Vascular endothelial growth factor inhibition: Conflicting roles in tumor growth

Angiogenesis, the physiological process of sprouting of new blood vessels from pre-existing ones, is a key biological feature of almost all cancers. Among the multitude of factors driving tumor angiogenesis, vascular endothelial growth factor (VEGF) is the most potent, exerting myriad effects on vascular pruning and sprouting, permeability, network formation, proliferation, and cell death. Despite the initial unimpressive clinical performance of anti-VEGF antibody (bevacizumab) as cancer monotherapy, clear improvements in clinical outcomes following combination bevacizumab and chemotherapy regimens and multi-targeted VEGF receptor tyrosine kinase inhibitors (sorafenib and sunitinib) in select tumor types have established VEGF-targeted agents as an effective means of controlling cancer growth. Prolongation of overall survival and cure with these agents, however, remains elusive. Moreover, recent data has revealed key differences in the therapeutic and biological tumor response to antibody versus receptor kinase VEGF inhibitors and suggested, at least pre-clinically, that VEGF blockade in certain circumstances may actually promote more aggressive tumor growth. Given the diverse mechanisms and potentially opposing roles of VEGF neutralization in cancer biology, identification of novel biomarkers predictive of in vivo angiogenic responses may hold the key to optimizing therapeutic outcomes of anti-VEGF therapy in future cancer patients.     

Playing only one instrument may be not enough: limitations and future of the antiangiogenic treatment of cancer

Angiogenesis plays an essential role in tumor growth, invasion and metastasis. After initial pessimism about the usefulness of the antiangiogenic therapeutic approach for cancer, interest has increased in the development of antiangiogenic compounds after the first clinical approval of an antiangiogenic therapy. The anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has recently been approved for use in combination with chemotherapy for the treatment of metastatic colorectal and non-small cell lung cancer patients. However, no survival benefit has been demonstrated in anti-VEGF monotherapy trials, probably due to the high complexity of tumor angiogenesis regulation. Experimental and clinical data, including the approval of the multitargeted drugs sunitinib and sorafenib, indicate that exciting results, including tumor regression, can be expected from the combined targeting of different pathways in the tumor angiogenesis scenario. Several obstacles, including the high cost of new molecular targeted drugs make this therapeutic approach difficult.     

Dll4 Blockade Potentiates the Anti-Tumor Effects of VEGF Inhibition in Renal Cell Carcinoma Patient-Derived Xenografts

Background

The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC).

Methods and Results

Severe combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36–62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38–54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72–80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model.

Conclusions

Overall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.     

CXCR2 Inhibition Combined with Sorafenib Improved Antitumor and Antiangiogenic Response in Preclinical Models of Ovarian Cancer

Antiangiogenic therapy is important for the treatment of gynecological cancer. However, the therapeutic benefit derived from these treatments is transient, predominantly due to the selective activation of compensatory proangiogenic pathways that lead to rapid development of resistance. We aimed to identify and target potential alternative signaling to anti-vascular endothelial growth factor (VEGF) therapy, with a view toward developing a combination of antiangiogenic agents to provide extended therapeutic benefits. We developed a preclinical in vivo phenotypic resistance model of ovarian cancer resistant to antiangiogenic therapy. We measured dynamic changes in secreted chemokines and angiogenic signaling in tumors and plasma in response to anti-VEGF treatment, as tumors advanced from the initial responsive phase to progressive disease. In tumors that progressed following sorafenib treatment, gene and protein expression levels of proangiogenic CXC chemokines and their receptors were significantly elevated, compared with responsive tumors. The chemokine (C-X-C motif) ligand 8 (CXCL8), also known as interleukin-8 (IL-8) increase was time-dependent and coincided with the dynamics of tumor progression. We used SB225002, a pharmacological inhibitor of chemokine (C-X-C motif) receptor 2 (CXCR2), to disrupt the CXC chemokine-mediated functions of ovarian cancer cells in in vitro assays of cell growth inhibition, spheroid formation, and cell migration. The combination of CXCR2 inhibitor with sorafenib led to a synergistic inhibition of cell growth in vitro, and further stabilized tumor progression following sorafenib in vivo. Our results suggest that CXCR2-mediated chemokines may represent an important compensatory pathway that promotes resistance to antiangiogenic therapy in ovarian cancer. Thus, simultaneous blockage of this proangiogenic cytokine pathway using CXCR2 inhibitors and the VEGF receptor (VEGFR) pathway could improve the outcomes of antiangiogenic therapy.     

