Tumor angiogenesis: molecular pathways and ...

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Tumor angiogenesis: molecular pathways and therapeutic targets © 2011 Nature America, Inc. All rights reserved.

Sara M Weis & David A Cheresh As angiogenesis is essential for tumor growth and metastasis, controlling tumor-associated angiogenesis is a promising tactic in limiting cancer progression. The tumor microenvironment comprises numerous signaling molecules and pathways that influence the angiogenic response. Understanding how these components functionally interact as angiogenic stimuli or as repressors and how mechanisms of resistance arise is required for the identification of new therapeutic strategies. Achieving a durable and efficient antiangiogenic response will require approaches to simultaneously or sequentially target multiple aspects of the tumor microenvironment. Angiogenesis occurs during development and vascular remodeling as a controlled series of events leading to neovascularization, which supports changing tissue requirements1. Blood vessels and stromal components are responsive to pro- and antiangiogenic factors that allow vascular remodeling during development, wound healing and pregnancy. However, in pathological situations such as cancer, the same angiogenic signaling pathways are induced and exploited. Although an oncogenic event may allow tumor cells to evade surveillance or may enhance their survival, the large-scale growth of a tumor ultimately requires a blood supply2. To obtain this blood supply, tumor cells can tilt the balance toward stimulatory angiogenic factors to drive vascular growth by attracting and activating cells from within the microenvironment of the tumor. The magnitude and quality of the angiogenic response is ultimately determined by the sum of pro- and antiangiogenic signals and, more specifically, their unique activities on multiple cell types. Understanding how these various components are regulated is required for the design and development of effective antiangiogenic therapies for cancer.

has traditionally been defined as the sprouting of new vessels from preexisting vessels, it is becoming clear that the blood vessels that support tumor growth or tumor rebound from therapy-induced trauma can also originate from cells recruited from the bone marrow or can even differentiate from tumor stem cells (vascular mimicry) (Fig. 1 and Box 1). In cancer, multiple sources and modes of vascular remodeling contribute to disease progression. Targeting one aspect of this remodeling process may produce a short-term effect, but suppressing one pathway could promote another. The redundancy and diversity by which blood vessels can remodel might account for the poor efficacy or acquired resistance often observed in antiangiogenic therapies. Improving the therapeutic response will require consideration of the signaling pathways that regulate the multiple cell types involved in the vascular component of cancer. Identification of common downstream signaling hubs could offer a new therapeutic strategy in suppressing angiogenesis and tumor progression, especially if these putative targets are selectively activated in tumor cells, stromal cells or in the bone marrow–derived cells that potentiate the angiogenic response.

Blood vessel remodeling in cancer During embryonic development, blood vessels initially form through vasculogenesis, which involves de novo production of endothelial cells from angioblasts, which are recruited to differentiate in response to local cues3. Vasculogenesis is typically followed by classical sprouting angiogenesis, which involves the endothelial cells on preexisting vessels. A variety of angiogenic signals induce endothelial cells to adopt an activated phenotype: endothelial cells detach their junctional adhesions from their neighbors, sprout toward gradients of proangiogenic factors, proliferate to form provisional tubes, recruit perivascular cells to provide stability and maturation and, finally, remodel and prune to form a functional network1. Although tumor-associated angiogenesis

The tumor microenvironment governs angiogenesis The tumor microenvironment is composed of a variety of cell types that influence the angiogenic response to a tumor (Figs. 1 and 2). Once a tumor lesion exceeds a few millimeters in diameter, hypoxia and nutrient deprivation triggers an ‘angiogenic switch’ to allow the tumor to progress4. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to activate normal, quiescent cells around them and initiate a cascade of events that quickly becomes dysregulated. For example, tumor cell–released vascular endothelial growth factor (VEGF) stimulates the sprouting and proliferation of endothelial cells. Although the induction of angiogenesis may initially provide the tumor with more oxygen and nutrients, the ultimate response is poor, as the continuously remodeled tumor vasculature is leaky and tortuous, causing irregular blood flow5. Sites of basal lamina exposed during vascular leak recruit and activate platelets, which, in turn, release angiogenic and permeability factors into the local environment to further escalate the local response5. Increased amounts of platelet-derived growth factor (PDGF) released by platelets and activated endothelial cells recruit

Department of Pathology and Moores University of California–San Diego Cancer Center, University of California–San Diego, La Jolla, California, USA. Correspondence should be addressed to D.A.C. (dcheresh@ucsd.edu). Published online 7 November 2011; doi:10.1038/nm.2537

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© 2011 Nature America, Inc. All rights reserved.

review Figure 1 Multiple origins of tumor-induced Sprouting angiogenesis Vasculogenesis neovascularization. The combination Recruitment of EPCs Tumor cells of stimulatory signals within the tumor Stem cells, m cells, PCs EPCs microenvironment prompts changes in multiple Hypoxia cell types. Perivascular cells detach from the VEGF Notch1, Release of Cancer mature blood vessels, compromising their VEGFR2 soluble factors: stem cell cytokines, growth integrity, permitting their remodeling and differentiation factors, guidance promoting an activated phenotype. Once the molecules and MMPs vascular barrier is disrupted, multiple cell types Bona fide Tip cell EC are exposed to angiogenic and inflammatory ECM Intravasation remodeling stimuli to escalate the response. Platelets are Notch ligand, EPC recruited to sites of exposed basement membrane, Ang-2 where they become activated and release their Vascular mimicry Stalk cell stores of stimulatory factors into the tumor microenvironment. Endothelial progenitor cells Platelet (EPCs) and myeloid cells from the bone marrow Circulating Vascular activation tumor cell leak move to the perceived wound, where they release even more soluble factors locally. Cancer stem cells can differentiate to become bona fide endothelial cells, or tumor cells can physically participate in the formation of new vessels through vascular mimicry. However, the escalation of this response does not lead to the production of mature and proper blood vessels that improve the initial hypoxic situation because the tumor microenvironment is characterized by pockets of hypoxia amid the leaky and tortuous blood vessels. This environment also makes the tumor cells more invasive, allowing them to intravasate into the vasculature or lymphatics for metastasis to distant tissues. Effective strategies for cancer therapy must consider targets on multiple cell types and address issues of poor drug delivery in the leaky and poorly perfused tumor microenvironment.

and activate perivascular cells. Whereas perivascular coverage typically provides vascular stability and maturation, it does not lead to mature vessels with proper function, as the vessels are exposed to persistent stimulatory signals within the tumor microenvironment6. The remodeling response is further driven by recruitment of tumorassociated fibroblasts, which aberrantly deposit extracellular matrix (ECM) proteins and release stimulatory factors7–9. Matrix metalloproteinases (MMPs) cleave and remodel the ECM to form fragments or to expose previously hidden epitopes that function as endogenous inhibitors of angiogenesis10, for example, tumstatin or endoExtracellular matrix remodeling Extracellular matrix components, factors and enzymes

statin. Inflammatory cells move to this perceived wound, where they release factors that positively or negatively modulate the angiogenic response11,12 (Box 2). The observations regarding aberrant vasculature, inflammatory response, areas of necrosis and dense stroma led Harold Dvorak to describe tumors as “wounds that do not heal”13. Considering the variety of cell types and signals involved, there are many aspects of angiogenesis that can be targeted therapeutically. But identifying which steps and components of this process are the most susceptible to treatment with drugs (‘drugable’) is an important concern. The most effective therapies will probably involve targeting combinations of factors, as well as improving the efficiency of drug delivery to the tumor microenvironment. Hypoxia and inflammation

Breakdown of basement membrane ECM: COL IV, laminin, COL XVIII MMPs and TIMPs Hypoxia

ECM fragments and cryptic sites

Cytokines

α4β1

Integrins Growth factors

Blood vessel

Angiogenic integrins: COL–α1β1, α2β1 LN–α6β1 FN–α5β1, αvβ3

Deposition of provisional ECM (FN, COL, VN, LN)

Tumor cells release VEGFA, SD1α,TNF-α, IL-1β and IL-6.

Hypoxia CD11b myeloid cell

HIF1α

Figure 2 Tumor microenvironments that favor blood vessel growth. Environmental or genetic events transform normal epithelial cells into tumor cells, which grow and divide with little effect on their surroundings until their size exceeds 1–2 mm. At that point, hypoxia and nutrient deprivation trigger the requirement for angiogenesis. Tumor cells release soluble growth factors, chemokines and cytokines, which create a concentration gradient that initiates the sprouting and proliferation of formerly quiescent endothelial cells on nearby blood vessels and lymphatics. These signals also recruit fibroblasts that deposit a repertoire of ECM proteins and enzymes in an attempt to remodel and repair the site. Most tumors elicit an inflammatory response that attracts myeloid cells into the tumor microenvironment, and these cell types release their stores of soluble factors to escalate the angiogenic response. This microenvironment continually changes and evolves as the tumor grows, creating localized pockets of hypoxia, inflammation and ECM turnover that affect blood vessel growth, remodeling and maturation.

