1, TNF-� and IL-6 were unchanged in VEGFtumor-bearing mice (Fig. S8 D–F).

Tissue Hypoxia and Vascular Permeability. To study vascular func-tions in CASS-affected tissues, tissue hypoxia and vascular perme-ability were measured in various tissues using a Hypoxia Probe kit.Hypoxic regions were unevenly distributed throughout VEGF andvector control tumors (Fig. S9B). In contrast, the entire hepatictissue of VEGF tumor-bearing mice was exposed to severe hypoxia,whereas hypoxia was only detectable around a tiny area of thecentral vein in control vector tumor-bearing mice (Fig. S9B).Similarly, the cortex of the adrenal gland was also exposed to severehypoxia in VEGF tumor-bearing mice and tissue hypoxia wasundetectable in the cortex, except for low-level hypoxia in themedulla of control vector tumor-bearing mice (Fig. S9E); BM ofVEGF tumor-bearing mice exhibited a high level of hypoxiathroughout the entire tissue (Fig. S9D). Interestingly, the spleenshowed undetectable levels of hypoxia in VEGF or control vectortumor-bearing mice (Fig. S9C). Vascular permeability was increased intumors and livers of VEGF tumor-bearing mice (Fig. S9F). Thesefindings suggest that VEGF induced abnormal vessels in the affectedtissues and organs that are highly permeable and lack appropriate bloodperfusion, although they contain a high number of microvessels.

Expression of VEGFR-1 and VEGFR-2 in Various Organs. The formationof aberrant sinusoidal vasculature in various organs suggested thatVEGFRs are expressed in blood vessels in the tissues. Immuno-histochemical analysis was performed using two specific antibodiesagainst mouse VEGFR-1 (MF1) and VEGFR-2 (TO14). Blood

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Fig. 1. Vascular alterations in various organs. (A) Microvascular networks inliver, spleen, adrenal gland, and BM were revealed by immunohistochemicalstaining with anti-CD31. Arrows point to sinusoidal blood vessels. (B) Vascularnetworks in tumor, liver, and BM from the circulating levels of 0.8 ng/ml and 1.2ng/ml VEGF in mice were compared. (C and D) Vascular areas were quantified bymeasuring CD31-positive signals and the mean values are presented (� SD). (E)Blood corticosterone levels were measured on day 14 after tumor implantation.Cx � cortex; M � medulla. (Scale bars in A and B, 50 �m.)

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vessels in the hepatic tissue of nontumor- and tumor-bearing miceexpressed high levels of both VEGFR-1 and VEGFR-2 (Fig. S10A).Although the expression patterns of VEGFR-1 and VEGFR-2 inthe liver vasculature almost completely overlapped in nontumor-and tumor-bearing mice, VEGFR-2 was expressed at a higher leveland in a broader spectrum of vascular networks than VEGFR-1. Inspleen, VEGFR-2 expression was significantly higher in VEGFtumor-bearing mice as compared with control mice and VEGFR-1was barely detectable in the blood vessels (Fig. S10B). WhereasVEGFR-2 was mainly expressed in blood vessels, VEGFR-1 wasexpressed on non-ECs in the adrenal gland of VEGF tumor-bearingmice (Fig. S10C). Notably, VEGF induced accumulation ofVEGFR-1-positive cells in the cortex and medulla of the adrenalgland (Fig. S10C). BM showed an overlapping distribution ofVEGFR-1 and VEGFR-2 (Fig. S10D). Tumor blood vessels ex-pressed high levels of both VEGFR-1 and VEGFR-2, althoughVEGF tumors exhibited a significantly higher density of overlap-ping VEGFR-1 and -2 positive signals. These findings providemolecular targets of the tumor VEGF-induced CASS.

Reversal of VEGF-Induced CASS by Anti-VEGFR-2 but Not by Anti-VEGFR-1. To investigate whether anti-VEGF agents could reverseVEGF-induced CASS in VEGF tumor-bearing mice and to define