Cytokine Levels Correlate with Immune Cell Infiltration after Anti-VEGF Therapy in Preclinical Mouse Models of Breast Cancer

The effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. However, no studies have compared the effectiveness of mechanistically different anti-VEGF inhibitors with respect to changes in tumor growth and alterations in the tumor microenvironment. In this study we use three distinct breast cancer models, a MDA-MB-231 xenograft model, a 4T1 syngenic model, and a transgenic model using MMTV-PyMT mice, to explore the effects of various anti-VEGF therapies on tumor vasculature, immune cell infiltration, and cytokine levels. Tumor vasculature and immune cell infiltration were evaluated using immunohistochemistry. Cytokine levels were evaluated using ELISA and electrochemiluminescence. We found that blocking the activation of VEGF receptor resulted in changes in intra-tumoral cytokine levels, specifically IL-1β, IL-6 and CXCL1. Modulation of the level these cytokines is important for controlling immune cell infiltration and ultimately tumor growth. Furthermore, we demonstrate that selective inhibition of VEGF binding to VEGFR2 with r84 is more effective at controlling tumor growth and inhibiting the infiltration of suppressive immune cells (MDSC, Treg, macrophages) while increasing the mature dendritic cell fraction than other anti-VEGF strategies. In addition, we found that changes in serum IL-1β and IL-6 levels correlated with response to therapy, identifying two possible biomarkers for assessing the effectiveness of anti-VEGF therapy in breast cancer patients.     

The multifaceted activity of VEGF in angiogenesis – Implications for therapy responses

Vascular endothelial growth factor (VEGF) is a key growth factor driving angiogenesis (i.e. the formation of new blood vessels) in health and disease. Pharmacological blockade of VEGF signaling to inhibit tumor angiogenesis is clinically approved but the survival benefit is limited as patients invariably acquire resistance. This is partially mediated by the intrinsic flexibility of tumor cells to adapt to VEGF-blockade. However, it has become clear that tumor stromal cells also contribute to the resistance. Originally, VEGF was thought to specifically target endothelial cells (ECs) but it is now clear that many stromal cells also respond to VEGF signaling, making anti-VEGF therapy more complex than initially anticipated. A more comprehensive understanding of the complex responses of stromal cells to VEGF-blockade might inform the design of improved anti-angiogenic agents.     

Low‐dose angiostatic tyrosine kinase inhibitors improve photodynamic therapy for cancer: lack of vascular normalization

Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro‐angiogenic side effects, which may counteract its angio‐occlusive mechanism. The combination of PDT with anti‐angiogenic drugs offers a possibility for improved anti‐tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti‐VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low‐dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR‐2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib‐treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti‐angiogenic agents to further improve the efficacy of photodynamic anti‐cancer therapy.     

Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in vivo. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high affinity VEGF receptors. VEGF plays an essential role in developmental angiogenesis and is important also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with VEGF inhibitors in a variety of malignancies are ongoing. Recently, a humanized anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the FDA as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

Monitoring sunitinib-induced vascular effects to optimize radiotherapy combined with soy isoflavones in murine xenograft tumor

Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to monitor vascular changes induced by sunitinib within a murine xenograft kidney tumor, we previously determined a dose that caused only partial destruction of blood vessels leading to "normalization" of tumor vasculature and improved blood flow. In the current study, kidney tumors were treated with this dose of sunitinib to modify the tumor microenvironment and enhance the effect of kidney tumor irradiation. The addition of soy isoflavones to this combined antiangiogenic and radiotherapy approach was investigated based on our studies demonstrating that soy isoflavones can potentiate the radiation effect on the tumors and act as antioxidants to protect normal tissues from treatment-induced toxicity. DCE-MRI was used to monitor vascular changes induced by sunitinib and schedule radiation when the uptake and washout of the contrast agent indicated regularization of blood flow. The combination of sunitinib with tumor irradiation and soy isoflavones significantly inhibited the growth and invasion of established kidney tumors and caused marked aberrations in the morphology of residual tumor cells. DCE-MRI studies demonstrated that the three modalities, sunitinib, radiation, and soy isoflavones, also exerted antiangiogenic effects resulting in increased uptake and clearance of the contrast agent. Interestingly, DCE-MRI and histologic observations of the normal contralateral kidneys suggest that soy could protect the vasculature of normal tissue from the adverse effects of sunitinib. An antiangiogenic approach that only partially destroys inefficient vessels could potentially increase the efficacy and delivery of cytotoxic therapies and radiotherapy for unresectable primary renal cell carcinoma tumors and metastatic disease.     

Vascular endothelial growth factor: biology and therapeutic applications

While the development of anti-angiogenic therapy, as it pertains to cancer treatment, may still be in its infancy relative to well-established modalities such as chemotherapy, radiation, and surgery, major strides made in the past several decades have allowed translation of basic science discoveries in this field into clinical reality. The discovery of key molecular modulators of angiogenesis, notably vascular endothelial growth factor (VEGF), has catalyzed the development of numerous neutralizing therapeutic agents. The validity of VEGF inhibition as a therapeutic strategy has been well supported in randomized clinical trials, as well as U.S. Food and Drug Administration approval of the VEGF antagonists bevacizumab, sunitinib malate, sorafenib, pegaptinib and ranibizumab. Accordingly, this review will (1) briefly review the basic molecular biology of VEGF and (2) summarize recent progress in targeting the VEGF molecular pathway as therapy for angiogenic diseases such as cancer and age-related macular degeneration.     

Sunitinib Does Not Accelerate Tumor Growth in Patients with Metastatic Renal Cell Carcinoma

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Highlights? Drugs targeting the tumor vasculature are under investigation for cancer treatment ? However, changes in tumor vasculature may promote tumor growth and/or tumor spread ? Our analysis shows that sunitinib does not accelerate tumor growth in humans ? Sunitinib administration has positive effects and no negative effects when stopped     

PD-L1和VEGF双靶点联合阻断治疗的研究进展

免疫检查点抑制剂(immune checkpoint inhibitors,ICIs)通过阻断负调控免疫信号激活宿主抗肿瘤免疫反应。临床试验表明,ICIs的治疗能够明显引起部分晚期癌症患者的肿瘤消退。在临床实践中,ICIs治疗的一个主要问题是药物应答率低。尽管PD-L1表达、错配修复缺陷、肿瘤浸润性淋巴细胞状态等多种预测生物标志物已被用于筛选对治疗有应答的患者,但ICIs单药治疗的耐药性仍存在。近期研究表明,联合抗VEGF治疗可以减轻ICIs的耐药性。VEGF能抑制肿瘤生长和转移所必需的血管生成,同时能够对肿瘤免疫微环境进行重编程,减轻ICIs的耐药性。目前已针对此双靶点的联合治疗开展了很多临床试验,并获得了令人振奋的结果。对抗PD-L1联合抗VEGF治疗的作用机制以及PD-L1/VEGF联合阻断治疗的临床研究进行了综述汇总。     

Antiangiogenic cancer therapy: why do mouse and human patients respond in a different way to the same drug?