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Angiogenic signaling pathways at the crossroads Strategies to inhibit specific signaling pathways in vascular endothelial cells could have a major impact on endothelial cell function, but these strategies can also have effects on other compartments as well. The angiogenic process requires the participation of multiple cell types and can therefore be regulated by various independent and interdependent factors (Fig. 3). Guidance molecules control cell recruitment. Similar to neuronal sprouting, vascular sprouting requires relevant cell types to migrate, elongate or retract in response to local guidance cues. As is seen in angiogenic growth factors, each signaling family involved in guidance—Notch, Semaphorins, Ephrins and Slits—is comprised of multiple ligands, for example, SLIT1, SLIT2 and SLIT3, that are recognized by one or more of its receptors, for example, ROBO1, ROBO2, ROBO3 and ROBO4. A cell expressing a certain ligand can bind to a neighboring cell expressing the appropriate receptor to drive signaling through a paracrine mechanism or can activate signal-

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BOX 1 Beyond sprouting angiogenesis A form of vasculogenesis occurs in adults when bone marrow– derived endothelial precursor cells move to an angiogenic site and integrate into newly forming vessels. Isner and his colleagues first isolated putative progenitor endothelial cells from human peripheral blood that could differentiate into mature endothelial cells in vitro and participate in augmenting vessel growth in response to ischemic injury in vivo86. Rafii and his colleagues then established that impairing this recruitment of bone marrow–derived endothelial and hematopoietic precursor cells could block tumor angiogenesis and growth87, providing the first evidence that blood vessel growth in cancer involved both vasculogenesis and angiogenesis. In fact, it was shown that dual blockade of VEGFR1 and VEGFR2 limited the recruitment of multiple cell types from the bone marrow and their incorporation into the tumor vascular compartment87. More recent preclinical studies have established that endothelial cells can actively recruit bone marrow–derived cells in an angiocrine manner by producing and releasing multiple growth factors or Notch ligands that attract circulating stem and progenitor cells88,89. Circulating monocytes are attracted to the inflammatory signature in the tumor zone, where they can rapidly differentiate into mature tumor-associated macrophages that exert specific immunologic functions90. Angiopoietin-2 released by tumor-associated endothelial cells recruits a population of Tie2-expressing monocytes into the tumor microenvironment, where they associate with angiogenic blood vessels and prompt the endothelial cells to release additional factors that escalate the angiogenic response91,92. Although the functional contribution of these macrophages to the progression of human cancer is less clear from a clinical perspective, these cells may be an important factor to consider when evaluating therapeutic strategies. For example, patients with colorectal cancer treated with the EGFR-targeted cetuximab show an abundance of tumor-associated macrophages associated with the release of tumor-promoting factors such as interleukin-10 (IL-10) and VEGF93. Notably, preclinical mouse models would have not predicted this effect of cetuximab, which is attributed to an interaction between therapeutic monoclonal antibodies and Fc-binding receptors on human macrophages that are not conserved in mice93. Understanding why certain drug combinations are effective or ineffective may require analysis of bone marrow–derived cells within clinical specimens rather than relying solely on preclinical models. Preventing mobilization of the various bone marrow–derived cell types with antiangiogenic drugs or by genetic manipulation reduces the angiogenic response, sensitizes the tumor to therapy and limits metastatic spread in multiple preclinical models94–99. Thus, suppressing the recruitment of circulating cells to a tumor may reduce the resistance and/or improve the sensitivity to anti-

ing in an autocrine manner when the same cell expresses both the ligand and the receptor. Thus, the expression profile of the various guidance molecules and their receptors on the multiple cell types involved in the angiogenic response can provide many combinations of local and directional cues that dictate how cells respond to their environment and how they interact with neighboring cells to control tip cell sprouting, perivascular cell coverage, recruitment of progenitor cells and attraction of macrophages. In some instances, ligand-receptor binding may modulate other angiogenic pathways. For example, Slit ligands binding to the vascular-specific ROBO4 receptor can inhibit the signaling of VEGF14 and nature medicine volume 17 | number 11 | november 2011

vascular agents and chemotherapy agents as well as limit the triggers driving metastatic spread. Preventing the production and incorporation of these bone marrow–derived cell types into the tumor vasculature could also provide long-term benefit by disabling a key mechanism for tissue regeneration following anticancer therapy88. Several lines of evidence support the concept of vascular mimicry, which was first proposed by Hendrix and her colleagues and in which tumor-derived cells differentiate to form vascular channels that augment the angiogenic response100. Although the novelty and importance of this vasculogenic mimicry concept has been challenged101–105, an angiography study on an individual with melanoma showed the circulation of blood inside such tubes in the eye106 to validate the existence of this alternative vascular network, albeit in a rare tumor type. Although a number of recent studies have proposed the involvement of cancer stem cells in this process (see ref. 107), the existence and functional importance of vascular mimicry in cancer remains under debate. Whereas vascular mimicry occurs when tumor cells simply relocate to physically form vascular structures that resemble endothelial tubes, some malignant tumor cells can become bona fide endothelial cells. In particular, the ability of cancer stem cells to differentiate into vascular cell types has been reported in leukemia108, in breast109, renal110 and ovarian111 cancers, and most recently, in glioblastoma multiforme (GBM) tumors112,113. Neural stem cells were known to be able to differentiate into endothelial cells114, and GBM cancer stem cells apparently retain this ability, as human endothelial cells can be formed by human GBM tumors growing orthotopically in mice112,113. The ability to autonomously generate endothelium is not a quality of all tumor cells but, rather, is unique to certain cancer stem cells. In GBM mouse models, ablation of the stem cell population positive for the glycoprotein CD133 limits angiogenesis and induces tumor regression113. Mechanistically, blocking Notch1 prevents the transition of CD133+ cells into endothelial progenitors, and blocking VEGF or silencing VEGFR2 inhibits the maturation of tumor-derived endothelial progenitors into endothelium cells112. The Notch pathway also plays a crucial role in linking angiogenesis and cancer stem cell self renewal in GBM, further suggesting Notch as a potential therapeutic target for angiogenesis in GBM115. Despite the evidence from multiple preclinical models, the clinical importance of this mechanism in terms of the tumor vascular response has yet to be determined. Time will tell whether this tumor cell differentiation phenomenon is an important factor in terms of neovascularization and tumor progression and whether targeting this event in patients will recapitulate what has been observed in the preclinical models.

fibroblast growth factor (FGF)15 pathways. Similarly, neuropilin-1 is a co-receptor for both Semaphorin and VEGF ligands, and the state of its ligand binding can shift the balance of signaling between the respective receptors16. Semaphorin binding to the Plexin D1 receptor limits the angiogenic potential of VEGF and guides tip cell formation17. The situation can be even more complicated if there are multiple signaling pathways involved, as local VEGF expression can dictate the fate of a cell to the tip or the stalk by controlling the expression of the Plexin D1 receptor, which binds Semaphorin 3E and negatively regulates the Deltalike 4 (DLL4)-Notch signaling pathway18. Inhibition of one receptor or ligand may therefore have crossover effects on alternative signaling 1361


review Figure 3 Mediators of endothelial activation Fibroblast Tumor cell Myeloid cell and the tumor angiogenic response. Each cell in the tumor microenvironment is affected by its MMP-2 neighboring cells, as well as by the components of Deposit provisional or MMP-9 Oncogenic changes, tumor-specific the tumor stroma. By the nature of their oncogenic MT1-MMP Differentiate into TAMs hypoxia response ECM Hypoxia transformation or their response to hypoxia, tumor TIMPs and neutrophils cells release a variety of factors that begin to Activation and recruitment to tumor through transform their environment. These factors induce Extracellular Gr1, α4β1, PI3K p110γ matrix the remodeling of the ECM to accommodate tumor growth and progression. Fibroblasts are TUMS BMPs, TSPs FN, Col END prompted to deposit ECM proteins, which foster ADAMs, SPARC VN, LN CANS syndecans, perlecan Autocrine/paracrine factors: the remodeling required for tumor expansion fibrin Cryptic sites VEGF, FGF, PDGF, SDF-1α, Perivascular cell and also release enzymes that can break down TNF-α, IL-1β, IL-6, angiopoietins, coverage & vascular chemokines, semaphorins components of the quiescent basement membrane normalization to expose cryptic binding sites or to form VSMC fragments that affect the function of integrins on Basement membrane neighboring cells. The ECM compartment also Endothelial cell contains components such as bone morphogenetic Integrins Growth, cytokine and guidance receptors Integrins, Notch α5β1/FN proteins (BMPs), thrombospondins (TSPs), a VEGFRs, PDGFRs, FGFRs, Ang-1 and Ang-2, PDGFR, VEGFR αvβ5/VN chemokines, Notch, semaphorins, ephrins, Slits disintegrin and metalloprotease (ADAM) family αvβ3/many members, secreted protein, acidic and rich in cysteine (SPARC), syndecans and perlecan, which Crosstalk between receptors have the capacity to govern the sequestration Angiogenic programs: or release of soluble factors within the matrix. VEGFR2 VEGF/FGF Slit/Robo Tip cell vs. stalk cell, sprouting vs. quiescence c-Met Tumor cells and activated endothelial cells αvβ3 EPC & myeloid cell recruitment FGFR1 Semaphorin/ or Invasion & migration release a range of soluble factors that induce the VEGF/NRP-1 PDGFR NRP1/PlexinD1 α5β1 Survival & proliferation mobilization and homing of myeloid cells from the EGFR Increased expression of: Notch/DLL4 PlexinD1/Sema3E IGFR bone marrow and their differentiation into tumorp-Erk, p-STAT3, NCAM, MSF, αvβ3 α5β1 VEGFR2/αvβ3 Signaling Tie2 associated macrophages or neutrophils. Within this dynamic environment, integrins and receptors on the surface of endothelial cells recognize and bind factors that initiate intracellular signaling pathways, leading to phenotypic changes that promote the migration, invasion, survival and proliferation that are required for sprouting. Receptors from different pathways can crosstalk to either suppress or augment cellular activation. The interactions between endothelial cells and their environment regulate their association with perivascular cells or tumor-associated macrophages and determine, in a localized sense, whether they become a tip cell or a stalk cell. Therapeutically targeting angiogenesis requires consideration of these signaling components among the different cell types and how receptor crosstalk might affect the net result. These complicated interactions govern angiogenic remodeling and can contribute to de novo or acquired resistance to a targeted therapy. A successful antiangiogenic approach should consider the signaling effects within the interdependent cell types involved in the constantly changing tumor microenvironment. FN, fibronectin; Col, collagen; VN, vitronectin; LN, laminin; TUMs, tumstatin; END, endostatin; CANS, canstatin.