receptor signaling pathways involved in the development of CASS, twospecific neutralizing anti-mouse VEGFR-1 (MF1) and VEGFR-2(DC101) monoclonal antibodies at a low dose (800 �g/mouse) and ahigh dose (1600 �g/mouse) were administered to the tumor-bearingmice. After a 12-day treatment, the VEGF-induced tissue damage,including pathological changes in the liver, spleen, adrenal gland, BM,anemia, and ascites, could be completely prevented by the DC101antibody at both low and high doses (Fig. 2A and C). The liver andspleen weights were significantly reduced and reverted almost to thoseofnontumor-bearingmice(Fig.3EandF).Thedilatedsinusoidalbloodvessels in the liver appeared normal (Fig. 2C). Consistent with thesehistological changes, hematocrit, hemoglobin, erythrocytes, and liverfunction were all normalized by the anti-VEGFR-2 neutralizing anti-body (Tables S1–3). Surprisingly, anti-VEGFR-2 at the effective dosefor normalization of systemic tissues and organs did not show asignificant antitumor effect in our model (Fig. 2B). However, anincreased dose of anti-VEGFR-2 showed remarkable antitumor activ-ity and complete reversal of VEGF-induced CASS (Fig. 2B, C). Incontrast to anti-VEGFR-2, treatment with anti-VEGFR-1 at the samedose produced no effects on VEGF-induced systemic syndrome ortumor growth, demonstrating that VEGFR-1 is not the primary target

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Fig. 2. VEGF blocking and prolongation of survivals(A) At day 14 after treatment with MF1 and DC101, arepresentative mouse of each group was photo-graphed. Arrows point to nose/mouth and paws. As-terisks mark the abdomens of mice. (B) Tumor volumeswere measured at the indicated times to determinetumor growth rates. (D) The percentage of survivalanimals in each group is presented during a 15-day-treatment course. (E and F) After killing of animals onday 15 after treatment, livers and spleens wereweighed and mean values are presented. (C) At thesame time point, liver, spleen, adrenal gland, and BMof buffer-treated, MF1-treated, and DC101-treatedmice (n � 8/group) were stained with H&E (top foursets of images). PA � portal area; RP � red pulp; WP �white pulp; Cx � cortex; and M � medulla. Vascularnetworks in tumors and livers were revealed by stain-ing with a CD31 antibody (bottom two sets of images).(Scal bar, 50 �m.) (G) CD31 positive signals were quan-tified in tumor tissues. (H) VEGF tumors were allowedto grow into sizes of 0.8 cm3, followed by treatmentwith bevacizumab for 10 days. The mouth/nose andpaws from a representative mouse of each group wasphotographed. (I-K) Tumor growth rates, liver weight,and spleen weight were measured. (L) The percentagesof survival animals in bevacizumab- versus buffer-treated groups were presented during a 19-day exper-imental period.

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for CASS (Fig. 2 A–C, Tables S1–3). These findings demonstrate thatnontumor vasculature was more susceptible than tumor vasculature toanti-VEGF agents and that VEGFR-2-mediated signaling is crucial forcausing the systemic damage of multiple tissues and organs.

Improvement of Survival by Anti-VEGF Agents. Despite thefact that theanti-VEGFR-2 neutralizing antibody remarkably prevented the sys-temic VEGF syndrome, surprisingly, the tumor growth rate was notaffected by this treatment. Consistent with this finding, tumor bloodvessels were unaffected by this treatment (Fig. 2 C and G). Strikingly,the anti-VEGFR-2 treatment at an effective dose significantly im-proved the lifetime of VEGF tumor-bearing mice (Fig. 2D). These datashow that an anti-VEGFR-2 neutralizing agent at an optimally low dosecould significantly prolong the lifetime of VEGF tumor-bearing micewithout significantly compromising tumor growth.

Treatment with the humanized anti-VEGF antibody bevaci-zumab was evaluated to further validate the survival advantage ofanti-VEGF agents by improving CASS. At day 16 after tumorimplantation, approximately 50% of nontreated VEGF-expressingtumor-bearing mice (n � 8) died of CASS and the experiments hadto be terminated at the endpoint determined by ethical consider-ations (tumor volume �1.5 cm3) (Fig. 2I). At 5 mg/kg, bevacizumabsignificantly delayed the tumor growth rate (Fig. 2I). Interestingly,none of the bevacizumab-treated mice (n � 8) died during theprolonged period of experimentation (Fig. 2 I and L). Improvementof survival by bevacizumab was not due to suppression of tumorgrowth because none of the bevacizumab-treated mice died evenwhen the tumor reached the ethically determined endpoint (volume�1.5 cm3). These findings suggest that bevacizumab may prolongsurvival by improving CASS. Indeed, VEGF-induced anemia andhepatosplenomegaly were significantly improved by bevacizumab (Fig.2 H, J, and K). These data confirmed the survival advantage of theVEGFR-2 blockage by improving CASS, not by tumor inhibition per se.

