Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome

Yuan Xue; Piotr Religa; Renhai Cao; Anker Jon Hansen; Franco Lucchini; Bernt Jones; Yan Wu; Zhenping Zhu; Bronislaw Pytowski; Yuxiang Liang; Weide Zhong; Paolo Vezzoni; Björn Rozell; Yihai Cao

doi:10.1073/pnas.0807967105

New Research In

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

Abstract

The underlying mechanism by which anti-VEGF agents prolong cancer patient survival is poorly understood. We show that in a mouse tumor model, VEGF systemically impairs functions of multiple organs including those in the hematopoietic and endocrine systems, leading to early death. Anti-VEGF antibody, bevacizumab, and anti-VEGF receptor 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 by rescuing mice from CASS without significantly compromising tumor growth, suggesting that “off-tumor” VEGF targets are more sensitive than the tumor vasculature to anti-VEGF drugs. Similarly, VEGF-induced CASS occurred in a spontaneous breast cancer mouse model overexpressing neu. Clinically, VEGF expression and CASS severity positively correlated in various human cancers. These findings define novel therapeutic targets of anti-VEGF agents and provide mechanistic insights into the action of this new class of clinically available anti-VEGF cancer drugs.

Although various anti-VEGF agents delivered as monotherapy display significant anti-cancer effects in different experimental tumor models, their therapeutic efficacies in clinical settings have been often evaluated as adjuvant therapies to chemotherapeutic agents (1, 2). In contrast to mouse tumor studies in which tumor masses are monitored, the clinically therapeutic benefits are mainly determined based on prolonged survival time of cancer patients (1, 2). Intriguingly, anti-angiogenic drugs approved on the basis of a surrogate marker of tumor size do not always reduce mortality (3).

The underlying mechanisms by which VEGF antagonists confer survival advantages to cancer patients have not been fully elucidated. In combinatorial therapy regimens, anti-VEGF agents might modulate the efficacy of chemotherapeutic agents by normalization of tumor blood vessels (4). Most preclinical and clinical studies of anti-VEGF agents have focused on tumor vasculature or tumor growth, and little is known about the systemic effects of these therapeutic agents in the body. Most cancer patients at the advanced stage of disease encounter cancer-associated systemic syndrome (CASS), which significantly impairs the quality of life and shortens lifespan. Clinical manifestation of CASS includes a broad spectrum of symptoms including defective hematopoiesis, endocrine system, ascites, GI track disorders, muscular and adipose atrophy, and functional impairment of liver, spleen, and kidney (5). Here, we report that tumor-produced VEGF had extensively destructive effects on multiple organs/tissues in mice and that an anti-VEGF receptor 2 (VEGFR-2) agent significantly prolonged mouse life time by improving CASS. A similar correlation between VEGF expression and CASS has also been detected in patients with various cancers.

Results

Tumor-Derived VEGF Induced CASS in Immuno-Competent and -Deficient Mice.

Tumor-derived VEGF induces CASS affecting multiple tissues and organs in both immunocompetent and immunodeficient mice. See supporting information (SI) Text and Figs. S1–S4 for detailed results.

To define the threshold level at which VEGF induced CASS, different ratios of vector- and VEGF-transfected tumor cells were mixed to create a series of in vivo tumors expressing different levels of VEGF in the in vivo tumors. At a serum concentration of VEGF of 1.2 ng/ml, CASS was clearly manifested in liver, spleen, bone marrow (BM) and adrenal gland (Fig. 1B, Fig. S5). In contrast, 0.8 ng/ml of serum VEGF did not result in any obvious CASS phenotypes, indicating that approximately 1 ng/ml of serum VEGF was the threshold level required to cause CASS in this particular xenograft tumor model. Similar results were seen in mice bearing another VEGF-overexpressing tumor type, Lewis lung carcinoma tumors (Fig. S6). These findings show that the tumor-produced VEGF affects multiple healthy organs in mice.

Fig. 1.

Vascular alterations in various organs. (A) Microvascular networks in liver, spleen, adrenal gland, and BM were revealed by immunohistochemical staining with anti-CD31. Arrows point to sinusoidal blood vessels. (B) Vascular networks in tumor, liver, and BM from the circulating levels of 0.8 ng/ml and 1.2 ng/ml VEGF in mice were compared. (C and D) Vascular areas were quantified by measuring 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 μm.)