pathways, with opposing effects within distinct locations during the angiogenic response or both. It is important to consider whether the readout of angiogenesis in a given assay is representative of global or local effects. Is angiogenesis enhanced or suppressed by preventing tip cell formation? What are the effects of blocking the endothelial stalk cell phenotype? It will be crucial to understand whether preventing the recruitment of perivascular cells destabilizes newly formed vessels and sensitizes them to vascular disrupting agents, as well as how suppression of one pathway affects the activity of other pathways within distinct local venues. Translating these findings into new therapeutic strategies will require an understanding of how guidance molecules and angiogenic growth factors cooperate. The balance of power between such pathways will probably vary as a function of concentration, location and time during the angiogenic process, and certain tumor cells might have developed strategies to preferentially perturb one or more of these pathways by their secretion of soluble ligands, their expression of receptors or both. Therefore, tumors may show differential sensitivity to certain antivascular therapies. Potential therapies could include targeting the key upstream factors driving these pathways or the common downstream mediators. Integrins orchestrate tumor angiogenesis. The tumor stroma is composed of a variety of ECM components that regulate the angiogenic cascade19, and integrins are the primary cell-matrix adhesion molecules that integrate signals between the ECM and intracellular signaling pathways20. Each integrin is a heterodimer comprised of a and b subunits 1362

that combine to ligate one or several ECM proteins. In particular, av and a5b1 integrins recognize the Arg-Gly-Asp sequence in their respective ligands: a5b1 binds fibronectin, avb5 binds vitronectin, and avb3 binds many substrates, including fibronectin, vitronectin, von Willebrand factor, tenascin, osteopontin, fibrillin, fibrinogen and thrombospondin21. Once ligation to a specific matrix protein occurs, integrin-mediated signaling typically develops as a function of crosstalk between integrins and specific activated cytokine or growth-factor receptors. These signaling mechanisms can determine whether a cell is in an appropriate microenvironment and will accordingly affect cell survival, migration and invasion. A cell in an inappropriate environment may express unligated integrin, which can actively promote apoptosis, anoikis or integrin-mediated death22,23, whereas proteolytic degradation of certain ECM proteins can produce fragments that also act as endogenous inhibitors of angiogenesis by perturbing integrin function24,25. These cellular functions can be modulated by exposing cells to soluble factors, peptides or antibodies, which compete for binding with matrix ligands but do not result in full integrin activation. As such, these integrin antagonists are possible therapies for treating cancer by selectively disrupting integrin signaling on both tumor and stromal cells20. Nearly 20 years ago, the selective expression of integrin avb3 on angiogenic endothelium was reported26, and cyclic peptides or functionblocking antibodies targeting this integrin were shown to successfully block angiogenesis and promote the regression of preestablished tumors in vivo27. Integrin avb3 expression in various tumors was also associated with increased tumor growth and metastasis in preclinical models and in volume 17 | number 11 | november 2011 nature medicine

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BOX 2 Tumor-induced inflammatory responses recruit multiple cell types to support the angiogenic process Whereas the recruitment of myeloid cells initiates a repair and cleanup program during physiological wound healing, this system is exploited by the tumor to bathe the microenvironment in stimulatory factors that support tumor cell survival and angiogenesis. As a tumor proliferates, it rapidly outgrows its blood supply, and pockets of hypoxia arise. The induction of HIF1a expression in hypoxic tumor, endothelial and stromal cells drives their production and the release of angiogenic factors, including VEGF, PDGF, FGF, angiopoietins and SD1a116. Myeloid cells attracted to these pools of stimuli in the hypoxic areas can differentiate into tumor-associated macrophages or neutrophils, which closely associate with vascular endothelial cells and release their own supplies of cytokines and proangiogenic growth factors117–120. Over time, persistent invasion of myeloid cells into the tumor results in the accumulation of these cells (comprising as much as 25% of the tumor mass121) to fuel tumor progression. The recruitment of myeloid cells has been linked to poor outcome for a number of tumor types in the clinic122. For example, in individuals with classic Hodgkin’s lymphoma, a higher content of tumor-associated macrophages was associated with shortened survival time, and gene-expression profiling identified a particular gene signature associated with treatment failure that could provide a new biomarker for risk stratification123. Because refractoriness to anti-VEGF therapy is associated with

the clinic28,29. For example, glioblastomas are high-grade brain tumors with strong expression of both the matrix protein vitronectin and its receptor, avb3 (refs. 28,30). As brain tumors are highly dependent on angiogenesis for their growth and spread, a cyclic peptide antagonist of avb3 causes regression of orthotopic glioblastomas in mice31. However, the growth of the same brain tumor cells injected simultaneously into the subcutis of mice was not affected by the integrin antagonist, suggesting that the brain microenvironment is a crucial determinant of brain tumor susceptibility to the inhibition of growth by integrin avb3 antagonists31. This important distinction raises the question of whether the efficacy of blocking av integrin function in a glioblastoma is a function of inhibiting integrins on tumor cells, angiogenic blood vessels or both. These preclinical studies helped pave the way for the testing of cilengitide, a cyclic peptide targeting avb3 and avb5 integrins, in people with glioblastoma. In phase 1 and 2 clinical trials, cilengitide produced a survival benefit in individuals with late-stage glioblastoma, was well tolerated and had a favorable safety profile32. The results of the phase 3 Cilengitide in Combination With Temozolomide and Radiotherapy in Newly Diagnosed Glioblastoma Phase III Randomized Clinical Trial (CENTRIC)33,34, which is currently nearing completion, are anticipated with hopes that cilengitide will be the first integrin antagonist approved by the US Food and Drug Administration (FDA) for cancer therapy. Randomized controlled phase 2 studies with cilengitide are ongoing in non–small-cell lung cancer, prostate cancer and squamous cell carcinoma of the head and neck32. Additional therapies targeting proangiogenic integrins are currently in clinical trials for cancer therapy (see refs. 32,35), including blocking antibodies to av (DI-17E6 and IMGN388), avb3 (MEDI-522/Vitaxin), a5b1 (M200/volociximab and PF-04605412), avb3 and avb5 (CNTO 95/intetumumab), peptide inhibitors of a5b1 (ATN-161) and multiple integrins that bind Arg-Gly-Asp (GLPG 0187). If successful, these clinical trials will provide support for the idea that therapies with an antiangiogenic component are capable of providing a tangible survival benefit in individuals with highly aggressive cancer. However, the relanature medicine volume 17 | number 11 | november 2011

the infiltration of myeloid cells into the tumor, combining antiVEGF treatment with an antibody to GR1 that targets myeloid cell mobilization inhibits the growth of refractory tumors more effectively than anti-VEGF therapy alone124,125. A study using multiple mouse orthotopic tumor models showed that a range of chemoattractants (such as SDF-1a, VEGF-A, tumor necrosis factor-a (TNF-a), IL-1b and IL-6 (ref. 121)) are released by tumor cells that act to escalate tumor inflammation by converging to activate the PI3K isoform p110g in GR1+CD11b+ myeloid cells, promoting the inside-out activation of integrin a4b1 and the myeloid cell to the tumor endothelium, leading to invasion into the tumor121. Because these are common mediators of inflammation induced by a variety of chemokines, growth factors and cytokines, pharmacological or genetic blockade of either p110g or a4 integrin in myeloid cells markedly suppressed the expression of inflammatory and angiogenic factors and, thereby, the growth and metastasis of multiple epithelial cancers in preclinical models121. Taken together, these recent studies support the concept of targeting the recruitment, infiltration and retention of myeloid cells into the tumor as a means of avoiding bathing vascular and stromal cells in proangiogenic stimuli. In addition to suppressing angiogenesis, this strategy limits the release of factors that also promote tumor cell proliferation, invasion and survival.

tive benefit of targeting integrins in vascular cells compared to tumor cells will probably be determined by the particular tumor cell genetics and tissue microenvironment in each individual tumor. Growth factor receptor and integrin crosstalk. There are several prolific families of angiogenic growth factors that have been extensively studied. Among these families are VEGFs, FGFs, PDGFs and the angiopoietins (Ang-1 and Ang-2). Each family of growth factors (for example, the VEGFs) comprises specific members (for example, VEGF-A) and a series of splice variant isoforms (for example, VEGFA-121, VEGFA165, VEGFA-189 and VEGFA-206) among which each has a unique role in signaling cascades36. In general, these soluble or matrix-bound growth factors signal by binding to one or more specific receptors (for example, VEGF receptor 1 (VEGFR1), VEGFR2, neuropilin-1 or neuropilin-2) on the surface of target cells (for example, endothelial or perivascular cells)36. The affinity and avidity of the receptors for their respective ligands is often enhanced by the clustering and dimerization of the receptors within the plasma membrane, which can manifest as homodimers of the same receptor subtype or as heterodimers from the same family (for example, VEGFR1 and VEGFR2) or receptors from distinct families (for example, VEGFR2 and PDGF receptor b (PDGFRb)). Receptor clustering augments ligand binding, which triggers downstream signaling intermediates and often leads to receptor internalization or recycling to determine the magnitude and kinetics of the signaling response37–39. Optimal growth factor stimulation often relies on integrin-mediated adhesion to an appropriate ECM protein, and this adhesion is relevant for multiple cell types that are involved in the angiogenic response. Although certain integrins and growth factor–signaling pathways show crosstalk, how this phenomenon affects the function of antagonist molecules is unclear. Whereas avb3 interacts with a number of angiogenic growth factor receptors, including VEGFR2, the hepatocyte growth factor receptor c-Met, FGFR1, PDGFR, EGF receptors and insulin-like growth factor 1 receptor (IGF-1R), integrin a5b1 interacts with some of these same receptors, as well as the cell-surface receptor TIE2, which 1363

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BOX 3 Resistance to VEGF-targeted therapies Judah Folkman first proposed the strategy of starving a tumor of its blood supply more than 40 years ago126, and this approach has been experimentally and clinically tested in the years since. However, in both preclinical and clinical settings, resistance mechanisms limit the long-term benefit of VEGF-targeted strategies127–129. For example, anti-VEGF therapy leads to the upregulation of placenta growth factor (PlGF)130, which binds to VEGFR1 and leads to the transphosphorylation of VEGFR2 (ref. 131). Accordingly, treatment with a function-blocking antibody to PlGF suppresses tumor growth and angiogenesis and is able to enhance the anti-cancer activity of VEGFR blockade or VEGF-Trap therapy130,132. Notably, one mechanism of activity for therapies using antibodies to PlGF has been attributed to suppression of macrophage infiltration into the tumor130. But PlGF also simulates recruitment of bone marrow–derived endothelial progenitor cells that confer resistance to anti-VEGF therapies131. Thus, blocking PlGF activity probably improves sensitivity to antiVEGF therapies by several mechanisms. In addition, although multiple mouse models of cancer show initial sensitivity to VEGFR2 antagonists, they become more aggressive, invasive and metastatic after several weeks of treatment133,134. Notably, cessation of

binds angiopoietins40–43. If integrin and growth factor crosstalk promotes maximal signal transduction downstream of angiogenic growth factors, disabling these interactions by impairing both components might therefore be subject to less compensation or resistance. The association of avb3 with VEGFR2 promotes the full activation of each receptor, leading to maximal angiogenesis in response to VEGF, and this receptor crosstalk is facilitated by the kinase SRC44 and the small GTPase RAP1B45. Rather than being generic mediators of receptor crosstalk, it seems that SRC and RAP1B are selectively required for the interaction

treatment is not sufficient to reverse the increased malignancy, suggesting that the tumor has adapted new mechanisms to circumvent their requirement for the VEGF pathway and other angiogenic pathways. One feature of this adaptive response is the appearance of regions of increased hypoxia at both the tumor and metastatic sites, a consequence of the vascular regression induced by antiangiogenic therapy133. Targeting the response to hypoxia might therefore alleviate some of the acquired resistance to antiangiogenic therapies. It is clear that antiangiogenic therapy substantially changes how tumor cells interact with their microenvironment. This phenomenon is not unique for VEGF-directed or antiangiogenic therapies, and a similar activation of tumor cell phenotype occurs for breast cancer patients treated with the antibody trastuzumab (Herceptin), which blocks the function of EGF receptor 2; these patients showed an increased incidence of brain metastases135. In this setting, however, the positive effects of suppressing aggressive EGFR2-driven breast cancer using targeted therapy does improve the clinical outcome despite the higher incidence of metastasis in the brain135. It is possible that certain tumors could benefit similarly from anti-VEGF therapy, despite the presence of very measurable adverse effects.

