Anti-VEGF/VEGFR therapy for cancer: reassessing the target

Judah Folkman recognized that new blood vessel formation is important for tumor growth and proposed antiangiogenesis as a novel approach to cancer therapy. Discovery of vascular permeability factor VEGF-A as the primary tumor angiogenesis factor prompted the development of a number of drugs that targeted it or its receptors. These agents have often been successful in halting tumor angiogenesis and in regressing rapidly growing mouse tumors. However, results in human cancer have been less impressive. A number of reasons have been offered for the lack of greater success, and, here, we call attention to the heterogeneity of the tumor vasculature as an important issue. Human and mouse tumors are supplied by at least 6 well-defined blood vessel types that arise by both angiogenesis and arterio-venogenesis. All 6 types can be generated in mouse tissues by an adenoviral vector expressing VEGF-A(164). Once formed, 4 of the 6 types lose their VEGF-A dependency, and so their responsiveness to anti-VEGF/VEGF receptor therapy. If therapies directed against the vasculature are to have a greater impact on human cancer, targets other than VEGF and its receptors will need to be identified on these resistant tumor vessels.     

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al., Science (Washington DC), 279: 377-380, 1998]. We show here that the receptor for the NGR peptides in tumor vasculature is aminopeptidase N (APN; also called CD13). NGR phage specifically bound to immunocaptured APN and to cells engineered to express APN on their surface. Antibodies against APN inhibited in vivo tumor homing by the NGR phage. Immunohistochemical staining showed that APN expression is up-regulated in endothelial cells within mouse and human tumors. In another tissue that undergoes angiogenesis, corpus luteum, blood vessels also expressed APN, but APN was not detected in blood vessels of various other normal tissues stained under the same conditions. APN antagonists specifically inhibited angiogenesis in chorioallantoic membranes and in the retina and suppressed tumor growth. Thus, APN is involved in angiogenesis and can serve as a target for delivering drugs into tumors and for inhibiting angiogenesis.     

Immunotherapy against angiogenesis-associated targets: evidence and implications for the treatment of malignant glioma

Angiogenesis, the growth of new blood vessels from previously existing vasculature, is a requirement for tumor growth and metastasis. The first US FDA-approved drugs targeting angiogenesis have shown potential in the treatment of malignant gliomas. Immunotherapy as a treatment modality lends itself well to specifically targeting angiogenesis in tumors and may represent a powerful tool in the treatment of malignant gliomas. This review focuses on developments in immunotherapy targeting angiogenesis and tumor-vascular-specific endothelial cells using a variety of immunotherapeutic strategies including monoclonal antibodies and conjugated immunotoxins, as well as cellular, peptide, DNA and dendritic cell vaccines.     

VEGF as a key mediator of angiogenesis in cancer

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein with a molecular weight of approximately 45 kDa. It is the key mediator of angiogenesis (the formation of new blood vessels), and binds two VEGF receptors (VEGF receptor-1 and VEGF receptor-2), which are expressed on vascular endothelial cells. In healthy humans, VEGF promotes angiogenesis in embryonic development and is important in wound healing in adults. VEGF is the key mediator of angiogenesis in cancer, in which it is up-regulated by oncogene expression, a variety of growth factors and also hypoxia. Angiogenesis is essential for cancer development and growth: before a tumor can grow beyond 1-2 mm, it requires blood vessels for nutrients and oxygen. The production of VEGF and other growth factors by the tumor results in the 'angiogenic switch', where new vasculature is formed in and around the tumor, allowing it to grow exponentially. Tumor vasculature formed under the influence of VEGF is structurally and functionally abnormal. Blood vessels are irregularly shaped, tortuous, have dead ends and are not organized into venules, arterioles and capillaries. They are also leaky and hemorrhagic, which leads to high interstitial pressure. These characteristics mean that tumor blood flow is suboptimal, resulting in hypoxia and further VEGF production. This central role of VEGF in the production of tumor vasculature makes it a rational target for anticancer therapy.     