VEGF-Induced CASS in a Spontaneous Mouse Tumor Model. To studythe physiopathological relevance of our findings, a spontaneous tumormodel of a transgenic mouse line overexpressing the neu oncogeneunder the tissue-specific promoter of the mouse mammary tumor virus(MMTVneu) was used (7). Female CD1 mice carrying the neu onco-gene developed mammary tumors at the age of approximately twomonthsandthe tumorsgrewtoarelatively largesizeduring thenext twomonths. Strikingly, gross examination of these mice showed pale paws,suggesting that MMTVneu tumor-bearing mice suffered from anemia(Fig. 3A). Hematological analysis confirmed the severe anemic pheno-type, showing significantly reduced levels of hemoglobin, hematocrit,and erythrocytes in peripheral blood (Fig. 3 G–I). Similar to theVEGF-overexpressing xenograft tumor model, MMTVneu tumor-bearing mice also showed hepatosplenomegaly (Fig. 3 C–E). Histolog-ical analysis demonstrated that a high density of sinusoidal vasculaturefilled the entire liver and the adrenal cortex tissues (Fig. 3B). Indeed,anti-CD31 staining showed that the vasculature in the liver and adrenalgland of MMTVneu tumor-bearing mice mainly consisted of dilatedsinusoidal microvessels (Fig. 3B). In the spleen, margins of white pulp(WP) and red pulp (RP) disappeared and were replaced by expandinghematopoietic red pulp (Fig. 3B). In addition, the average tumor-freebody weight of the MMTVneu transgenic mice was significantly de-creased compared to that of wild-type mice (Fig. 3F). Consistent withdevelopment of CASS, the circulating VEGF level was also significantlyelevated in MMTVneu tumor-bearing mice (Fig. 3J). Remarkably,treatment of these spontaneous tumor-bearing mice with DC101 at thelow dose twice/wk almost completely reversed the systemic syndromeincludinganemiaandhepatosplenomegaly (Fig.3AandC–I).Similarly,histology of the liver, spleen and adrenal gland, and vascular networksin theseorgansshowedthat theywerecompletelynormalizedbyDC101treatment (Fig. 3B). It should be noted that there was a trend of tumorgrowth inhibition by DC101 treatment, although the spontaneoustumor sizes were heterogeneous and thus difficult to quantify. These

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Fig. 3. CASS in a spontaneous mouse tumor model.Spontaneous mammary tumors developed in MMTV-neu transgenic mice at 2-month age and mice werekilled when they reached 4 months old. One group ofmice (n � 6) received the anti-VEGFR-2 treatment at adose of 800 �g/mouse. Paws (A), liver and spleen (C)were photographed. (B) Liver, spleen, and adrenalgland were evaluated by H&E staining (top three setsof images). The arrow indicates a hematopoietic isletin the liver tissue. Arrowheads indicate dilated sinu-soidal blood vessels. Tissue sections of liver and adre-nal gland were stained with anti-CD31 (bottom twosets of images). CV, central vein; RP, red pulp; WP,white pulp; Cx, cortex; M, medulla. (Scale bars, 50 �m.)Liver weight (D), spleen weight (E), and net bodyweight (F) were measured. Blood samples were col-lected and hemoglobin (G), hematocrit (H), and eryth-rocytes (I) were determined. (J) The serum levels ofVEGF in various groups of mice were measured using asensitive ELISA.

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findings demonstrate that a spontaneous tumor mouse model that wasnotgeneticallypropagatedtooverexpressVEGFalsodevelopedCASS,which was correlated with an elevated level of circulating VEGF andreversed by an anti-VEGFR-2 agent.

Correlation of Circulating VEGF Levels with Development of CASS inHuman Cancer Patients. To further correlate our findings with clinicalrelevance, we analyzed blood samples derived from cancer patients. Weused exactly the same ELISA method for our mouse experiments tomeasure the circulating levels of VEGF. Interestingly, we found that thecirculating VEGF level was significantly higher in prostate and bladdercancer patients and renal cell carcinoma (RCC) patients as comparedwith healthy individuals (Fig. 4A). The circulating VEGF levels in thesepatients (average of 1–1.5 ng/ml) were in a range similar to those foundin our xenograft mouse tumor model (Fig. 1B, Fig. S5). Interestingly,RCC patients had slightly higher VEGF levels than prostate andbladder cancer patients. Circulating VEGF levels correlated well withseverity of hepatomegaly, splenomegamy and ascites (Fig. 4 B, D, andE). Consistent with this positive correlation, liver tissues in patients withhigh VEGF levels showed sinusoidal dilation of vascular networks andimpaired functions, including high levels of ALT and AST, and lowlevels of albumin (Fig. 4 C and G–I). Again, RCC patients showed asignificantly positive correlation between VEGF level and impairmentof liver function (Fig. 4 K–M). In contrast, hemoglobin levels weresignificantly decreased and reversely correlated with the circulatingVEGF levels in these cancer patients, particularly in RCC patients (Fig.4 F and J). These findings demonstrate an equivalent circulating VEGFlevel between human cancer patients and VEGF tumor-bearing mice.Furthermore, VEGF levels were positively correlated with the severityof CASS in human cancer patients.