between avb3 and VEGFR2 but not for several other pairings between integrins and growth receptors44,45. Some integrins can also directly bind growth factor ligands. Endothelial cells can adhere and spread in vitro on immobilized VEGF ligands mediated by direct integrin-VEGF interactions that occur independently of VEGF receptor binding46, and integrins such as a5b1 and avb3 can directly bind angiopoietins47,48. Thus, tumor or stromal cells that lack receptors for these growth factors may function differently in their presence or when they are targeted with function-blocking antibodies.

Figure 4 Intracellular signaling effectors of the angiogenic response. In general, an antiangiogenic Guidance molecules Angiogenic factors Extracellular matrix & receptors & receptors components & integrins strategy can target one or more different signaling components. At the extracellular level, the transmission of signals from the tumor microenvironment can be blocked by impairing the binding of soluble or matrix-bound factors or the activation of cell-surface receptors. As suppressing a single pathway at this level invites Membrane proximal Ras, Src, FAK, PKC, resistance or compensation by parallel pathways, PI3K, Shc, PAK signaling molecules some approaches target multiple receptors or adhesion molecules. The receptor-initiated signals could be blocked by the inhibition of Downstream signaling Raf, MEK, Akt, PTEN, mTOR, eNOS, p38MAPK, p53 various classes of membrane-proximal signaling intermediates microRNAs molecules. Because these signaling molecules are often downstream of multiple receptors, blocking miR-126, Erk, Jnk, Plk-1, Aurora A, p21, Common effector a single component may have a more noticeable miR-130a, miR-132, 14-3-3, NFkB, Erk5, Ral, Cyclins, signaling hubs effect. Likewise, blocking downstream signaling CDKs, Caspases, Bad, ASK-1, NO miR-210, miR-296, intermediates or common effector signaling hubs miR-221, miR-222 may provide a robust antiangiogenic response Elk, CREB, HIF-1α, HIF-1β, VEGF, that is less susceptible to resistance mechanisms. Gene transcription chemoattractants for EPC and TAM miRNAs are a potential strategy to suppress the expression of one or more proangiogenic signaling components or to change the balance of signaling Proliferation, migration, by driving the expression of negative regulators. cell cycle, survival Successful development of new antiangiogenic strategies should consider the network of intracellular signaling effectors in an attempt to identify components that could be targeted alone or in combination to shift from an activated to a quiescent endothelial cell phenotype. However, these same pathways exist in other cells (including stromal constituents and tumor cells). Thus, manipulation of a molecular target could have opposing effects on different cell types or on cells in distinct locations within the tumor microenvironment.

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review Regardless of the mechanism, interactions between integrins and growth factors should be considered as a part of the equation when targeting angiogenic signaling pathways. Understanding how to target these crosstalk events could improve the efficacy of current antiangiogenic strategies and could potentially block a mechanism that contributes to resistance to therapy. ECM remodeling controls angiogenic potential. The tumor stroma undergoes continuous remodeling to support angiogenesis, and MMPs that proteolytically degrade ECM components are crucial in this process. The effect of any MMP is counteracted by the expression of its natural antagonists (tissue inhibitors of MMPs) and regulated by the availability of its substrate. MMPs such as MMP-2, MMP-9 and MT1-MMP contribute to the angiogenic process by remodeling the basement membrane to allow sprouting, liberating matrix-bound angiogenic factors and cleaving matrix proteins into antiangiogenic fragments49,50. A particular MMP has the capacity to cleave a given matrix component at certain sites to create fragments that can be recognized by specific receptors. It is therefore not unexpected that a certain MMP may act in either a proor antiangiogenic capacity in different environments49. MMP-mediated degradation of collagen can lead to the exposure of cryptic Arg-Gly-Asp sites that can be recognized by the integrin avb3 (ref. 51), which probably allows newly sprouting endothelium that express avb3 to selectively interact with the remodeling ECM. In fact, antibodies to these ArgGly-Asp sites suppressed angiogenesis and tumor growth in preclinical models52. Understanding the context of the tumor microenvironment is therefore crucial for assigning the functional contribution of MMPs and tissue inhibitors of MMPs and for addressing the functions of these enzymes as a part of therapeutic strategies to target tumor angiogenesis. MicroRNAs as intracellular angiogenic switches. Recent studies have shown the regulation of vascular development and angiogenesis by microRNAs (miRNAs), which are small noncoding RNAs that bind to the 3ʹ untranslated region of mRNAs, recruit a silencing complex and block translation53. For an additional level of control, a variety of RNA-

binding proteins affect miRNA function by regulating their biogenesis, localization, degradation and activity54. The relationship between a given miRNA and its pairing target is not exclusive, and depending on what genes it suppresses, a given miRNA could have either a positive or negative role in the regulation of angiogenesis. For example, a handful of miRNAs function both coordinately and competitively to regulate VEGF expression55. In the past decade, specific angiogenic regulatory miRNAs termed angiomiRs have been identified, and some of their gene targets have been validated in order to provide mechanistic insight into their activity56–59; miR-126, miR-130a, miR-210 and miR-296 promote angiogenesis, whereas miR-221 and miR-222 block angiogenesis56. These angiomiRs can inhibit a range of target genes and, thus, function by regulating a variety of mechanisms such as migration, survival and response to hypoxia. If gene delivery of miRNAs or inhibitors of miRNAs (‘anti-miRs’) can be optimized, targeting angiomiRs may ultimately be a powerful approach in manipulating cancer progression. Unique miRNA signatures were observed for the distinctive stages of multistep tumorigenesis in the RIP1-Tag2 mouse model of pancreatic cancer60, which is a model used to study the angiogenic switch. In particular, 11 miRNAs were differentially regulated during the angiogenic switch from hyperproliferative islets to angiogenic islets. Notably, the signature of the angiogenic stage primarily involved the upregulation of miRNAs, including the known angiomiR miR-126 (ref. 60). One interpretation of this data is that the miRNA-regulated switch to the angiogenic state largely involves suppression of target genes, analogous to releasing a brake. Thus, treatment with the corresponding anti-miRNA inhibitors could counteract these increases in miRNA expression, allow the expression of antiangiogenic target genes and suppress the angiogenic response. For example, exposing endothelial cells to VEGF or basic FGF induces the expression of miR-132, which, by suppressing p120RasGAP (the Ras GTPase activating protein) expression, leads to increased Ras activity and endothelial cell proliferation61. Expression of its inhibitor, anti-miR-132, preserves p120RasGAP expression, sup-

BOX 4 The potential of ligands to selectively target tumor endothelium In 1994, we found that integrin avb3 was highly upregulated on the tumor vasculature relative to quiescent vessels in normal tissues26,27. This led to the development of avb3-targeted nanoparticles to successfully image136 and treat tumors growing in vivo72. Specifically, Sipkins et al.136 developed gadoliniumlabeled nanoparticles coated with a monoclonal antibody specific to avb3 (LM609) that could home to the vasculature of carcinomas growing in rabbits136. Tumors were then imaged by magnetic resonance imaging, which revealed angiogenic ‘hotspots’ in locations in which tumor growth could not yet be detected. By targeting a mutant RAF gene or using cytotoxic drugs to avb3 on tumor blood vessels, it was possible to produce a strong antitumor activity with few, if any, side effects71,72. In fact, positron emission tomography with avb3-targeted tracers can detect the tumor response to antiangiogenic therapy before any substantial volumetric changes are observed137,138. Pasqualini and Ruoslahti pioneered a method to screen phage display libraries for peptides that home specifically to tumor vasculature139. Since then, the resulting ‘vascular zip codes’140 have proven useful in directing therapeutic agents to tumors in mice141 and have shown promise in monitoring response to therapy, as a new bevacizumabresponsive peptide that binds to tumor-associated vasculature allows non-invasive visualization of early responses to anti-VEGF therapy142. When human tumor-derived endothelial cells are


isolated and grown in mice, the same phage-display screening approaches identified markers specific for human renal-tumor– associated blood vessels143. A new peptide that binds collagen IV only when proteolytically modified by matrix metalloproteinase-2 during tumor blood vessel remodeling accumulates in tumors and blocks angiogenesis144. This approach has also shown potential for targeted gene delivery, as the electrostatic complex of an avb1-binding peptide with the anti-cancer TP53 gene homes to tumor endothelial cells in vivo, delivers the TP53 gene and induces apoptosis145. Tumor-associated endothelial cells and perivascular cells can also be targeted with separate peptides that, when combined, produce enhanced anti-tumor efficacy with nearly total ablation of both cell types within the tumor microenvironment146. The unique ‘iRGD peptide’ targets the Arg-Gly-Aps sequence on avb3-expressing angiogenic endothelial cells within tumors, whereas its C-end rule (CendR) sequence mediates neuropilin-1–dependent penetration into cells in the tumor microenvironment147–149. These properties allow the iRGD peptide to improve tumor-targeted drug delivery for agents that are either conjugated to the peptide or which are simply coadministered150. Peptides identified using phage-display library screening approaches therefore have potential applications for delivering drugs, genes or imaging agents selectively to cells associated with the activated tumor vasculature.

