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Tumor angiogenesis and accessibility: Role of vascular endothelial growth factor
Tumor Angiogenesis and Accessibility: Role of Vascular Endothelial Growth Factor Rakesh K. Jain Solid tumors consist of several components, including normal and stromal cells, extracellular matrix, and vasculature. To grow and metastasize, tumors must stimulate the development of new vasculature through a process known as angiogenesis. Unlike normal blood vessels, tumor blood vessels are chaotic, irregular, and leaky, which leads to uneven delivery of nutrients and therapeutic agents to the tumor. Conventional therapies target neoplastic cells within a tumor; however, tumor vasculature is emerging as an important target for anticancer therapy. Antiangiogenic therapy offers several potential advantages as an approach to cancer treatment, notably physical accessibility and genetic stability of target cells. Vascular endothelial growth factor (VEGF), a central mediator of angiogenesis, has emerged as an important target for antiangiogenic therapy. In preclinical studies, treatment of human tumor xenografts in immunodeficient mice with the antiVEGF monoclonal antibody A4.6.1 led to reduced tumor vessel permeability and caused vascular regression. The reduced vascular permeability, resulting from inhibition of VEGF, led to increased delivery of oxygen and therapeutic agents to tumors. Anti-VEGF therapy was effectively combined with other treatment modalities, including radiation, antihormonal, antibody, and chemotherapies in multiple preclinical models. Currently, several phase 3 clinical trials in various cancer types are under way to establish the efficacy of antiangiogenic therapy with a recombinant humanized anti-VEGF monoclonal antibody, bevacizumab (Avastin, rhuMAb-VEGF; Genentech, South San Francisco, CA), in combination with chemotherapeutic agents. Semin Oncol 29 (suppl 16):3-9. Copyright 2002, Elsevier Science (USA). All rights reserved.
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OLID TUMORS are multicompartmentalized structures, consisting of three major components: cancer and stromal cells, the extracellular matrix (ECM), and the vasculature.1 The volumes of each of these components vary depending on the origin and size of the tumor and the organ in which the primary tumor develops.1 Tumors require vasculature to gain access to oxygen and other nutrients, allowing growth and metastasis.2 While most anticancer therapies target neoplastic cells, novel approaches that target other tumor compartments may be potentially useful additions to the armamentarium of antitumor agents. Specifically, the development of new blood vessels (angiogenesis) is essential for tumor growth and metastasis, and several unique features of tumor Seminars in Oncology, Vol 29, No 6, Suppl 16 (December), 2002: pp 3-9
vasculature offer attractive targets for anticancer therapy. VASCULAR AND EXTRAVASCULAR BARRIERS IN TUMORS
Unlike normal angiogenesis, which is an ordered and highly regulated process that occurs during fetal and early postnatal development and during wound repair in the adult, the development of tumor vasculature is chaotic and irregular.2 Tumor blood vessels are functionally abnormal; their diameter is variable, they are excessively branched and twisted, and they are often dilated. Additionally, the walls of tumor vessels have many openings (endothelial fenestrae and transcellular holes), widened interendothelial junctions, and a defective basement membrane. The structural and functional abnormalities of tumor vasculature result in variable blood flow in the tumor, which in turn leads to hypoxic and acidic regions in tumors.1,2 The abnormal and chaotic architecture of tumor vasculature is one of several physiologic barriers that impede the effective delivery of therapeutic agents to target cells.3,4 To reach the cancer cells, any systemically administered therapeutic agent must enter the tumor via the vascular compartment, cross the vessel wall, and move through the interstitial compartment. In addition to abnormal features of tumor vasculature, the ECM also impedes movement of therapeutic agents toward target cells. The total barrier strength varies not only across different tumor types but also for the same tumor implanted in different body locations.5
From the Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Harvard Medical School and Massachusetts General Hospital, Boston, MA. Supported in part by an unrestricted educational grant from Genentech BioOncology and by grants from the National Cancer Institute. Address reprint requests to Rakesh K. Jain, PhD, Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Harvard Medical School and Massachusetts General Hospital, 100 Blossom St – Box 7, Boston MA 02114. Copyright 2002, Elsevier Science (USA). All rights reserved. 0093-7754/02/2906-1602$35.00/0 doi:10.1053/sonc.2002.37265 3
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Elevated interstitial fluid pressure (IFP) is a key barrier that prevents effective transport of therapeutic agents into tumors and thus reduces the efficacy of those agents. This is of particular concern with high–molecular-weight anticancer agents, such as monoclonal antibodies, because of their low diffusibility. The elevation of tumor IFP to the level seen in microvasculature is thought to be the result of the high vascular permeability of tumor blood vessels and the absence of a functional lymphatic circulation system.6,7 Our studies show that the oncotic pressure within human tumor xenografts is equivalent to that of plasma.3 In contrast, under normal conditions, IFP is lower than the pressure of the surrounding vasculature, allowing convective movement of macromolecules across the vessel wall and into the interstitial space. The composition of the ECM also influences the penetration of a drug into a solid tumor. Both tumor and stromal cells secrete components of the ECM, such as collagens and proteoglycans. Studies with fluorescently labeled macromolecules showed a direct correlation between ECM composition and macromolecular transport resistance. Specifically, greater mechanical stiffness of the tumor, a property dependent on fibrillar collagen networks and their interactions with proteoglycans, was associated with increased resistance to macromolecular penetration. Collagen contributes to ECM stiffness, and our studies showed that interstitial diffusion rates were increased by treating penetration-resistant tumors with collagenase.8 ANTIANGIOGENIC THERAPY