Vascular Phenotypes.

Immunohistochemical analysis of xenograft tumor models using anti-CD31 antibody showed that blood vessels in the liver, spleen, BM, and adrenal cortex of VEGF tumor-bearing mice appeared as primitive and dilated sinusoidal vascular structures, which consisted of disorganized, tortuous, and interconnected vascular plexuses (Fig. 1A). Quantification analysis showed that although the vessel density in the spleen was remarkably increased, the total vessel density in the liver was significantly decreased (Fig. 1 C and D). In addition to the cortex, the adrenal medulla developed a high density of vascular plexuses (Fig. 1A). Consistent with structural alterations of the adrenal cortex, the serum corticosterone level in VEGF tumor-bearing mice was consequentially reduced (Fig. 1E). The reduction of corticosterone levels was reminiscent of hypoadrenocorticism found in Addison's disease (6).

Hepatic Necrosis, Apoptosis Endothelial Cells (ECs) in the Sinusoidal Blood Vessels.

The pathological hepatic changes induced by the tumor-produced VEGF led to regional necrosis in the liver tissue (Fig. S7A). Ki67, a proliferating marker, staining showed that ECs in the hepatic sinusoidal blood vessels were actively proliferating (Fig. S7B). There was an approximately sixfold increase in proliferating ECs in the liver of VEGF tumor-bearing mice as compared with the control group (Fig. S7D). TUNEL staining showed an approximately sixfold increase in hepatocyte death in the VEGF group compared with the vector control (Fig. S7 B and E). These findings demonstrate that VEGF-induced hyperproliferation of ECs in the sinusoidal blood vessels leads to an elevated apoptotic rate accompanied by liver necrosis.

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

Impairment of Liver Function.

VEGF-induced hepatic tissue damage resulted from the high rate of hepatocyte apoptosis and necrosis; the expansion of sinusoidal blood vessels also suggested impairment of liver function. Indeed, measurement of serum transaminases showed that levels of alanine transaminase (ALT) and asparate transaminase (AST) were already considerably elevated at day 14 after tumor implantation in VEGF tumor-bearing mice (Table S1). In contrast, serum levels of other parameters reflecting hepatic function such as cholesterol and albumin remained unchanged during the entire 14-day period, probably due to the highly compensatory capacity of the remaining hepatocytes (Table S1).

Severe Anemia in VEGF Tumor-Bearing Mice.

Depletion of hematopoietic cells from BM suggested an anemic phenotype in VEGF tumor-bearing mice. Gross examination of these mice revealed a severe anemic phenotype, which manifested as considerable paleness of several hairless regions of the mouse body, including the paws, mouth, nose, and genitals (Fig. S1C). Hematological analysis of the peripheral blood showed a significant decrease in hematocrit in both immunocompetent and immunoincompetent mice at day 14 after VEGF tumor implantation (Table S2). The level of hemoglobin and the number of erythrocytes in the peripheral blood were significantly decreased (Table S2). These results showed that VEGF tumor-bearing mice suffered from a severe anemia. In addition, the total number of white blood cells was also significantly decreased, suggesting defective myelogenesis (Table S3). Taken together, depletion of BM hematopoietic cells and decreased numbers of red blood cells and white blood cells demonstrate that tumor-produced VEGF results in severe anemia in mice.

Extramedullary Hematopoiesis and Mobilization of BM Cells.

Hepatomegaly and splenomegaly, as well as infiltration of hematopoietic cells, suggested that in VEGF-expressing tumor-bearing mice exhibited active extramedullary hematopoiesis occurred in these organs. Immunohistochemical analysis with a specific anti-erythroblast antibody (Ter119) demonstrated a high density of erythroblasts and reticulocytes in the liver and spleen tissues of VEGF tumor-bearing mice as compared with those of control mice. These erythroblasts formed clusters, which appeared as hematopoietic islets (Fig. S7 B, C, F, and G). BM transplantation of syngeneic EGFP⁺ cells to irradiated recipient mice showed significant mobilization of GFP⁺ BM-derived cells to the liver and spleen tissues (Fig. S8 A and B). These findings demonstrate that active extramedullary hematopoiesis occurs in livers and spleens of mice with VEGF-expressing tumors, which mobilized BM cells to these sites.