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review presses Ras activity and blocks angiogenesis downstream of multiple growth factors to drive vascular quiescence61. Other miRNAs have been shown to signal in tumor cells and affect the vascular response within the tumor microenvironment. For example, miR-20b modulates VEGF expression by targeting hypoxiainducible factor 1a (HIF-1a) and signal transducer and activator of transcription 3 (STAT3) in breast cancer cells62. Overexpression of miR-93 in U87 glioblastoma cells decreases integrin avb8 expression, resulting in increased growth and tube formation of co-cultured endothelial cells in vitro and enhanced tumor angiogenesis in vivo63. The tumor suppressor p53 dampens the cellular response to hypoxia by suppressing HIF-1b expression through miR-107 (ref. 64). Thus, overexpression of miR-107 in tumor cells suppresses tumor angiogenesis, tumor growth and the expression of VEGF in mouse tumors64. miR591c, which is expressed in individuals with cancer who have a better prognosis, targets HIF-1a and blocks tumor angiogenesis65. Targeting miRNA signaling pathways in tumor cells as well as in angiogenic endothelial cells opens new therapeutic avenues to suppress tumorassociated angiogenesis. The ultimate application of any miRNA or anti-miRNA approach to block angiogenesis will require the development of new gene delivery methods, as the miRNAs or anti-miRNAs used are short RNA sequences that must be expressed in the target cell types. Whereas this approach is somewhat feasible in preclinical models, translating this approach to humans is more complicated because the miRNA or anti-miRNA used needs to be effectively delivered to and taken up by the relevant cell type in vivo. One organ that readily takes up systemically delivered miRNA reagents is the liver, providing a viable therapeutic option for liver cancer or cancers that metastasize to the liver66. Approaches using adeno-associated virus for miRNA delivery have also shown promise in the liver67 and brain68. miRNAs can be delivered to metastatic tumor cells in bone using an atelocollagen delivery method69 or to tumor cells in the lung using a neutral lipid emulsion70. There is initial proof of concept that an anti-miRNA could be delivered to tumor endothelium using targeted nanoparticles61, as angiogenic endothelial cells endocytose avb3-targeted nanoparticles

along with their payloads71,72. Although there are a number of potential methods to deliver miRNAs or anti-miRNAs to either tumor cells or tumor-associated blood vessels, substantial progress must still be made before any of these options can progress to the clinic. Considerations for targeting angiogenesis Is starving a tumor of its blood supply really a rational approach to suppress cancer progression, considering that some antiangiogenic approaches are likely to target other vascular processes as well? A tumor effectively hijacks and exploits various physiological functions already in place and, in some instances, prompts quiescent cell types to revert back to their embryonic state of proliferation and differentiation. In addition, the same angiogenic signaling cascades that occur in tumors also regulate the normal physiological vascular remodeling that occurs during pregnancy and wound healing. These caveats apply to nearly every therapeutic target, ranging from proangiogenic growth factors, integrins, ECM molecules and bone marrow–derived myeloid cells to miRNAs. Further, recent evidence showed how VEGF-targeted therapies can change how tumor cells interact with their environment by converting them to a more aggressive and metastatic phenotype (Box 3). Although there are certain pitfalls involved in targeting these pathways, there are few safe alternatives, and it is often the case that treating a lifethreatening tumor with the potential for metastatic spread trumps other risks. The field of angiogenic research has identified hundreds of new potential therapeutic targets, but researchers from the field have generally not succeeded in translating these ideas into safe and effective antiangiogenic strategies for treating cancer patients. This section highlights several issues for which there are currently no clear solutions but which warrant consideration when developing new therapeutic strategies. Blocking a single pathway may have opposing effects on multiple cell types. As the generation of functional blood vessels requires some level of pruning and vascular maturation, cells providing perivascular coverage of nascent vessels are crucial. For example, culturing endothelial cells in the presence of vascular smooth muscle cells (VSMCs) inhibits endothelial cell proliferation73. In fact, tumors overexpressing VSMC mitogen PDGF showed increased perivascular cell content and

BOX 5 New approaches to image angiogenesis Assessing angiogenesis in cancer patients can use functional measures (for example, blood flow, blood volume or vascular permeability) detected using standard imaging modalities, such as dynamic contrast-enhanced computed tomography or magnetic resonance imaging. Recently, a number of new targeted techniques to image the angiogenic content of a tumor and its response to antiangiogenic therapy have been developed and tested in the preclinical and clinical settings. VEGF positron emission tomography (PET) imaging allows serial analysis of angiogenic changes in different areas within a tumor treated with sunitinib151. Similarly, VEGF single photon emission computed tomography imaging with a tracer dose of 111In-bevacizumab can detect the presence of VEGF in stage 3 and 4 melanoma lesions152, just as PET imaging can detect tumor angiogenesis using a radioimmunoconjugate 86Y-CHX-A’’-DTPA-bevacizumab153. Integrin avb3 expression on angiogenic blood vessels (as well as on some tumor types) can be assessed and quantified noninvasively using radiolabeled Arg-Gly-Asp peptides for PET154. Monitoring the tumor response to sunitinib in mice using PET tracer 18F-fluciclatide (which is selective for avb3 and avb5 integrins) showed a reduction in tracer uptake only two days after starting antiangiogenic

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treatment138. Similarly, the 64Cu-DOTA-RGD tracer for PET could identify the activation status of integrin avb3 after sunitinib treatment155. These studies support the concept of imaging angiogenesis with targeted tracers for VEGF or avb3 to monitor the course of antiangiogenic therapy and to inform about the response noninvasively over time in the clinic. Optical frequency domain imaging is an alternative imaging approach capable of high-resolution, wide-field, deep imaging of tumor vasculature and allows for geometrical and fractal analysis of the imaging data sets to quantify vessel size and shape and the density of the vascular network156. Notably, this method can be used to assess drug activity in the form of angiograms acquired at different time points before or after VEGFR2 blockade, which indicates antiangiogenic activity in vivo156. This new technology bridges the gap between magnetic resonance imaging and computed tomography technologies, which can image greater depths at less resolution, and multiphoton optical microscopes, which can only reach subcellular resolution. Imaging the tumor microenvironment before and after exposure to antiangiogenic therapy would provide a new means to assess a drug’s doseresponse effects and to assess drug resistance.



review decreased tumor size73, consistent with the idea that perivascular cells promote the maturation or quiescence of their neighboring endothelial cells. Accordingly, treating PDGF-expressing tumors with the PDGFreceptor inhibitor imatinib resulted in decreased pericyte content and increased tumor growth73. This finding is in contrast with other studies, which have shown the antitumor properties of imatinib, probably because of its inhibition of tumor cell PDGF receptor activity rather than an effect on the perivascular cell component. Results from that study provide an example of how identifying and targeting a specific pathway in tumor cells may have opposing effects on tumor outcome because of ‘off-target effects’ on other cell types. This may also be true for other therapies, such as those targeting particular integrins or miRNAs, which may upset the interaction between different cell types or the balance of positive to negative regulators of an intracellular signaling pathway. A similar dichotomy exists between VEGF signaling in vascular compared to perivascular cell types during the angiogenic response. Exposing VSMC to PDGF stimulates migration and proliferation to promote coverage and stabilization of newly formed blood vessels, whereas exposure to VEGF inhibits VSMC function and suppresses PDGF-induced angiogenesis74. At the molecular level, VEGF-mediated activation of VEGFR2 suppresses PDGFRb signaling in VSMCs through the assembly of a VEGR2-PDGFRb complex. As such, inhibition of VEGFR2 or elimination of tumor cell–derived VEGF prevents assembly of this receptor complex, causing activation of VSMCs and leading to increased development of a mature vascular supply. This study shows the ability of VEGF and VEGFR2 signaling to promote endothelial cell function while suppressing VSMC function, suggesting that proangiogenic growth factors and antiangiogenic therapeutic strategies might involve similar balances in power between different cell types. A similar effect on the angiogenic switch in solid tumors was shown for inflammatory-cell–derived VEGF75. When myeloid cells move to a tumor, they release a host of progrowth and proinflammatory factors into the tumor microenvironment. It is therefore counterintuitive that knocking out myeloid-cell–derived VEGF would accelerate tumor growth and progression, which is characterized by reduced hypoxia and tumor cell death75. These results were attributed to a normalized vasculature with enhanced perivascular cell coverage, again illustrating the ability of VEGF to differentially activate or suppress signaling among different cell types. Tumor-associated endothelial cells have unique properties. It is widely appreciated that endothelial cells in tumors have a unique activated phenotype compared to those in normal quiescent tissues. Tumorassociated blood vessels are exposed to a distinct set of stimuli in their local environment, they are governed by abnormal signaling pathways, and there are obvious differences in their structure and function. This has been a recent area of focus76, and a number of new methods have been developed to isolate, culture and analyze the unique properties of tumor-associated endothelial cells from mouse or human tumors. In general, a pure population of live endothelial cells can be obtained from fresh tumor tissue after dissociation into a single-cell suspension using collagenases followed by flow cytometry or magnetic bead separation using endothelial-specific markers. Compared with those isolated from normal adjacent tissue, endothelial cells isolated from human tumors showed enhanced angiogenic capabilities and increased survival, adhesion to tumor cells, motility and chemoresistance77,78. These endothelial cells express distinct markers, for example, neural-cell adhesion molecules78, which might represent new approaches to selectively modulate their function. An antibody-based functional proteomics screen identified an antibody that targets migration-stimulating factor (an isoform of fibronectin), which suppressed cell migration and adhesion of human endothelial cells in esophageal cancer79. In tumors nature medicine volume 17 | number 11 | november 2011