Changes in blood vessel maturation in the fibrous cap of the tumor rim

It is widely accepted that blood vessels in the tumor microenvironment are immature because mural cell (MC) adhesion to endothelial cells (ECs) is broadly lacking. Hyperpermeability of the tumor vasculature then results in interstitial hypertension that mitigates against penetration of anticancer drugs into the depths of the tumor. It has been suggested that treatment with angiogenesis inhibitors normalizes blood vessels, resulting in restoration of normal permeability and improved drug delivery. However, recent reports suggest that cancer cell invasion is induced from the edge of the tumor into peripheral areas after treatment with angiogenesis inhibitors. Therefore, it is important to assess the status of blood vessels in the fibrous cap at the tumor rim after antiangiogenesis therapy. In the present study, we found that mature blood vessels in which ECs are covered with MCs are present in the fibrous cap. After treatment with angiogenesis inhibitors, immature blood vessels were destroyed and vascular function was significantly improved, but maturing blood vessels in which ECs were covered with MCs remained visible. These maturing blood vessels showed a less dilated character after treatment with the angiogenesis inhibitors. It is widely accepted that well-matured blood vessels are sheathed in extracellular matrix (ECM) and that cancer cells migrate along tracks made of ECM collagen fibers. Therefore, our data indicate the importance of destroying maturing blood vessels outside the tumor parenchyma to prevent cancer cell invasion.     

Significance of blood vessel leakiness in cancer

Despite major advances in the field of tumor angiogenesis, relatively little attention has been paid to the permeability of blood vessels in tumors. The leakiness of tumor vessels is well documented in experimental tumor models and in human cancer, but the mechanism is poorly understood, as are the implications to the rate of cancer growth, predisposition to metastasis, and delivery of macromolecular therapeutics to tumor cells. Sixteen experts in the fields of cancer biology and vascular biology gathered at the William Guy Forbeck "Focus on the Future" Conference to discuss this topic. The meeting was the first of its kind focused on the significance of blood vessel leakiness in tumors. The participants discussed the cellular basis of tumor vessel leakiness, endothelial barrier function of blood vessels, monitoring tumor vessel leakiness, mediators of endothelial leakiness, consequences of tumor vessel leakiness, genomic analysis of vascular targets, targeting drugs to tumor vessels, and therapeutic manipulation of tumor vessels. The group concluded that a more complete understanding of the basic biology of tumor vessels will be necessary to fully appreciate the consequences of vessel leakiness in cancer. New research tools such as intravital measurements of tumor blood flow and vessel leakiness, in vivo phage display, magnetic resonance imaging, and use of selective angiogenesis inhibitors will contribute to this understanding.     

Tumor angiogenesis—characteristics of tumor endothelial cells

Tumor blood vessels provide nutrition and oxygen to the tumor, resulting in tumor progression. They also act as gatekeepers, inducing tumor metastasis. Thus, targeting tumor blood vessels is an important strategy in cancer therapy. Tumor endothelial cells (TECs), which line the inner layer of blood vessels of the tumor stromal tissue, are the main targets of anti-angiogenic therapy. Because new tumor blood vessels generally sprout from pre-existing vasculature, they have been considered to be the same as normal blood vessels. However, tumor blood vessels demonstrate a markedly abnormal phenotype that includes several important morphological changes. The degree of angiogenesis is determined by the balance between the angiogenic stimulators and inhibitors released by the tumor and host cells. Recent studies have revealed that TECs also exhibit altered characteristics which depend on the tumor microenvironment. Here, we review recent studies on TEC abnormalities and heterogeneity with respect to tumor progression and consider their therapeutic implications.     

Targeted therapies of cancer: angiogenesis inhibition seems not enough

The therapeutic potential of targeting tumor endothelium to induce tumor regression is now widely recognized. Tumors obtain their blood supply by the formation of new vasculature and the incorporation of pre-existent vessels. Since anti-angiogenic therapy prevents formation of neovasculature, vessels in more matured stages are not affected by such therapies. Therefore, additional vascular targeting therapy, which aim at regression of existent tumor vasculature, seems an attractive approach to effectively deprive tumors from blood supply. In this review we present an overview of different strategies to target tumor endothelium. In addition, we discuss the pitfalls of anti-angiogenic therapies in clinical settings.     