Antiangiogenic therapy offers several potential advantages over conventional key cytotoxic therapies, most notably with respect to physical accessibility and genetic stability of target cells. Unlike antitumor agents, whose target cells are often inaccessible and genetically unstable, antiangiogenic drugs target vascular endothelial cells, which are easily accessible and genetically stable. Furthermore, because most vasculature in adults is relatively quiescent, the potential for adverse effects with antiangiogenic therapy is reduced. Antiangiogenic therapy may be particularly effective in combination with other treatment modalities, such as conventional chemotherapy, antihor-
monal therapy, immunotherapy, and radiation therapy.9 VASCULAR ENDOTHELIAL GROWTH FACTOR AND ANGIOGENESIS
The focus of our studies is the vascular endothelial growth factor (VEGF, also known as vascular permeability factor [VPF], or VEGF-A), a critical and central regulator of angiogenesis. The other members of the VEGF family (VEGF-B, VEGF-C, VEGF-D, and PlGF) also play a role in angiogenesis. Vascular endothelial growth factor is both a vascular growth factor and a vascular permeability factor. Its expression is upregulated in many tumors,10,11 and it can upregulate expression of adhesion molecules on the vascular endothelium.12 It is required to initiate the formation of blood vessels during development, as demonstrated by the fact that disruption of one copy of the VEGF gene in mice leads to embryonic lethality because of severe vascular abnormalities. It is also required for normal vascular development for the first few weeks postnatally, while its inactivation in older (adult) animals affects only those structures that undergo vascular remodeling, such as the corpus luteum and bone growth plates.2,10 Chronic anti-VEGF downregulation during embryonic development may also lead to neurologic and lung impairment.2,13 ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN TUMOR ANGIOGENESIS
Bevacizumab (Avastin, rhuMAb-VEGF; Genentech, South San Francisco, CA) is a humanized mouse monoclonal antibody that neutralizes human, but not mouse, VEGF. Bevacizumab is derived from A4.6.1, the mouse monoclonal antibody directed against human VEGF (VEGF-A), and has the same high affinity and biological properties as A4.6.1.14 Both bevacizumab and A4.6.1 recognize all spliced variants of VEGF, but no other members of the VEGF family of proteins.14 Preclinical studies showed that inhibition of VEGF with A4.6.1 could alter and ultimately decrease tumoral blood supply. When human cancers (glioblastoma, melanoma, and colon adenocarcinoma) were transplanted into immunodeficient mice, intravenous treatment with A4.6.1 produced a rapid decrease in tumor vessel permeability
TUMOR ANGIOGENESIS AND ACCESSIBILITY
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Fig 1. Four requirements for intravital microscopy. (a) An appropriate animal model. Three different models are shown: a chronic transparent window in which a tumor grown on the inner surface of the skin can be viewed through a coverslip; an in situ preparation in which an optical tracer is injected into the interstitial space of the tail; and an acute liver model in which the animal is surgically prepared to reveal a tumor in the internal organ. (b) A molecular probe (usually fluorescent) that can be imaged. Two examples are shown: green fluorescent protein (GFP) that is expressed in genetically engineered cells/animals and controlled by the promoter of interest; and an optical probe that is activated by a specific enzyme or microenvironment. (c) A microscope equipped with an excitation source and a detection system (such as photomultiplier tube (PMT) or charged-coupled device/silicon-intensified target (SIT) camera), which passes the images to a computer acquisition and analysis workstation. (d) Image processing and analysis algorithms are then applied to extract quantitative data. (Reprinted with permission from Nature Reviews Cancer, copyright 2002 Macmillan Magazines Ltd.1)
(within 6 hours) and led to vascular regression.15 These effects persisted for 72 hours following a single intravenous dose of A4.6.1. Intraperitoneal administration of a single dose of A4.6.1 produced a decrease in vascular permeability in glioma after 6 days, whereas multiple doses over an 11-day period were associated with the regression of preformed vessels.15 This study also showed the specificity of A4.6.1 for human cancers, as the vascularization and vascular permeability of a transplanted murine breast cancer cell line that produces murine VEGF was unaffected by treatment with A4.6.1. In another study, treatment with A4.6.1 significantly reduced tumor IFP, leading to an increase in oxygen levels in some tumors.16 The antiangiogenic effect of bevacizumab on human tumor xenografts was further confirmed by multiphoton laser scanning intravital microscopy.17 This technique allows tumor images to be continuously captured by a video camera attached to a microscope that visualizes images through a