generated by co-injecting both tumor-associated endothelial cells and the corresponding tumor cells, the antibody that specifically targets migration-stimulating factor moved to the humanized endothelial cells in the tumor and acted to suppress angiogenesis in vivo79. These examples highlight the importance of studying tumor angiogenesis and testing the effects of antiangiogenic strategies using tumorderived endothelial cells, but there is also a need to move to in vivo models to confirm and validate the relevance of these strategies in distinct tumor microenvironments. In particular, studies that focus only on cultured endothelial cells have a somewhat limited relevance to the three-dimensional environment in vivo, which is comprised of many cell types that interact on some level to remodel and change during the vascular response to cancer. Identifying new pathways that are different in normal compared to tumor-associated blood vessels is an important step in order to enable the design of new strategies to image tumor angiogenesis or to deliver drugs selectively to pathological blood vessels (Box 4). Strategies to monitor the response to antiangiogenic therapy in vivo. Several antiangiogenic therapies are currently approved by the FDA for cancer, including the humanized antibody Avastin (bevacizumab), which targets VEGF-A, the tyrosine kinase inhibitor sorafenib, which targets Raf and VEGF and PDGF receptors, and the tyrosine kinase inhibitor sunitinib, which targets VEGF and PDGF receptors80. In the case of multi-kinase inhibitors, it is probable that the observed anti-tumor effect represents the combined result of blocking angiogenic signaling pathways as well as blocking signaling in tumor cells and other cell types. Understanding how antiangiogenic drugs function in vivo and what types of drugs could be combined to produce better results in certain patients are key steps to improving the success of antiangiogenic strategies. Currently, however, there are no validated biomarkers for appropriately selecting individuals with cancer that is suited for antiangiogenic therapy81. It might be necessary to think outside the box to establish new markers suitable for gauging the efficacy of antiangiogenic strategies. For example, a number of newly developed imaging strategies have shown promise in preclinical models (Box 5). Given that therapy-induced hypertension is associated with better outcome in some cancers82–84, a rapid hypertensive response to any therapy that has a component that targets VEGF could potentially indicate drug activity in vivo. However, this is a concept that might not provide much clinical value considering that hypertension can be induced by a number of different pathological or therapeutic stimuli. If tumor biopsies are available, analysis of vascular signaling events might be a useful histological biomarker for angiogenesis in vivo. For example, vascular staining of phosphorylated extracellular signal–regulated kinase (ERK) and STAT3 is specifically elevated in tumor endothelium, and activity is suppressed when mice are treated with VEGF blockade85. Staining biopsies for such phosphorylation events could provide a new option to gauge the response to antiangiogenic therapy. Although preclinical mouse models can represent many facets of cancer progression, the most efficient means to develop and validate new biomarkers might require careful imaging and analysis of patient tissues before treatment and at multiple time points during and after treatment to capture the initial response to therapy as well as the transition (or lack of transition) to resistance. Future perspectives Successful manipulation of angiogenesis in cancer will require the transition from bench to bedside and back. Strategies must be evaluated for their effects on not only tumor growth but also on endothelial tip cell sprouting, vascular maturation, recruitment of endothelial progenitor cells, hypoxia and more. How does each therapeutic target affect angiogenesis locally and globally? How does this process change the tumor 1367


review microenvironment and the aggressive nature of tumor cells? How can we best predict the response to and gauge the efficacy of a given approach? It is also worthwhile to investigate how the angiogenic switch is engaged and maintained in cancer. Do tumors share a common mechanism to drive the angiogenic switch? Considering that a burst of angiogenic growth factor is sufficient to drive sustained angiogenesis, uncovering what extracellular and intracellular components promote sustained neovascularization during tumor growth is needed. Could angiomiRs have a role in the sustained response by changing gene expression of signaling pathways to lift multiple brakes on cell activation? Even though tumor angiogenesis is a dynamic process, it is typically studied by taking a ‘snapshot’ of its progression at discrete time points. New imaging techniques may offer a way to obtain angiograms at multiple time points during tumor blood vessel remodeling. Given that tumors use multiple growth factors to initiate angiogenesis, should we be surprised that anti-VEGF therapy is not more effective as a single agent? In fact, there are only a few examples supporting the efficacy of any antiangiogenic monotherapy, most notably in renal and liver cancers1. It is unclear how much of the impact of anti-VEGF therapy is caused by a true antiangiogenic effect rather than vascular normalization. Anti-VEGF therapy is typically combined with chemotherapeutic agents to produce an anti-tumor effect. Perhaps anti-VEGF therapy has not really taught us what we would expect to learn from a true antiangiogenic drug. A closer look at the properties of the tumor vasculature in renal or liver cancers that do respond to antiangiogenic therapies is warranted, and understanding why these cancers have a different vascular response compared with other epithelial cancers, such as breast or colon cancer, could provide some perspective about the therapeutic window for such therapy and how vascular regression and vascular normalization might cooperate to limit tumor growth. Ultimately, it might be best to consider pharmacological targets that are downstream of multiple angiogenic stimuli (Fig. 4). There are also questions regarding whether attacking the perivascular compartment of tumors would be beneficial. If perivascular cells are crucial for vessel maturation and optimal blood flow, we could be reducing the efficacy of systemically delivered anticancer agents. Selectively disabling the deposition of VEGF by myeloid cells is sufficient to cause large changes on the remodeling blood vessels75. As such, a strategy to locally attack the appropriate pool of VEGFs may provide a better long-term response than global therapy. For example, nanoparticles or peptides that target the tumor endothelium could deliver a drug payload to a particular tumor microenvironment. A number of strategies could be used to target tumor-associated angiogenesis. As there is a substantial degree of redundant signaling, compensation and resistance mechanisms that limit the long-term and short-term benefit of blocking a particular upstream receptor, inhibiting multiple pathways may be more efficient (Fig. 4). Cocktails of multikinase inhibitors with less specificity could provide blanket coverage to decrease the odds of igniting the intracellular signaling pathways leading to cellular activation and angiogenesis; however, if just one pathway is left unprotected, it might be enough to drive angiogenesis. Alternatively, one could target the activity of pivotal signaling hubs that act as convergent nodes for multiple growth factors, integrins and cytokine receptors. For example, targeting Ras and Raf in endothelial cells or targeting phosphoinositide 3-kinase (PI3K) p110g in myeloid cells may provide a more durable approach that is less prone to compensation and resistance. As is often the case for chemotherapy regimens, multiple approaches can be combined in parallel or in series. Notably, any new approaches will probably be tested initially in combination with the current standard of care for a given cancer type or subtype. From the perspective of an oncologist, each cancer type or 1368

subtype is unique, and only certain agents are approved by the FDA for patient care. Going forward, the bench scientist will need to work closely with the clinician to develop antiangiogenic strategies that will be compatible or that will synergize with the existing therapy standards and to customize approaches for specific cancers. ACKNOWLEDGMENTS D.A.C. is supported by grants from the US National Institutes of Health (grants R37 CA50286, R01 CA95262, R01 CA45726, P01 HL57900 and R01 HL103956). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29. 30.

Carmeliet, P. & Jain, R.K. Molecular mechanisms and clinical applications of angiogenesis. Nature 473, 298–307 (2011). Folkman, J. Role of angiogenesis in tumor growth and metastasis. Semin. Oncol. 29, 15–18 (2002). Herbert, S.P. & Stainier, D.Y.R. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat. Rev. Mol. Cell Biol. 12, 551–564 (2011). Folkman, J. & Hanahan, D. Switch to the angiogenic phenotype during tumorigenesis. Princess Takamatsu Symp. 22, 339–347 (1991). Weis, S.M. & Cheresh, D.A. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 437, 497–504 (2005). Hellberg, C., Ostman, A. & Heldin, C.H. PDGF and vessel maturation. Recent Results Cancer Res. 180, 103–114 (2010). Franco, O.E., Shaw, A.K., Strand, D.W. & Hayward, S.W. Cancer associated fibroblasts in cancer pathogenesis. Semin. Cell Dev. Biol. 21, 33–39 (2010). Gonda, T.A., Varro, A., Wang, T.C. & Tycko, B. Molecular biology of cancer-associated fibroblasts: can these cells be targeted in anti-cancer therapy? Semin. Cell Dev. Biol. 21, 2–10 (2010). Xing, F., Saidou, J. & Watabe, K. Cancer associated fibroblasts (CAFs) in tumor microenvironment. Front. Biosci. 15, 166–179 (2010). Sund, M. et al. Function of endogenous inhibitors of angiogenesis as endotheliumspecific tumor suppressors. Proc. Natl. Acad. Sci. USA 102, 2934–2939 (2005). Demaria, S. et al. Cancer and inflammation: promise for biologic therapy. J. Immunother. 33, 335–351 (2010). Grivennikov, S.I., Greten, F.R. & Karin, M. Immunity, inflammation, and cancer. Cell 140, 883–899 (2010). Dvorak, H.F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J. Med. 315, 1650–1659 (1986). Jones, C.A. et al. Robo4 stabilizes the vascular network by inhibiting pathological angiogenesis and endothelial hyperpermeability. Nat. Med. 14, 448–453 (2008). Park, K.W. et al. Robo4 is a vascular-specific receptor that inhibits endothelial migration. Dev. Biol. 261, 251–267 (2003). Rizzolio, S. & Tamagnone, L. Multifaceted role of neuropilins in cancer. Curr. Med. Chem. 18, 3563–3575 (2011). Zygmunt, T. et al. Semaphorin-plexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1. Dev. Cell 21, 301–314 (2011). Kim, J., Oh, W.J., Gaiano, N., Yoshida, Y. & Gu, C. Semaphorin 3E-Plexin-D1 signaling regulates VEGF function in developmental angiogenesis via a feedback mechanism. Genes Dev. 25, 1399–1411 (2011). Campbell, N.E. et al. Extracellular matrix proteins and tumor angiogenesis. J. Oncol. 2010, 586905 (2010). Desgrosellier, J.S. & Cheresh, D.A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010). Humphries, J.D., Byron, A. & Humphries, M.J. Integrin ligands at a glance. J. Cell Sci. 119, 3901–3903 (2006). Stupack, D.G. & Cheresh, D.A. Get a ligand, get a life: integrins, signaling and cell survival. J. Cell Sci. 115, 3729–3738 (2002). Stupack, D.G., Puente, X.S., Boutsaboualoy, S., Storgard, C.M. & Cheresh, D.A. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J. Cell Biol. 155, 459–470 (2001). Nyberg, P., Xie, L. & Kalluri, R. Endogenous inhibitors of angiogenesis. Cancer Res. 65, 3967–3979 (2005). Ribatti, D. Endogenous inhibitors of angiogenesis: a historical review. Leuk. Res. 33, 638–644 (2009). Brooks, P.C., Clark, R.A. & Cheresh, D.A. Requirement of vascular integrin a v b 3 for angiogenesis. Science 264, 569–571 (1994). Brooks, P.C. et al. Integrin a v b 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157–1164 (1994). Gladson, C.L. & Cheresh, D.A. Glioblastoma expression of vitronectin and the a v b 3 integrin. Adhesion mechanism for transformed glial cells. J. Clin. Invest. 88, 1924–1932 (1991). Desgrosellier, J.S. et al. An integrin avb3-c-Src oncogenic unit promotes anchorageindependence and tumor progression. Nat. Med. 15, 1163–1169 (2009). Gladson, C.L. Expression of integrin a v b 3 in small blood vessels of glioblastoma tumors. J. Neuropathol. Exp. Neurol. 55, 1143–1149 (1996).


review 31. 32. 33. 34. 35. 36. 37. 38. 39.