The merits of vascular targeting for gynecologic malignancies

Neovascularization is an early and critical step in tumor development and progression. Tumor vessels are distinct from their normal counterparts morphologically as well as at a molecular level. Recent studies on factors involved in tumor vascular development have identified new therapeutic targets for inhibiting tumor neovascularization and thus tumor progression. However, the process of tumor blood vessel formation is complex, and each tumor exhibits unique features in its vasculature. An understanding of the relative contribution of various pathways in the development of tumor vasculature is critical for developing effective and selective therapeutic approaches. Several such agents are currently in clinical trials, and many others are under development. In this review, the mechanisms and factors involved in tumor blood vessel formation are discussed. In addition, selected novel classes of antivascular therapies, including those targeting tumor endothelial cells and other components of the tumor vasculature, are summarized.     

Molecular mechanisms of tumor angiogenesis

Tumors have been recently recognized as aberrant organs composed of a complex mixture of highly interactive cells that in addition to the cancer cell include stroma (fibroblasts, adipocytes, and myofibroblasts), inflammatory (innate and adaptive immune cells), and vascular cells (endothelial and mural cells). While initially cancer cells co-opt tissue-resident vessels, the tumor eventually recruits its own vascular supply. The process of tumor neovascularization proceeds through the combined output of inductive signals from the entire cellular constituency of the tumor. During the last two decades, the identification and mechanistic outcome of signaling pathways that mediate tumor angiogenesis have been elucidated. Interestingly, many of the genes and signaling pathways activated in tumor angiogenesis are identical to those operational during developmental vascular growth, but they lack feedback regulatory control and are highly affected by inflammatory cells and hypoxia. Consequently, tumor vessels are abnormal, fragile, and hyperpermeable. The lack of hierarchy and inconsistent investment of mural cells dampen the ability of the vessels to effectively perfuse the tumor, and the resulting hypoxia installs a vicious cycle that continuously perpetuates a state of vascular inefficiency. Pharmacological targeting of blood vessels, mainly through the VEGF signaling pathway, has proven effective in normalizing tumor vessels. This normalization improves perfusion and distribution of chemotherapeutic drugs with resulting tumor suppression and moderate increase in overall survival. However, resistance to antiangiogenic therapy occurs frequently and constitutes a critical barrier in the inhibition of tumor growth. A concrete understanding of the chief signaling pathways that stimulate vascular growth in tumors and their cross-talk will continue to be essential to further refine and effectively abort the angiogenic response in cancer.     

Expression of vascular endothelial growth factor (VEGF) and VEGF receptors in tumor angiogenesis and malignancies

Angiogenesis is a process by which new blood vessels are formed from preexisting vessels. New blood vessel formation by angiogenesis involves the degradation of extra-cellular matrix combined with sprouting and migration of endothelial cells from preexisting capillaries. Solid tumors consist of several components, including normal and stromal cells, extracellular matrix, and vasculature. To grow and metastasize, tumors must stimulate the development of new vasculature through angiogenesis. Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine regeneration. VEGF is both a vascular growth factor and a vascular permeability factor. Its expression can upregulate several proangiogenic and prometa-static molecules. As a central mediator of angiogenesis, VEGF has emerged as an important target for antiangiogenic therapy. In this review, the authors describe the essential characteristics of VEGF and the VEGF family of ligands and their receptors. They also provide an overview of the central role of VEGF in physiologic and pathologic angiogenesis, directly or indirectly. This review sheds light on the importance of VEGF-targeted antiangiogenic therapy based on the monoclonal antibodies against VEGF, small interfering RNA, and therapy directed against VEGF-VEGFR kinase. It also gives a brief overview of the natural products or dietary compounds that could be used as antiangiogenic agents. Therapeutic inhibition of vessel formation could be best suited to preventive strategies aimed at the suppression of angiogenesis in primary tumors in subjects at risk or of micrometastases after surgical removal of primary tumor.     