DiscussionHere, we show that tumor-produced VEGF induces CASS by damag-ing the structures and functions of multiple tissues and organs. VEGF-induced CASS was manifested as severe anemia, hepatic dysfunction

and necrosis, ascites, loss of body weight, and low serum levels ofcorticosterone. The severity of these systemic changes was generallywell correlatedwith thecirculatingVEGFlevel inbothmiceandhumancancer patients. VEGF-induced CASS resembles cancer cachexia andparaneoplastic syndromes, which manifests functional failures of mul-tiple organs often at an advanced stage of the malignancy (8). Cancercachexia and paraneoplastic syndrome are the primary causes ofmortality in cancer patients. Although a few cytokines including TNF-�and IL-6 contribute to CASS, its underlying molecular mechanismsremain unknown (8, 9). Our present study provides a novel mechanisticinsight into the role of tumor-derived VEGF in the development ofCASS.

In the xenograft VEGF tumor model, we were able to determine thethreshold level of VEGF that causes CASS by mixing VEGF-producingand vector-transfected tumor cells in different ratios. Similar circulatingVEGF levels were detected in various cancer patients including RCC,prostate cancer and bladder cancer patients. In fact, the averagecirculating VEGF level in RCC patients was approximately 1.5 ng/ml.Intriguingly, most of these cancer patients developed obvious CASSincluding severe anemia, hepatosplenomegaly, and ascites. These clin-ical data correlated well with our VEGF tumor model in mice. It isestimated that at the time of diagnosis, the rate of CASS is �7–10% ofpatients with malignancy and that as many as 50% of all cancer patientsmayexperiencesuchasyndromeat sometimeduring thecourseof theirillness (5). Similar to our present findings in mouse tumors and humanpatients, autopsies of RCC patients revealed that �20% of patients hadsinusoidal dilation in the liver, spleen, and adrenals (10). The sinusoidaldilation of these organs is considered to be a nonmetastatic tumor-specific manifestation, although the etiology remains unclear. It shouldbe mentioned that VEGF-induced vascular leakage might be involvedin the axis of the reactive oxygen-rac-angiopoietin-2 pathway (11).

CASS is defined as a constellation of symptoms in associationwith the presence of an actively growing tumor that releases anunknown factor in excess into the circulation. The identity of thisunknown factor has not been characterized. Our present study with

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Fig. 4. CASS in human cancer patients.Clinical samples were collected from RCC,bladder and prostate cancer patients. (A)Circulating levels of VEGF were measuredby ELISA. (C) Histological micrographs ofH&E and CD31 immunohistochemicalstaining of livers from RCC patients at ahigh (1.2 ng/ml) and a low (0.3 ng/ml) cir-culating VEGF level are presented. (Scalebars, 50 �m.) The development of hepato-megaly (B) and splenomegaly (D) were cor-related with circulating VEGF levels. (E) Per-centage of patients with ascites wascorrelated with the average circulatingVEGF level. Levels of blood hemoglobin (F),ALT (G), AST (H), and albumin (I) were mea-sured and correlated with circulating VEGFexpression levels (J–M). HI, healthy individ-uals; PC, prostate cancer patients; BC, blad-der cancer patients; RCC, renal cell carci-noma patients. Statistic analyses wereindicated as in figures.

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the VEGF-expressing tumor model in mice resembles an equivalentsituation in cancer patients possessing advanced VEGF-secretingtumors and demonstrates that the unknown factor is likely to beVEGF. In addition to RCC, sinusoidal dilation of tissues has alsobeen observed in other tumors. For example, nine patients withdifferent cancers including Hodgkin’s disease, stomach cancer,lymphosarcoma, and gastric carcinoma had dilation of hepaticsinusoidal blood vessels (12). A similar systemic syndrome is alsopresent in patients with malignant histiocytosis, pediatric Wilm’stumor, and pancreatic cancers (13–15). The percentage of patientswith highly dilated hepatic sinusoidal blood vessels is probably muchhigher than that reported in the literature because almost all caseswere encountered at autopsy, which is not routinely performed.