40. 41.


42. 43. 44.

45. 46.

47.

48.

49.

50. 51.

52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68.

MacDonald, T.J. et al. Preferential susceptibility of brain tumors to the antiangiogenic effects of an a(v) integrin antagonist. Neurosurgery 48, 151–157 (2001). Reardon, D.A. et al. Cilengitide: an RGD pentapeptide anb3 and anb5 integrin inhibitor in development for glioblastoma and other malignancies. Future Oncol. 7, 339–354 (2011). Tabatabai, G. et al. Targeting integrins in malignant glioma. Target. Oncol. 5, 175– 181 (2010). Reardon, D.A., Nabors, L.B., Stupp, R. & Mikkelsen, T. Cilengitide: an integrintargeting arginine-glycine-aspartic acid peptide with promising activity for glioblastoma multiforme. Expert Opin. Investig. Drugs 17, 1225–1235 (2008). Avraamides, C.J., Garmy-Susini, B. & Varner, J.A. Integrins in angiogenesis and lymphangiogenesis. Nat. Rev. Cancer 8, 604–617 (2008). Ferrara, N., Gerber, H.P. & LeCouter, J. The biology of VEGF and its receptors. Nat. Med. 9, 669–676 (2003). Sawamiphak, S. et al. Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis. Nature 465, 487–491 (2010). Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010). Meyer, R.D. et al. PEST motif serine and tyrosine phosphorylation controls vascular endothelial growth factor receptor 2 stability and downregulation. Mol. Cell. Biol. 31, 2010–2025 (2011). Serini, G., Napione, L., Arese, M. & Bussolino, F. Besides adhesion: new perspectives of integrin functions in angiogenesis. Cardiovasc. Res. 78, 213–222 (2008). Eliceiri, B.P. Integrin and growth factor receptor crosstalk. Circ. Res. 89, 1104–1110 (2001). Alam, N. et al. The integrin-growth factor receptor duet. J. Cell. Physiol. 213, 649– 653 (2007). Somanath, P.R., Ciocea, A. & Byzova, T.V. Integrin and growth factor receptor alliance in angiogenesis. Cell Biochem. Biophys. 53, 53–64 (2009). Mahabeleshwar, G.H., Feng, W., Reddy, K., Plow, E.F. & Byzova, T.V. Mechanisms of integrin vascular endothelial growth factor receptor cross-aactivation in angiogenesis. Circ. Res. 101, 570–580 (2007). Lakshmikanthan, S. et al. Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin avb3. Blood 118, 2015–2026 (2011). Hutchings, H., Ortega, N. & Plouet, J. Extracellular matrix-bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrin ligation. FASEB J. 17, 1520–1522 (2003). Cascone, I., Napione, L., Maniero, F., Serini, G. & Bussolino, F. Stable interaction between a5b1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1. J. Cell Biol. 170, 993–1004 (2005). Carlson, T.R., Feng, Y., Maisonpierre, P.C., Mrksich, M. & Morla, A.O. Direct cell adhesion to the angiopoietins mediated by integrins. J. Biol. Chem. 276, 26516–26525 (2001). Deryugina, E.I. & Quigley, J.P. Pleiotropic roles of matrix metalloproteinases in tumor angiogenesis: contrasting, overlapping and compensatory functions. Biochim. Biophys. Acta 1803, 103–120 (2010). Sounni, N.E., Paye, A., Host, L. & Noel, A. MT-MMPS as regulators of vessel stability associated with angiogenesis. Front. Pharmacol. 2, 111 (2011). Davis, G.E., Bayless, K.J., Davis, M.J. & Meininger, G.A. Regulation of tissue injury responses by the exposure of matricryptic sites within extracellular matrix molecules. Am. J. Pathol. 156, 1489–1498 (2000). Xu, J. et al. Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo. J. Cell Biol. 154, 1069–1079 (2001). Pillai, R.S. MicroRNA function: multiple mechanisms for a tiny RNA? RNA 11, 1753–1761 (2005). van Kouwenhove, M., Kedde, M. & Agami, R. MicroRNA regulation by RNA-binding proteins and its implications for cancer. Nat. Rev. Cancer 11, 644–656 (2011). Hua, Z. et al. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia. PLoS ONE 1, e116 (2006). Fish, J.E. & Srivastava, D. MicroRNAs: opening a new vein in angiogenesis research. Sci. Signal. 2, pe1 (2009). Wang, S. & Olson, E.N. AngiomiRs—key regulators of angiogenesis. Curr. Opin. Genet. Dev. 19, 205–211 (2009). Anand, S. & Cheresh, D.A. MicroRNA-mediated regulation of the angiogenic switch. Curr. Opin. Hematol. 18, 171–176 (2011). Bonauer, A., Boon, R.A. & Dimmeler, S. Vascular microRNAs. Curr. Drug Targets 11, 943–949 (2010). Olson, P. et al. MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer. Genes Dev. 23, 2152–2165 (2009). Anand, S. et al. MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nat. Med. 16, 909–914 (2010). Cascio, S. et al. miR-20b modulates VEGF expression by targeting HIF-1a and STAT3 in MCF-7 breast cancer cells. J. Cell. Physiol. 224, 242–249 (2010). Fang, L. et al. MicroRNA miR-93 promotes tumor growth and angiogenesis by targeting integrin-b8. Oncogene 30, 806–821 (2011). Yamakuchi, M. et al. P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis. Proc. Natl. Acad. Sci. USA 107, 6334–6339 (2010). Cha, S.T. et al. MicroRNA-519c suppresses hypoxia-inducible factor-1a expression and tumor angiogenesis. Cancer Res. 70, 2675–2685 (2010). Huynh, C. et al. Efficient in vivo microRNA targeting of liver metastasis. Oncogene 30, 1481–1488 (2011). Kota, J. et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 137, 1005–1017 (2009). Christensen, M., Larsen, L.A., Kauppinen, S. & Schratt, G. Recombinant adeno-


associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo. Front Neural Circuits 3, 16 (2010). 69. Takeshita, F. et al. Systemic delivery of synthetic microRNA-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes. Mol. Ther. 18, 181–187 (2010). 70. Trang, P. et al. Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol. Ther. 19, 1116–1122 (2011). 71. Murphy, E.A. et al. Nanoparticle-mediated drug delivery to tumor vasculature suppresses metastasis. Proc. Natl. Acad. Sci. USA 105, 9343–9348 (2008). 72. Hood, J.D. et al. Tumor regression by targeted gene delivery to the neovasculature. Science 296, 2404–2407 (2002). 73. McCarty, M.F. et al. Overexpression of PDGF-BB decreases colorectal and pancreatic cancer growth by increasing tumor pericyte content. J. Clin. Invest. 117, 2114–2122 (2007). 74. Greenberg, J.I. et al. A role for VEGF as a negative regulator of pericyte function and vessel maturation. Nature 456, 809–813 (2008). 75. Stockmann, C. et al. Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis. Nature 456, 814–818 (2008). 76. Bussolati, B., Grange, C. & Camussi, G. Tumor exploits alternative strategies to achieve vascularization. FASEB J. 25, 2874–2882 (2011). 77. Xiong, Y.Q. et al. Human hepatocellular carcinoma tumor-derived endothelial cells manifest increased angiogenesis capability and drug resistance compared with normal endothelial cells. Clin. Cancer Res. 15, 4838–4846 (2009). 78. Bussolati, B. et al. Neural-cell adhesion molecule (NCAM) expression by immature and tumor-derived endothelial cells favors cell organization into capillary-like structures. Exp. Cell Res. 312, 913–924 (2006). 79. Hu, H. et al. Antibody library-based tumor endothelial cells surface proteomic functional screen reveals migration-stimulating factor as an anti-angiogenic target. Mol. Cell. Proteomics 8, 816–826 (2009). 80. Chung, A.S., Lee, J. & Ferrara, N. Targeting the tumour vasculature: insights from physiological angiogenesis. Nat. Rev. Cancer 10, 505–514 (2010). 81. Jain, R.K. et al. Biomarkers of response and resistance to antiangiogenic therapy. Nat. Rev. Clin. Oncol. 6, 327–338 (2009). 82. Österlund, P. et al. Hypertension and overall survival in metastatic colorectal cancer patients treated with bevacizumab-containing chemotherapy. Br. J. Cancer 104, 599–604 (2011). 83. Dahlberg, S.E., Sandler, A.B., Brahmer, J.R., Schiller, J.H. & Johnson, D.H. Clinical course of advanced non-small-cell lung cancer patients experiencing hypertension during treatment with bevacizumab in combination with carboplatin and paclitaxel on ECOG 4599. J. Clin. Oncol. 28, 949–954 (2010). 84. Maitland, M.L. et al. Ambulatory monitoring detects sorafenib-induced blood pressure elevations on the first day of treatment. Clin. Cancer Res. 15, 6250–6257 (2009). 85. Lassoued, W. et al. Effect of VEGF and VEGF Trap on vascular endothelial cell signaling in tumors. Cancer Biol. Ther. 10, 1326–1333 (2011). 86. Asahara, T. et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964–967 (1997). 87. Lyden, D. et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat. Med. 7, 1194–1201 (2001). 88. Butler, J.M., Kobayashi, H. & Rafii, S. Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors. Nat. Rev. Cancer 10, 138–146 (2010). 89. Kobayashi, H. et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat. Cell Biol. 12, 1046–1056 (2010). 90. Solinas, G., Germano, G., Mantovani, A. & Allavena, P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J. Leukoc. Biol. 86, 1065–1073 (2009). 91. De Palma, M. et al. Tumor-targeted interferon-a delivery by Tie2-expressing monocytes inhibits tumor growth and metastasis. Cancer Cell 14, 299–311 (2008). 92. Mazzieri, R. et al. Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells. Cancer Cell 19, 512–526 (2011). 93. Pander, J. et al. Activation of tumor-promoting type 2 macrophages by EGFR-targeting antibody cetuximab. Clin. Cancer Res. 17, 5668–5673 (2011). 94. Shaked, Y. et al. Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell 14, 263–273 (2008). 95. Kerbel, R.S. Improving conventional or low dose metronomic chemotherapy with targeted antiangiogenic drugs. Cancer Res. Treat. 39, 150–159 (2007). 96. Shaked, Y. & Kerbel, R.S. Antiangiogenic strategies on defense: on the possibility of blocking rebounds by the tumor vasculature after chemotherapy. Cancer Res. 67, 7055–7058 (2007). 97. Shaked, Y. et al. Therapy-induced acute recruitment of circulating endothelial progenitor cells to tumors. Science 313, 1785–1787 (2006). 98. Gao, D. et al. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science 319, 195–198 (2008). 99. Daenen, L.G. et al. Low-dose metronomic cyclophosphamide combined with vascular disrupting therapy induces potent antitumor activity in preclinical human tumor xenograft models. Mol. Cancer Ther. 8, 2872–2881 (2009). 100. Maniotis, A.J. et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am. J. Pathol. 155, 739–752 (1999). 101. Bissell, M.J. Tumor plasticity allows vasculogenic mimicry, a novel form of angiogenic switch. A rose by any other name? Am. J. Pathol. 155, 675–679 (1999).