Targeting mutant p53 protein and the tumor vasculature: an effective combination therapy for advanced breast tumors

Breast cancer progression depends upon the elaboration of a vasculature sufficient for the nourishment of the developing tumor. Breast tumor cells frequently contain a mutant form of p53 (mtp53), a protein which promotes their survival. The aim of this study was to determine whether combination therapy targeting mtp53 and anionic phospholipids (AP) on tumor blood vessels might be an effective therapeutic strategy for suppressing advanced breast cancer. We examined the therapeutic effects, singly, or in combination, of p53 reactivation and induction of massive apoptosis (PRIMA-1), which reactivates mtp53 and induces tumor cell apoptosis, and 2aG4, a monoclonal antibody that disrupts tumor vasculature by targeting AP on the surface of tumor endothelial cells and causes antibody-dependent destruction of tumor blood vessels, leading to ischemia and tumor cell death. Xenografts from two tumor cell lines containing mtp53, BT-474 and HCC-1428, were grown in nude mice to provide models of advanced breast tumors. After treatment with PRIMA-1 and/or 2aG4, regressing tumors were analyzed for vascular endothelial growth factor (VEGF) expression, blood vessel loss, and apoptotic markers. Individual drug treatment led to partial suppression of breast cancer progression. In contrast, combined treatment with PRIMA-1 and 2aG4 was extremely effective in suppressing tumor growth in both models and completely eradicated approximately 30% of tumors in the BT-474 model. Importantly, no toxic effects were observed in any treatment group. Mechanistic studies determined that PRIMA-1 reactivated mtp53 and also exposed AP on the surface of tumor cells as determined by enhanced 2aG4 binding. Combination treatment led to significant induction of tumor cell apoptosis, loss of VEGF expression, as well as destruction of tumor blood vessels. Furthermore, combination treatment severely disrupted tumor blood vessel perfusion in both tumor models. The observed in vitro PRIMA-1-induced exposure of tumor epithelial cell AP might provide a target for 2aG4 and contribute to the increased effectiveness of such combination therapy in vivo. We conclude that the combined targeting of mtp53 and the tumor vasculature is a novel effective strategy for combating advanced breast tumors.     

Mouse models for studying angiogenesis and lymphangiogenesis in cancer

The formation of new blood vessels (angiogenesis) is required for the growth of most tumors. The tumor microenvironment also induces lymphangiogenic factors that promote metastatic spread. Anti-angiogenic therapy targets the mechanisms behind the growth of the tumor vasculature. During the past two decades, several strategies targeting blood and lymphatic vessels in tumors have been developed. The blocking of vascular endothelial growth factor (VEGF)/VEGF receptor-2 (VEGFR-2) signaling has proven effective for inhibition of tumor angiogenesis and growth, and inhibitors of VEGF-C/VEGFR-3 involved in lymphangiogenesis have recently entered clinical trials. However, thus far anti-angiogenic treatments have been less effective in humans than predicted on the basis of pre-clinical tests in mice. Intrinsic and induced resistance against anti-angiogenesis occurs in patients, and thus far the clinical benefit of the treatments has been limited to modest improvements in overall survival in selected tumor types. Our current knowledge of tumor angiogenesis is based mainly on experiments performed in tumor-transplanted mice, and it has become evident that these models are not representative of human cancer. For an improved understanding, angiogenesis research needs models that better recapitulate the multistep tumorigenesis of human cancers, from the initial genetic insults in single cells to malignant progression in a proper tissue environment. To improve anti-angiogenic therapies in cancer patients, it is necessary to identify additional molecular targets important for tumor angiogenesis, and to get mechanistic insight into their interactions for eventual combinatorial targeting. The recent development of techniques for manipulating the mammalian genome in a precise and predictable manner has opened up new possibilities for the generation of more reliable models of human cancer that are essential for the testing of new therapeutic strategies. In addition, new imaging modalities that permit visualization of the entire mouse tumor vasculature down to the resolution of single capillaries have been developed in pre-clinical models and will likely benefit clinical imaging.     

Notch signaling in developmental and tumor angiogenesis

The discovery that Notch, a key regulator of cell fate determination, is functional in the vasculature has greatly improved our understanding of differentiation and specialization of vessels. Notch signaling has been proven to be critical for arterial specification, sprouting angiogenesis, and vessel maturation. In newly forming vascular sprouts, Notch promotes the distinction between the leading "tip" endothelial cell and the growing "stalk" cell, the endothelial cells that eventually form a new capillary. Notch signaling has also been implicated in vessel stability by regulating vascular mural cell function. More recently, macrophages carrying an activated Notch have been implicated in shaping the course of new sprout formation. Tumor vessels abide by similar principles and use Notch signaling in similar ways. An exciting discovery, made by several researchers, shows that blocking Notch function in tumor vasculature provides a means by which to suppress tumor growth. The authors discuss the developmental and physiological role of Notch in the vasculature and apply this knowledge to an overview of how Notch targeting in the tumor environment can affect tumor angiogenesis and growth.     