The tumor vasculature is an increasingly attractive target for development of anticancer drugs. The fundamental principle for antiangiogenic cancer therapy is based on the inhibitory effect of chemical compounds, proteins or nucleotides on tumor angiogenesis. Indeed, in almost all preclinical tumor models, antiangiogenic monotherapy with different agents shows potent effects on suppression of tumor growth. However, antiangiogenic monotherapy has barely produced any clinical benefits in cancer patients. Although in combination with chemotherapy some antiangiogenic drugs demonstrate survival improvement in patients with certain types of cancers, the overall benefits by addition of antiangiogenic drugs (ADs) to chemotherapy remain modest. The disparity of AD responses between preclinical models and clinical cancer patients has raised important issues, which include: 1) Are current animal tumor models appropriate for assessing the therapeutic efficacy of ADs for clinical development? 2) What are the key differences between mouse tumor models and human cancer patients? 3) Are anti-VEGF drugs off target in cancer patients? 4) What are alternative options for improvement of the clinical benefits of ADs? In this short review, I discuss these critical issues in relation to the clinical practice of ADs.     

Dynamic contrast-enhanced magnetic resonance imaging of sunitinib-induced vascular changes to schedule chemotherapy in renal cell carcinoma xenograft tumors

In an attempt to develop better therapeutic approaches for metastatic renal cell carcinoma (RCC), the combination of the antiangiogenic drug sunitinib with gemcitabine was studied. Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), we have previously determined that a sunitinib dosage of 20 mg/kg per day increased kidney tumor perfusion and decreased vascular permeability in a preclinical murine RCC model. This sunitinib dosage causing regularization of tumor vessels was selected to improve delivery of gemcitabine to the tumor. DCE-MRI was used to monitor regularization of vasculature with sunitinib in kidney tumors to schedule gemcitabine. We established an effective and nontoxic schedule of sunitinib combined with gemcitabine consisting of pretreatment with sunitinib for 3 days followed by four treatments of gemcitabine at 20 mg/kg given 3 days apart while continuing daily sunitinib treatment. This treatment caused significant tumor growth inhibition resulting in small residual tumor nodules exhibiting giant tumor cells with degenerative changes, which were observed both in kidney tumors and in spontaneous lung metastases, suggesting a systemic antitumor response. The combined therapy caused a significant increase in mouse survival. DCE-MRI monitoring of vascular changes induced by sunitinib, gemcitabine, and both combined showed increased tumor perfusion and decreased vascular permeability in kidney tumors. These findings, confirmed histologically by thinning of tumor blood vessels, suggest that both sunitinib and gemcitabine exert antiangiogenic effects in addition to cytotoxic antitumor activity. These studies show that DCE-MRI can be used to select the dose and schedule of antiangiogenic drugs to schedule chemotherapy and improve its efficacy.     

VEGF-targeted cancer therapy strategies: current progress, hurdles and future prospects

Despite setbacks, the clinical development of antiangiogenic agents has accelerated remarkably over the past 3-4 years. Consequently, there are currently three direct inhibitors of the VEGF pathway approved for use in cancer therapy. Other agents that block the VEGF pathway are in advanced stages of clinical development and have shown promising results. With these exciting developments come crucial questions regarding the use of these new molecular-targeted agents, alone or in combination with standard cytotoxic or targeted agents. Importantly, the mechanisms of action of anti-VEGF therapy remain unknown. Here, we discuss several potential mechanisms of action such as tumor vascular normalization, bone marrow-derived cell recruitment blockade and cytostatic effects of anti-VEGF therapy. We review the current progress, the major stumbling blocks and the future directions for anti-cancer therapy using anti-VEGF agents, emphasizing clarification of the underlying molecular mechanisms of action and biomarker identification and validation.     

Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells

Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRb and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.     

Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells

Vascular endothelial growth factor (VEGF) is an essential regulator of normal and abnormal blood vessel growth. A monoclonal antibody (mAb) that targets VEGF suppresses tumor growth in murine cancer models and human patients. We investigated cellular and molecular events that mediate refractoriness of tumors to anti-angiogenic therapy. Inherent anti-VEGF refractoriness is associated with infiltration of the tumor tissue by CD11b+Gr1+ myeloid cells. Recruitment of these myeloid cells is also sufficient to confer refractoriness. Combining anti-VEGF treatment with a mAb that targets myeloid cells inhibits growth of refractory tumors more effectively than anti-VEGF alone. Gene expression analysis in CD11b+Gr1+ cells isolated from the bone marrow of mice bearing refractory tumors reveals higher expression of a distinct set of genes known to be implicated in active mobilization and recruitment of myeloid cells. These findings indicate that, in our models, refractoriness to anti-VEGF treatment is determined by the ability of tumors to prime and recruit CD11b+Gr1+ cells.     

Vascular endothelial growth factor-dependent and -independent regulation of angiogenesis

Angiogenesis, the formation of blood vessels, is essential for preparing a closed circulatory system in the body, and for supplying oxygen and nutrition to tissues. Major diseases such as cancer, rheumatoid arthritis, and atherosclerosis include pathological angiogenesis in their malignant processes, suggesting anti-angiogenic therapy to be a new strategy for suppression of diseases. However, until the 1970s, the molecular basis of angiogenesis was largely unknown. In recent decades, extensive studies have revealed a variety of angiogenic factors and their receptors, including vascular endothelial growth factor (VEGF)-VEGFRs, Angiopoietin-Tie, Ephrin-EphRs and Delta-Notch to be the major regulators of angiogenesis in vertebrates. VEGF and its receptors play a central role in physiological as well as pathological angiogenesis, and functional inhibitors of VEGF and VEGFRs such as anti-VEGF neutralizing antibody and small molecules that block the tyrosine kinase activity of VEGFRs have recently been approved for use to treat patients with colorectal, lung, renal and liver cancers. These drugs have opened a novel field of cancer therapy, i.e. anti-angiogenesis therapy. However, as yet they cannot completely cure patients, and cancer cells could become resistant to these drugs. Thus, it is important to understand further the molecular mechanisms underlying not only VEGF-VEGFR signaling but also the VEGF-independent regulation of angiogenesis, and to learn how to improve anti-angiogenesis therapy.     

Specific blockade of VEGF and HER2 pathways results in greater growth inhibition of breast cancer xenografts that overexpress HER2

We have previously reported that breast cancer cells which overexpress HER2 produce higher levels of VEGF than cells with low levels of HER2. This study tested the hypothesis that dual targeting of the VEGF (with VEGF-Trap) and HER2 (with trastuzumab) pathways would result in greater growth inhibition of HER2-overexpressing breast cancer xenografts than either agent alone. In this study we found that human and murine endothelial cells expressed high levels of VEGF receptors (VEGFR1, VEGFR2, & VEGFR3). VEGF-Trap decreased levels of secreted VEGF derived from both human and murine cells and effectively blocked VEGF-induced tyrosine phosphorylation of VEGFR2. VEGF-Trap as a single treatment inhibited tumor microvessel density (MVD), tumor vasculature, cell proliferation, and tumor growth of BT474 xenografts in a dose-dependent manner from 2.5 mg/kg to 25 mg/kg. VEGF-Trap decreased levels of both human VEGF and PlGF protein in vivo. Trastuzumab as a single agent effectively inhibited BT474 tumor growth in a dose-dependent manner, associated with a decrease in human VEGF, tumor MVD and tumor cell proliferation. Treatment with a combination of VEGF-Trap (2.5-10 mg/kg) and trastuzumab (1 mg/kg) produced significantly greater inhibition of BT474tumor growth than either individual agent, associated with greater inhibition of tumor MVD and tumor cell proliferation. Thus, VEGF-Trap in combination with trastuzumab produces superior growth inhibition of tumor xenografts which overexpress HER2, which may result from inhibition of both tumor angiogenesis and proliferation. Similar mechanisms may contribute to the clinical anti-tumor activity of trastuzumab in combination with inhibitors of VEGF signaling pathway in women with breast cancers which overexpress HER2.