1369


review 102. Fausto, N. Vasculogenic mimicry in tumors. Fact or artifact? Am. J. Pathol. 156, 359 (2000). 103. Folberg, R., Hendrix, M.J. & Maniotis, A.J. Vasculogenic mimicry and tumor angiogenesis. Am. J. Pathol. 156, 361–381 (2000). 104. McDonald, D.M., Munn, L. & Jain, R.K. Vasculogenic mimicry: how convincing, how novel, and how significant? Am. J. Pathol. 156, 383–388 (2000). 105. Shubik, P. & Warren, B.A. Additional literature on “vasculogenic mimicry” not cited. Am. J. Pathol. 156, 736 (2000). 106. Frenkel, S. et al. Demonstrating circulation in vasculogenic mimicry patterns of uveal melanoma by confocal indocyanine green angiography. Eye (Lond.) 22, 948–952 (2008). 107. Yao, X.H., Ping, Y.F. & Bian, X.W. Contribution of cancer stem cells to tumor vasculogenic mimicry. Protein Cell 2, 266–272 (2011). 108. Shen, R. et al. Precancerous stem cells can serve as tumor vasculogenic progenitors. PLoS ONE 3, e1652 (2008). 109. Bussolati, B., Grange, C., Sapino, A. & Camussi, G. Endothelial cell differentiation of human breast tumour stem/progenitor cells. J. Cell. Mol. Med. 13, 309–319 (2009). 110. Bussolati, B., Bruno, S., Grange, C., Ferrando, U. & Camussi, G. Identification of a tumor-initiating stem cell population in human renal carcinomas. FASEB J. 22, 3696–3705 (2008). 111. Alvero, A.B. et al. Stem-like ovarian cancer cells can serve as tumor vascular progenitors. Stem Cells 27, 2405–2413 (2009). 112. Wang, R. et al. Glioblastoma stem-like cells give rise to tumour endothelium. Nature 468, 829–833 (2010). 113. Ricci-Vitiani, L. et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 468, 824–828 (2010). 114. Wurmser, A.E. et al. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage. Nature 430, 350–356 (2004). 115. Hovinga, K.E. et al. Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate. Stem Cells 28, 1019–1029 (2010). 116. Du, R. et al. HIF1a induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell 13, 206–220 (2008). 117. Biswas, S.K. & Mantovani, A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat. Immunol. 11, 889–896 (2010). 118. Lin, E.Y. et al. Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res. 66, 11238–11246 (2006). 119. Pollard, J.W. Trophic macrophages in development and disease. Nat. Rev. Immunol. 9, 259–270 (2009). 120. Grunewald, M. et al. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells. Cell 124, 175–189 (2006). 121. Schmid, M.C. et al. Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3K3, a single convergent point promoting tumor inflammation and progression. Cancer Cell 19, 715–727 (2011). 122. Ruhrberg, C. & De Palma, M. A double agent in cancer: deciphering macrophage roles in human tumors. Nat. Med. 16, 861–862 (2010). 123. Steidl, C. et al. Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma. N. Engl. J. Med. 362, 875–885 (2010). 124. Shojaei, F. et al. Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells. Nat. Biotechnol. 25, 911–920 (2007). 125. Shojaei, F. et al. Bv8 regulates myeloid-cell-dependent tumour angiogenesis. Nature 450, 825–831 (2007). 126. Folkman, J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 285, 1182–1186 (1971). 127. Bergers, G. & Hanahan, D. Modes of resistance to anti-angiogenic therapy. Nat. Rev. Cancer 8, 592–603 (2008). 128. Ellis, L.M. & Hicklin, D.J. Pathways mediating resistance to vascular endothelial growth factor-targeted therapy. Clin. Cancer Res. 14, 6371–6375 (2008). 129. Ebos, J.M., Lee, C.R. & Kerbel, R.S. Tumor and host-mediated pathways of resistance and disease progression in response to antiangiogenic therapy. Clin. Cancer Res. 15, 5020–5025 (2009). 130. Fischer, C. et al. Anti-PlGF inhibits frowth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell 131, 463–475 (2007).

1370

131. Loges, S., Schmidt, T. & Carmeliet, P. “Antimyeloangiogenic” therapy for cancer by inhibiting PlGF. Clin. Cancer Res. 15, 3648–3653 (2009). 132. Van de Veire, S. et al. Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease. Cell 141, 178–190 (2010). 133. Pàez-Ribes, M. et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15, 220–231 (2009). 134. Ebos, J.M. et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 15, 232–239 (2009). 135. Park, Y.H. et al. Trastuzumab treatment improves brain metastasis outcomes through control and durable prolongation of systemic extracranial disease in HER2overexpressing breast cancer patients. Br. J. Cancer 100, 894–900 (2009). 136. Sipkins, D.A. et al. Detection of tumor angiogenesis in vivo by aVb3-targeted magnetic resonance imaging. Nat. Med. 4, 623–626 (1998). 137. Beer, A.J. et al. Positron emission tomography using [18F]Galacto-RGD identifies the level of integrin a(v)b3 expression in man. Clin. Cancer Res. 12, 3942–3949 (2006). 138. Battle, M.R., Goggi, J.L., Allen, L., Barnett, J. & Morrison, M.S. Monitoring tumor response to antiangiogenic sunitinib therapy with 18F-fluciclatide, an 18F-labeled aVb3-integrin and aV b5-integrin imaging agent. J. Nucl. Med. 52, 424–430 (2011). 139. Pasqualini, R. & Ruoslahti, E. Organ targeting in vivo using phage display peptide libraries. Nature 380, 364–366 (1996). 140. Ruoslahti, E. Vascular zip codes in angiogenesis and metastasis. Biochem. Soc. Trans. 32, 397–402 (2004). 141. Ruoslahti, E., Bhatia, S.N. & Sailor, M.J. Targeting of drugs and nanoparticles to tumors. J. Cell Biol. 188, 759–768 (2010). 142. Cao, Q. et al. Phage display peptide probes for imaging early response to bevacizumab treatment. Amino Acids published online, doi:10.1007/s00726-010-0548-9 (16 March 2010). 143. Bussolati, B. et al. Targeting of human renal tumor-derived endothelial cells with peptides obtained by phage display. J. Mol. Med. 85, 897–906 (2007). 144. Mueller, J., Gaertner, F.C., Blechert, B., Janssen, K.P. & Essler, M. Targeting of tumor blood vessels: a phage-displayed tumor-homing peptide specifically binds to matrix metalloproteinase-2-processed collagen IV and blocks angiogenesis in vivo. Mol. Cancer Res. 7, 1078–1085 (2009). 145. Samanta, S., Sistla, R. & Chaudhuri, A. The use of RGDGWK-lipopeptide to selectively deliver genes to mouse tumor vasculature and its complexation with p53 to inhibit tumor growth. Biomaterials 31, 1787–1797 (2010). 146. Loi, M. et al. Combined targeting of perivascular and endothelial tumor cells enhances anti-tumor efficacy of liposomal chemotherapy in neuroblastoma. J. Control. Release 145, 66–73 (2010). 147. Sugahara, K.N. et al. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science 328, 1031–1035 (2010). 148. Teesalu, T., Sugahara, K.N., Kotamraju, V.R. & Ruoslahti, E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration. Proc. Natl. Acad. Sci. USA 106, 16157–16162 (2009). 149. Sugahara, K.N. et al. Tissue-penetrating delivery of compounds and nanoparticles into tumors. Cancer Cell 16, 510–520 (2009). 150. Sugahara, K.N. et al. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science 328, 1031–1035 (2010). 151. Nagengast, W.B. et al. VEGF-PET imaging is a noninvasive biomarker showing differential changes in the tumor during sunitinib treatment. Cancer Res. 71, 143–153 (2011). 152. Nagengast, W.B. et al. VEGF-SPECT with (111)In-bevacizumab in stage III/IV melanoma patients. Eur. J. Cancer 47, 1595–1602 (2011). 153. Nayak, T.K., Garmestani, K., Baidoo, K.E., Milenic, D.E. & Brechbiel, M.W. PET imaging of tumor angiogenesis in mice with VEGF-A-targeted (86)Y-CHX-A’’-DTPAbevacizumab. Int. J. Cancer 128, 920–926 (2011). 154. Niu, G. & Chen, X. PET imaging of angiogenesis. PET Clin. 4, 17–38 (2009). 155. Dumont, R.A. et al. Noninvasive imaging of aVb3 function as a predictor of the antimigratory and antiproliferative effects of dasatinib. Cancer Res. 69, 3173–3179 (2009). 156. Vakoc, B.J. et al. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nat. Med. 15, 1219–1223 (2009).