Consistent with extramedullary hematopoiesis, plasma levels of erythropoietin (EPO) were significantly elevated in VEGF tumor-bearing mice (Fig. S8C). Surprisingly, high levels of EPO were unable to initiate active BM hematopoiesis, suggesting a defective response of BM hematopoietic cells to EPO. Although a significant decrease in circulating soluble VEGFR-2 was detected, levels of soluble VEGFR-1, TNF-α and IL-6 were unchanged in VEGF tumor-bearing mice (Fig. S8 D–F).

Tissue Hypoxia and Vascular Permeability.

To study vascular functions in CASS-affected tissues, tissue hypoxia and vascular permeability were measured in various tissues using a Hypoxia Probe kit. Hypoxic regions were unevenly distributed throughout VEGF and vector control tumors (Fig. S9B). In contrast, the entire hepatic tissue of VEGF tumor-bearing mice was exposed to severe hypoxia, whereas hypoxia was only detectable around a tiny area of the central vein in control vector tumor-bearing mice (Fig. S9B). Similarly, the cortex of the adrenal gland was also exposed to severe hypoxia in VEGF tumor-bearing mice and tissue hypoxia was undetectable in the cortex, except for low-level hypoxia in the medulla of control vector tumor-bearing mice (Fig. S9E); BM of VEGF tumor-bearing mice exhibited a high level of hypoxia throughout the entire tissue (Fig. S9D). Interestingly, the spleen showed undetectable levels of hypoxia in VEGF or control vector tumor-bearing mice (Fig. S9C). Vascular permeability was increased in tumors and livers of VEGF tumor-bearing mice (Fig. S9F). These findings suggest that VEGF induced abnormal vessels in the affected tissues and organs that are highly permeable and lack appropriate blood perfusion, although they contain a high number of microvessels.

Expression of VEGFR-1 and VEGFR-2 in Various Organs.

The formation of aberrant sinusoidal vasculature in various organs suggested that VEGFRs are expressed in blood vessels in the tissues. Immunohistochemical analysis was performed using two specific antibodies against mouse VEGFR-1 (MF1) and VEGFR-2 (TO14). Blood vessels in the hepatic tissue of nontumor- and tumor-bearing mice expressed high levels of both VEGFR-1 and VEGFR-2 (Fig. S10A). Although the expression patterns of VEGFR-1 and VEGFR-2 in the liver vasculature almost completely overlapped in nontumor- and tumor-bearing mice, VEGFR-2 was expressed at a higher level and in a broader spectrum of vascular networks than VEGFR-1. In spleen, VEGFR-2 expression was significantly higher in VEGF tumor-bearing mice as compared with control mice and VEGFR-1 was barely detectable in the blood vessels (Fig. S10B). Whereas VEGFR-2 was mainly expressed in blood vessels, VEGFR-1 was expressed on non-ECs in the adrenal gland of VEGF tumor-bearing mice (Fig. S10C). Notably, VEGF induced accumulation of VEGFR-1-positive cells in the cortex and medulla of the adrenal gland (Fig. S10C). BM showed an overlapping distribution of VEGFR-1 and VEGFR-2 (Fig. S10D). Tumor blood vessels expressed high levels of both VEGFR-1 and VEGFR-2, although VEGF tumors exhibited a significantly higher density of overlapping VEGFR-1 and -2 positive signals. These findings provide molecular 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 reverse VEGF-induced CASS in VEGF tumor-bearing mice and to define receptor signaling pathways involved in the development of CASS, two specific neutralizing anti-mouse VEGFR-1 (MF1) and VEGFR-2 (DC101) monoclonal antibodies at a low dose (800 μg/mouse) and a high dose (1600 μg/mouse) were administered to the tumor-bearing mice. 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 DC101 antibody at both low and high doses (Fig. 2 A and C). The liver and spleen weights were significantly reduced and reverted almost to those of nontumor-bearing mice (Fig. 3 E and F). The dilated sinusoidal blood vessels in the liver appeared normal (Fig. 2C). Consistent with these histological changes, hematocrit, hemoglobin, erythrocytes, and liver function were all normalized by the anti-VEGFR-2 neutralizing antibody (Tables S1–3). Surprisingly, anti-VEGFR-2 at the effective dose for normalization of systemic tissues and organs did not show a significant antitumor effect in our model (Fig. 2B). However, an increased dose of anti-VEGFR-2 showed remarkable antitumor activity and complete reversal of VEGF-induced CASS (Fig. 2B, C). In contrast to anti-VEGFR-2, treatment with anti-VEGFR-1 at the same dose produced no effects on VEGF-induced systemic syndrome or tumor growth, demonstrating that VEGFR-1 is not the primary target for CASS (Fig. 2 A–C, Tables S1–3). These findings demonstrate that nontumor vasculature was more susceptible than tumor vasculature to anti-VEGF agents and that VEGFR-2-mediated signaling is crucial for causing the systemic damage of multiple tissues and organs.