Tumor endothelial markers: new targets for cancer therapy

PURPOSE OF REVIEW: Targeting the endothelial cells that line tumor infiltrating blood vessels is a new anticancer strategy that has gained widespread support from biologists and clinicians. Here we highlight different approaches currently being used to target tumor endothelium and discuss new avenues for intervention that have been opened through the recent identification of tumor endothelial markers (TEMs). RECENT FINDINGS: The ability of Avastin to prolong survival in a Phase III clinical trial of human colorectal cancer has established the validity of the anti-angiogenic approach. However, realization of the full potential of a vascular targeting strategy may require the exploitation of molecules which are highly restricted in expression to tumor endothelium. Here we explore the potential of TEMs as new targets for cancer therapy. Current knowledge of these markers and their relation to other family members in the context of tumor angiogenesis is discussed. In particular, we highlight those molecules which, by virtue of their structure, cell-surface location and expression pattern, appear to hold promise as targets for future drug development. The identification of TEM8 as the anthrax toxin receptor and the successful targeting of this receptor in preclinical tumor models make this molecule a particularly attractive candidate for future vascular targeting studies. SUMMARY: Technological advances in cellular fractionation and genomics enabled the identification of several markers preferentially expressed on human tumor endothelium. Studies of these TEMs are expected to aid in our understanding of angiogenesis and could lead to the development of new imaging and diagnostic agents for cancer.     

Tumor response to ionizing radiation combined with antiangiogenesis or vascular targeting agents: exploring mechanisms of interaction.

Recent preclinical studies have suggested that radiotherapy in combination with antiangiogenic/vasculature targeting agents enhances the therapeutic ratio of ionizing radiation alone. Because radiotherapy is one of the most widely used treatments for cancer, it is important to understand how best to use these two modalities to aid in the design of rational patient protocols. The mechanisms of interaction between antiangiogenic/vasculature targeting agents and ionizing radiation are complex and involve interactions between the tumor stroma and vasculature and the tumor cells themselves. Vascular targeting agents are aimed specifically at the existing tumor vasculature. Antiangiogenic agents target angiogenesis or the new growth of tumor vessels. These agents can decrease overall tumor resistance to radiation by affecting both tumor cells and tumor vasculature, thereby breaking the codependent cycle of tumor growth and angiogenesis. The hypoxic microenvironment of the tumor also contributes to the mechanisms of interactions between antiangiogenic/vasculature targeting agents and ionizing radiation. Hypoxia stimulates up-regulation of angiogenic and tumor cell survival factors, giving rise to tumor proliferation, radioresistance, and angiogenesis. Preclinical evidence suggests that antiangiogenic agents reduce tumor hypoxia and provides a rationale for combining these agents with ionizing radiation. Optimal scheduling of combined treatment with these agents and ionizing radiation will ultimately depend on understanding how tumor oxygenation changes as tumors regress and regrow during exposure to these agents. This review article explores the complex interactions between antiangiogenic/vasculature targeting agents and radiation and offers insight into the mechanisms of interaction that may be responsible for improved tumor response to radiation.     

Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels

Tumor blood vessels normalized by antiangiogenic therapy may provide improved delivery of chemotherapeutic agents during a window of time but it is unknown how protein expression in tumor vascular endothelial cells changes. We evaluated the distribution of RGD-4C phage, which binds alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins on tumor blood vessels before and after antiangiogenic therapy. Unlike the control phage, fd-tet, RGD-4C phage homed to vascular endothelial cells in spontaneous tumors in RIP-Tag2 transgenic mice in a dose-dependent fashion. The distribution of phage was similar to alpha(v)beta(3) and alpha(5)beta(1) integrin expression. Blood vessels that survived treatment with AG-013736, a small molecule inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptors, had only 4% as much binding of RGD-4C phage compared with vessels in untreated tumors. Cellular distribution of RGD-4C phage in surviving tumor vessels matched the alpha(5)beta(1) integrin expression. The reduction in integrin expression on tumor vessels after antiangiogenic therapy raises the possibility that integrin-targeted delivery of diagnostics or therapeutics may be compromised. Efficacious delivery of drugs may benefit from identification by in vivo phage display of targeting peptides that bind to tumor blood vessels normalized by antiangiogenic agents.     

Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular function, and promotes tumor growth in vivo

The vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. However, clinical trials targeting the VEGF pathway are often ineffective, suggesting that other factors/pathways are also important in tumor angiogenesis. We have previously shown that the Notch ligand Delta-like 4 (DLL4) is up-regulated in tumor vasculature. Here, we show that DLL4, when expressed in tumor cells, functions as a negative regulator of tumor angiogenesis by reducing the number of blood vessels in all five types of xenografts, but acts as a positive driver for tumor growth in two of them (human glioblastoma and prostate cancer). The growth of in vivo models was not related to the effects on growth in vitro. DLL4 expressed in the tumor cells activated Notch signaling in host stromal/endothelial cells, increased blood vessel size, and improved vascular function within tumors. The promotion of tumor growth was, to some extent, due to a reduction of tumor hypoxia and apoptosis. DLL4-expressing tumor cells responded to anti-VEGF therapy with bevacizumab. A soluble form of DLL4 (D4ECD-Fc) blocked tumor growth in both bevacizumab-sensitive and bevacizumab-resistant tumors by disrupting vascular function despite increased tumor vessel density. In addition, we show that DLL4 is up-regulated in tumor cells and tumor endothelial cells of human glioblastoma. Our findings provide a rational basis for the development of novel antiangiogenic strategies via blockade of DLL4/Notch signaling and suggest that combined approaches for interrupting both DLL4 and VEGF pathways may improve antiangiogenic therapy.     

Crosstalk between angiogenesis and lymphangiogenesis in tumor progression.

Extensive studies have identified reliable markers of lymphatic endothelial cells, and mechanisms and molecules that regulate development and growth of the lymphatic vessels. Vascular endothelial growth factors (VEGF)-C and VEGF-D, and their cognate receptor tyrosine kinase, VEGF receptor-3 (VEGFR-3), are critical regulators of lymphangiogenesis. By binding to its endothelial cell surface receptors VEGFR-1 and VEGFR-2, VEGF-A mediates vascular leakage, endothelial proliferation and migration. Angiopoietin-2 (Ang-2) is expressed at sites of blood vessel remodeling and invasion, and factors that induce angiogenesis in vivo, such as VEGF-A, upregulate Ang-2 in endothelial cells. In this review, we summarize the literature concerning the crosstalk between angiogenesis and lymphangiogenesis in tumor progression, that is, involvement of VEGF-C, VEGF-D and VEGFR-3 in angiogenesis, and the role played by VEGF-A and Ang-2 in lymphangiogenesis, respectively.     

Bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells do not contribute to vasculogenesis in Lewis lung carcinoma

The development of tumor vasculature is thought to occur through two complementary processes: sprouting angiogenesis from preexisting blood vessels of the host, and vasculogenesis, which involves the spontaneous development of vessels through specific recruitment, differentiation, and vascular incorporation of circulating endothelial cells (EC), endothelial progenitor cells (EPC), or potentially bone marrow-derived cells. Recent reports, however, have challenged the belief that bone marrow-derived cells contribute to tumor neovascularization, claiming an exclusive role for sprouting angiogenesis in tumor blood vessel development.In the present study, we explored the recruitment behavior of bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells to subcutaneously implanted Lewis lung carcinoma in a syngeneic bone marrow transplantation model. We observed that although lin(-)c-kit(+)Sca-1+ and their derived cells demonstrate significant recruitment to carcinomas in vivo, they do not appear to functionally contribute to tumor neovascularization. Furthermore, our results support the hypothesis that new vessel formation in carcinomas occurs primarily through endothelialization from adjacent and preexisting vasculature.