Intriguingly, high VEGF levels induced CASS in both Lewis lung andfibrosarcoma models, suggesting that the VEGF-induced systemiceffect is independent of tumor type. Indeed, continuous injection ofpurified VEGF protein into nontumor-bearing mice could also causehepatosplenomegaly in mice (Fig. S11). In addition to high VEGF-producing tumor models, a spontaneous breast cancer mouse model,which is not genetically propagated to express VEGF, also developeda similar systemic syndrome as manifested by severe anemia, hepato-splenomegaly, ascites, and loss of body weight. The circulating VEGFlevels in these spontaneous tumor-bearing mice were lower than that ofmice with VEGF xenograft tumors, and the development and growthrate of these spontaneous tumors were considerably slower than forxenograft tumors. VEGF may accumulate in various tissues and organsover a relatively long period of tumor development. Persistent exposureof these organs to VEGF might result in initiation of vascular growthand impairment of vascular function. Indeed, vascular networks in liver,spleen, and adrenal glands of spontaneous tumor-bearing mice exhib-ited a high degree of disorganization, dilation, and tortuous architec-ture. Importantly, anti-VEGFR-2 could completely reverse vascularabnormalities and tissue structures in MMTVneu tumor mice. Takentogether, this finding demonstrates that VEGF plays an important rolein initiation, progression and maintenance of CASS in spontaneoustumor-bearing mice.

Surprisingly, BM hematopoietic cells were virtually completelyeradicated by VEGF in mice. Due to a lack of a sufficient numberof hematopoietic stem cells in BM, both red blood cells and whiteblood cells in the peripheral blood were dramatically decreased.Development of anemia is unlikely due to the direct inhibitoryeffect of VEGF on hematopoiesis because extramedullary hema-topoiesis in the liver and spleen was stimulated by VEGF.

Overall, our studies demonstrate that in both xenograft and spon-taneous tumor-bearing mice, tumor-expressed VEGF induces CASS,which resembles cachexia and paraneoplastic syndromes in human

cancer patients. Circulating VEGF levels correlated well with CASSseverity in tumor-bearing mice and human cancer patients. We suggestthat nontumor tissues are important therapeutic targets for improve-ment in cancer patient survival. The functional and pathologicalchanges in tissues and organs might serve as useful noninvasive markersfor the effectiveness of anti-VEGF therapy in improving cancer patientsurvival rates. Thus, these results provide molecular insight into theglobal impact of tumor-produced VEGF in cancer patients and suggestthat combinatorial therapies of anti-VEGF agents with other drugs toimprove tissue and organ function will produce immense benefits forcancer patients.

Experimental ProceduresAnimals, Human Materials, and Mouse Tumor Model. All animal studies werereviewed and approved by the animal care and use committees of the local animalboard. All human studies were approved by the Chinese Medical Information Com-mittee. Detailed methods and criteria of patient selection are described in SI Text.

Tissue and Organ Collection, ELISA, and Blood Sample Analysis. See SI Text fordetails.

Tissue Hypoxia Analysis and Vascular Permiability Assay. Tissue hypoxia in tumortissues, liver, spleen, BM, and adrenal glands was measured according to a standardprotocol using HypoxyprobeTM-1 Plus kit (Chemicon). See SI Text for details.

Bone Marrow Transplantation and Tumor Implantation. See SI Text for details.

Histological Studies, Whole-Mount Staining and Immunofluorescent Staining.Malignant and nonmalignant paraffin-embedded tissues were sectioned in 5 �mthickness and stained with hematoxylin-eosin (H&E) according to our previouslydescribed methods (18). Paraffin sections of BM tissues were stained with theanti-mouse CD31 antibody and positive signal were developed using DAB as thesubstrate. Whole-mount staining was performed according to previously pub-lished methods (19). See SI Text for details.

Statistical Analysis. Statisticalanalysiswasperformedusingthestudent’s t testbyaMicrosoftExcelprogram.Datawerepresentedasmeansofdeterminants (�SD)and p-values � 0.05 were considered as statistically significant. The Kaplan-Meiersurvival curve was generated using Statistica 5.0 (Statsoft).

ACKNOWLEDGMENTS. We thank Dr. Rolf Brekken at the University of TexasSouthwestern Medical Center for supplying the anti-VEGFR-2 polyclonal antibody.This work was supported by the laboratory of Y.C. through research grants from theSwedish Research Council, the Swedish Heart and Lung Foundation, the SwedishCancerFoundation,theKarolinskaInstituteFoundation,andtheTorstenandRagnarSöderberg’s Foundation and by European Union Integrated Projects of Angiotar-geting Contract 504743 (to Y.C.) and VascuPlug Contract STRP 013811 (to Y.C.), andsupported in part by a grant from Fondazione Cariplo (N.O.B.E.L Project) (to P.V.).

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