Fig. 2.

VEGF blocking and prolongation of survivals (A) At day 14 after treatment with MF1 and DC101, a representative mouse of each group was photographed. Arrows point to nose/mouth and paws. Asterisks mark the abdomens of mice. (B) Tumor volumes were measured at the indicated times to determine tumor growth rates. (D) The percentage of survival animals in each group is presented during a 15-day-treatment course. (E and F) After killing of animals on day 15 after treatment, livers and spleens were weighed and mean values are presented. (C) At the same time point, liver, spleen, adrenal gland, and BM of buffer-treated, MF1-treated, and DC101-treated mice (n = 8/group) were stained with H&E (top four sets of images). PA = portal area; RP = red pulp; WP = white pulp; Cx = cortex; and M = medulla. Vascular networks in tumors and livers were revealed by staining with a CD31 antibody (bottom two sets of images). (Scal bar, 50 μm.) (G) CD31 positive signals were quantified in tumor tissues. (H) VEGF tumors were allowed to grow into sizes of 0.8 cm³, followed by treatment with bevacizumab for 10 days. The mouth/nose and paws from a representative mouse of each group was photographed. (I-K) Tumor growth rates, liver weight, and spleen weight were measured. (L) The percentages of survival animals in bevacizumab- versus buffer-treated groups were presented during a 19-day experimental period.

Improvement of Survival by Anti-VEGF Agents.

Despite the fact that the anti-VEGFR-2 neutralizing antibody remarkably prevented the systemic VEGF syndrome, surprisingly, the tumor growth rate was not affected by this treatment. Consistent with this finding, tumor blood vessels were unaffected by this treatment (Fig. 2 C and G). Strikingly, the anti-VEGFR-2 treatment at an effective dose significantly improved the lifetime of VEGF tumor-bearing mice (Fig. 2D). These data show that an anti-VEGFR-2 neutralizing agent at an optimally low dose could significantly prolong the lifetime of VEGF tumor-bearing mice without significantly compromising tumor growth.

Treatment with the humanized anti-VEGF antibody bevacizumab was evaluated to further validate the survival advantage of anti-VEGF agents by improving CASS. At day 16 after tumor implantation, approximately 50% of nontreated VEGF-expressing tumor-bearing mice (n = 8) died of CASS and the experiments had to be terminated at the endpoint determined by ethical considerations (tumor volume >1.5 cm³) (Fig. 2I). At 5 mg/kg, bevacizumab significantly delayed the tumor growth rate (Fig. 2I). Interestingly, none of the bevacizumab-treated mice (n = 8) died during the prolonged period of experimentation (Fig. 2 I and L). Improvement of survival by bevacizumab was not due to suppression of tumor growth because none of the bevacizumab-treated mice died even when the tumor reached the ethically determined endpoint (volume >1.5 cm³). These findings suggest that bevacizumab may prolong survival by improving CASS. Indeed, VEGF-induced anemia and hepatosplenomegaly were significantly improved by bevacizumab (Fig. 2 H, J, and K). These data confirmed the survival advantage of the VEGFR-2 blockage by improving CASS, not by tumor inhibition per se.

VEGF-Induced CASS in a Spontaneous Mouse Tumor Model.

To study the physiopathological relevance of our findings, a spontaneous tumor model of a transgenic mouse line overexpressing the neu oncogene under the tissue-specific promoter of the mouse mammary tumor virus (MMTVneu) was used (7). Female CD1 mice carrying the neu oncogene developed mammary tumors at the age of approximately two months and the tumors grew to a relatively large size during the next two months. 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 phenotype, showing significantly reduced levels of hemoglobin, hematocrit, and erythrocytes in peripheral blood (Fig. 3 G–I). Similar to the VEGF-overexpressing xenograft tumor model, MMTVneu tumor-bearing mice also showed hepatosplenomegaly (Fig. 3 C–E). Histological analysis demonstrated that a high density of sinusoidal vasculature filled the entire liver and the adrenal cortex tissues (Fig. 3B). Indeed, anti-CD31 staining showed that the vasculature in the liver and adrenal gland of MMTVneu tumor-bearing mice mainly consisted of dilated sinusoidal microvessels (Fig. 3B). In the spleen, margins of white pulp (WP) and red pulp (RP) disappeared and were replaced by expanding hematopoietic red pulp (Fig. 3B). In addition, the average tumor-free body weight of the MMTVneu transgenic mice was significantly decreased compared to that of wild-type mice (Fig. 3F). Consistent with development of CASS, the circulating VEGF level was also significantly elevated in MMTVneu tumor-bearing mice (Fig. 3J). Remarkably, treatment of these spontaneous tumor-bearing mice with DC101 at the low dose twice/wk almost completely reversed the systemic syndrome including anemia and hepatosplenomegaly (Fig. 3 A and C–I). Similarly, histology of the liver, spleen and adrenal gland, and vascular networks in these organs showed that they were completely normalized by DC101 treatment (Fig. 3B). It should be noted that there was a trend of tumor growth inhibition by DC101 treatment, although the spontaneous tumor sizes were heterogeneous and thus difficult to quantify. These findings demonstrate that a spontaneous tumor mouse model that was not genetically propagated to overexpress VEGF also developed CASS, which was correlated with an elevated level of circulating VEGF and reversed by an anti-VEGFR-2 agent.

Fig. 3.

CASS in a spontaneous mouse tumor model. Spontaneous mammary tumors developed in MMTVneu transgenic mice at 2-month age and mice were killed when they reached 4 months old. One group of mice (n = 6) received the anti-VEGFR-2 treatment at a dose of 800 μg/mouse. Paws (A), liver and spleen (C) were photographed. (B) Liver, spleen, and adrenal gland were evaluated by H&E staining (top three sets of images). The arrow indicates a hematopoietic islet in the liver tissue. Arrowheads indicate dilated sinusoidal blood vessels. Tissue sections of liver and adrenal gland were stained with anti-CD31 (bottom two sets of images). CV, central vein; RP, red pulp; WP, white pulp; Cx, cortex; M, medulla. (Scale bars, 50 μm.) Liver weight (D), spleen weight (E), and net body weight (F) were measured. Blood samples were collected and hemoglobin (G), hematocrit (H), and erythrocytes (I) were determined. (J) The serum levels of VEGF in various groups of mice were measured using a sensitive ELISA.

Correlation of Circulating VEGF Levels with Development of CASS in Human Cancer Patients.

To further correlate our findings with clinical relevance, we analyzed blood samples derived from cancer patients. We used exactly the same ELISA method for our mouse experiments to measure the circulating levels of VEGF. Interestingly, we found that the circulating VEGF level was significantly higher in prostate and bladder cancer patients and renal cell carcinoma (RCC) patients as compared with healthy individuals (Fig. 4A). The circulating VEGF levels in these patients (average of 1–1.5 ng/ml) were in a range similar to those found in our xenograft mouse tumor model (Fig. 1B, Fig. S5). Interestingly, RCC patients had slightly higher VEGF levels than prostate and bladder cancer patients. Circulating VEGF levels correlated well with severity of hepatomegaly, splenomegamy and ascites (Fig. 4 B, D, and E). Consistent with this positive correlation, liver tissues in patients with high VEGF levels showed sinusoidal dilation of vascular networks and impaired functions, including high levels of ALT and AST, and low levels of albumin (Fig. 4 C and G–I). Again, RCC patients showed a significantly positive correlation between VEGF level and impairment of liver function (Fig. 4 K–M). In contrast, hemoglobin levels were significantly decreased and reversely correlated with the circulating VEGF levels in these cancer patients, particularly in RCC patients (Fig. 4 F and J). These findings demonstrate an equivalent circulating VEGF level between human cancer patients and VEGF tumor-bearing mice. Furthermore, VEGF levels were positively correlated with the severity of CASS in human cancer patients.

Fig. 4.

CASS in human cancer patients. Clinical samples were collected from RCC, bladder and prostate cancer patients. (A) Circulating levels of VEGF were measured by ELISA. (C) Histological micrographs of H&E and CD31 immunohistochemical staining of livers from RCC patients at a high (1.2 ng/ml) and a low (0.3 ng/ml) circulating VEGF level are presented. (Scale bars, 50 μm.) The development of hepatomegaly (B) and splenomegaly (D) were correlated with circulating VEGF levels. (E) Percentage of patients with ascites was correlated with the average circulating VEGF level. Levels of blood hemoglobin (F), ALT (G), AST (H), and albumin (I) were measured and correlated with circulating VEGF expression levels (J–M). HI, healthy individuals; PC, prostate cancer patients; BC, bladder cancer patients; RCC, renal cell carcinoma patients. Statistic analyses were indicated as in figures.

Discussion

Here, we show that tumor-produced VEGF induces CASS by damaging 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 of corticosterone. The severity of these systemic changes was generally well correlated with the circulating VEGF level in both mice and human cancer patients. VEGF-induced CASS resembles cancer cachexia and paraneoplastic syndromes, which manifests functional failures of multiple organs often at an advanced stage of the malignancy (8). Cancer cachexia and paraneoplastic syndrome are the primary causes of mortality in cancer patients. Although a few cytokines including TNF-α and IL-6 contribute to CASS, its underlying molecular mechanisms remain unknown (8, 9). Our present study provides a novel mechanistic insight into the role of tumor-derived VEGF in the development of CASS.

In the xenograft VEGF tumor model, we were able to determine the threshold level of VEGF that causes CASS by mixing VEGF-producing and vector-transfected tumor cells in different ratios. Similar circulating VEGF levels were detected in various cancer patients including RCC, prostate cancer and bladder cancer patients. In fact, the average circulating VEGF level in RCC patients was approximately 1.5 ng/ml. Intriguingly, most of these cancer patients developed obvious CASS including severe anemia, hepatosplenomegaly, and ascites. These clinical data correlated well with our VEGF tumor model in mice. It is estimated that at the time of diagnosis, the rate of CASS is ≈7–10% of patients with malignancy and that as many as 50% of all cancer patients may experience such a syndrome at some time during the course of their illness (5). Similar to our present findings in mouse tumors and human patients, autopsies of RCC patients revealed that ≈20% of patients had sinusoidal dilation in the liver, spleen, and adrenals (10). The sinusoidal dilation of these organs is considered to be a nonmetastatic tumor-specific manifestation, although the etiology remains unclear. It should be mentioned that VEGF-induced vascular leakage might be involved in the axis of the reactive oxygen-rac-angiopoietin-2 pathway (11).

CASS is defined as a constellation of symptoms in association with the presence of an actively growing tumor that releases an unknown factor in excess into the circulation. The identity of this unknown factor has not been characterized. Our present study with the VEGF-expressing tumor model in mice resembles an equivalent situation in cancer patients possessing advanced VEGF-secreting tumors and demonstrates that the unknown factor is likely to be VEGF. In addition to RCC, sinusoidal dilation of tissues has also been observed in other tumors. For example, nine patients with different cancers including Hodgkin's disease, stomach cancer, lymphosarcoma, and gastric carcinoma had dilation of hepatic sinusoidal blood vessels (12). A similar systemic syndrome is also present in patients with malignant histiocytosis, pediatric Wilm's tumor, and pancreatic cancers (13–15). The percentage of patients with highly dilated hepatic sinusoidal blood vessels is probably much higher than that reported in the literature because almost all cases were encountered at autopsy, which is not routinely performed.

Intriguingly, high VEGF levels induced CASS in both Lewis lung and fibrosarcoma models, suggesting that the VEGF-induced systemic effect is independent of tumor type. Indeed, continuous injection of purified VEGF protein into nontumor-bearing mice could also cause hepatosplenomegaly 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 developed a similar systemic syndrome as manifested by severe anemia, hepatosplenomegaly, ascites, and loss of body weight. The circulating VEGF levels in these spontaneous tumor-bearing mice were lower than that of mice with VEGF xenograft tumors, and the development and growth rate of these spontaneous tumors were considerably slower than for xenograft tumors. VEGF may accumulate in various tissues and organs over a relatively long period of tumor development. Persistent exposure of these organs to VEGF might result in initiation of vascular growth and impairment of vascular function. Indeed, vascular networks in liver, spleen, and adrenal glands of spontaneous tumor-bearing mice exhibited a high degree of disorganization, dilation, and tortuous architecture. Importantly, anti-VEGFR-2 could completely reverse vascular abnormalities and tissue structures in MMTVneu tumor mice. Taken together, this finding demonstrates that VEGF plays an important role in initiation, progression and maintenance of CASS in spontaneous tumor-bearing mice.

Surprisingly, BM hematopoietic cells were virtually completely eradicated by VEGF in mice. Due to a lack of a sufficient number of hematopoietic stem cells in BM, both red blood cells and white blood cells in the peripheral blood were dramatically decreased. Development of anemia is unlikely due to the direct inhibitory effect of VEGF on hematopoiesis because extramedullary hematopoiesis in the liver and spleen was stimulated by VEGF.

Overall, our studies demonstrate that in both xenograft and spontaneous tumor-bearing mice, tumor-expressed VEGF induces CASS, which resembles cachexia and paraneoplastic syndromes in human cancer patients. Circulating VEGF levels correlated well with CASS severity in tumor-bearing mice and human cancer patients. We suggest that nontumor tissues are important therapeutic targets for improvement in cancer patient survival. The functional and pathological changes in tissues and organs might serve as useful noninvasive markers for the effectiveness of anti-VEGF therapy in improving cancer patient survival rates. Thus, these results provide molecular insight into the global impact of tumor-produced VEGF in cancer patients and suggest that combinatorial therapies of anti-VEGF agents with other drugs to improve tissue and organ function will produce immense benefits for cancer patients.

Experimental Procedures

Animals, Human Materials, and Mouse Tumor Model.

All animal studies were reviewed and approved by the animal care and use committees of the local animal board. All human studies were approved by the Chinese Medical Information Committee. Detailed methods and criteria of patient selection are described in SI Text.

Tissue and Organ Collection, ELISA, and Blood Sample Analysis.

See SI Text for details.

Tissue Hypoxia Analysis and Vascular Permiability Assay.

Tissue hypoxia in tumor tissues, liver, spleen, BM, and adrenal glands was measured according to a standard protocol using Hypoxyprobe^TM-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 μm thickness and stained with hematoxylin-eosin (H&E) according to our previously described methods (18). Paraffin sections of BM tissues were stained with the anti-mouse CD31 antibody and positive signal were developed using DAB as the substrate. Whole-mount staining was performed according to previously published methods (19). See SI Text for details.

Statistical Analysis.

Statistical analysis was performed using the student's t test by a Microsoft Excel program. Data were presented as means of determinants (± SD) and p-values < 0.05 were considered as statistically significant. The Kaplan-Meier survival curve was generated using Statistica 5.0 (Statsoft).

Acknowledgments

We thank Dr. Rolf Brekken at the University of Texas Southwestern Medical Center for supplying the anti-VEGFR-2 polyclonal antibody. This work was supported by the laboratory of Y.C. through research grants from the Swedish Research Council, the Swedish Heart and Lung Foundation, the Swedish Cancer Foundation, the Karolinska Institute Foundation, and the Torsten and Ragnar Söderberg's Foundation and by European Union Integrated Projects of Angiotargeting Contract 504743 (to Y.C.) and VascuPlug Contract STRP 013811 (to Y.C.), and supported in part by a grant from Fondazione Cariplo (N.O.B.E.L Project) (to P.V.).

Footnotes

¹To whom correspondence should be addressed. E-mail: yihai.cao{at}ki.se
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. contributed new 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.
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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(2006) Hepatocyte growth factor is a lymphangiogenic factor with an indirect mechanism of action. Blood 107:3531–3536.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 105 (47)

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Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome

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