surgically implanted glass window near the tumor locus. Using a high-powered multiphoton microscope and computer-assisted analysis of captured images, multiple parameters of the tumor vasculature (vessel morphology, hemodynamics, pH, pO2, vascular permeability, leukocyte adhesion, and molecular distribution patterns) can be monitored (Fig 1).1,17 In addition, fluorescent dyes or live fluorescent reporters, such as green fluorescent protein, can be used to visualize vasculature or gene expression patterns. In intravital microscopy studies, A4.6.1 and an anti-VEGF receptor-2 antibody, DC101 (ImClone, New York, NY) inhibited neovascularization and growth of implanted tumors.15,18 Intravital microscopy was also used to determine the relative contribution of host stromal cells to tumoral VEGF levels. Transgenic mice expressing green fluorescent protein under the control of the VEGF promoter show fluorescence around the healing margins and throughout the granulation tissue of superficial wounds, a finding consistent
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with the role of VEGF in wound healing.19 Implantation of a non– green fluorescent protein-producing tumor cell line in dorsal skin chambers of the transgenic mice induced VEGF promoter activity and led to an accumulation of VEGF promoter-directed fluorescence. With time, the fluorescent cells (predominantly fibroblasts) invaded the tumor and could be seen throughout the tumor mass, indicating that normal stromal cells actively participate in tumor vascularization.17,19 We further examined the tumoral VEGF contribution of normal cells by transplantation of VEGF-/- tumors into VEGF⫹/⫹ mice. VEGF-/- tumors contained approximately 50% of VEGF levels found in wild-type (VEGF⫹/⫹) tumors, showing that host stromal cells contribute significantly to the VEGF levels found in tumors.19,20 Tumor cells have previously been shown to have elevated VEGF levels.10,11 HYPOXIA AND ANGIOGENESIS
Physiologic adaptation to hypoxia is a necessity for organisms having an oxygen-based metabolism. In mammals, these adjustments include vasodilatation, angiogenesis, upregulation of glucose transport and activation of glycolysis, and apoptosis (programmed cell death). By limiting tumor blood flow and oxygen supply, antiangiogenic therapy may select tumor cells that have acquired an unusual degree of tolerance to chronic hypoxia. To determine if tumors could acquire tolerance to chronic hypoxia, we examined the effect of inactivation of a key factor in the hypoxia-induced signaling cascade. Hypoxia inducible factor-1 alpha (HIF-1␣) is a transcription factor that mediates hypoxia-induced responses, including apoptosis, and VEGF gene regulation.21 We evaluated angiogenesis, tumor growth, and apoptosis in tumors that lacked HIF-1␣ (HIF-1␣-/-) and in wild type tumors. Whereas, the growth of the two tumor types was similar, HIF-1␣-/-tumors had reduced angiogenesis (and VEGF levels)20 and also decreased apoptosis.21 Thus, the decreased apoptosis in HIF-1␣-/- tumors effectively compensates for reduced vascularization and allows normal tumor growth. In other words, HIF-1␣-/- tumors show tolerance to chronic hypoxia, and their growth is not substantially affected by oxygen deprivation. Moreover, analysis of heterogeneous tumors established from mixtures of HIF-1␣⫹/⫹ and HIF-1␣-/-
viable cells showed that the proportion of cells expressing HIF-1␣ was increased in perivascular areas and decreased in distal tumor regions, indicating increased tolerance to hypoxia by HIF-1␣-/cells. In other words, HIF-1␣⫹/⫹ cells were highly dependent on proximity to blood vessels for their growth and survival in vivo, whereas HIF-1␣-/cells were not.17 This finding confirms the potential of antiangiogenic therapy to select tumor cells tolerant to hypoxia, which may have important implications for the long-term efficacy of antiangiogenic therapy as a single treatment modality. COMBINATION OF ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR APPROACHES WITH OTHER THERAPEUTIC MODALITIES
Although A4.6.1 and DC101 suppress tumor vascularization and growth, they do not appear to be curative. Therefore, it is important to evaluate their role in combination with other treatment modalities such as radiation, antihormonal, antibody, and chemotherapies. Both A4.6.1 and DC101 reduce tumor vascular permeability and IFP15,16,18 and thus “normalize” the abnormal tumor vasculature (Fig 2).9 This normalization may translate into enhanced intratumoral delivery of other drugs (eg, antitumor antibodies, cytotoxic agents). Additionally, this normalization may increase local oxygen level in some tumors, which may result in enhanced efficacy of antitumor radiation.9,16,22 The effect of anti-VEGF or VEGF receptor-2 antibody in combination with radiation therapy on the growth of human tumor xenografts was evaluated under normal and hypoxic conditions. Radiation treatment showed a dose-dependent increase in tumor growth delay, a finding that was sensitive to hypoxia (less tumor growth delay under hypoxic conditions). Treatment with A4.6.1 alone also increased tumor growth delay under both normoxic and hypoxic conditions. Treatment with A4.6.1 augmented radiation-induced tumor growth delay under both normal and hypoxic conditions.16 A similarly enhanced therapeutic effect was reported with the combined use of anti-(murine)VEGF monoclonal antibody and radiation therapy in murine tumor models, such as the Lewis lung carcinoma.23 Other inhibitors of the VEGF signaling pathway also have demon-
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Fig 2. Imaging therapeutic responses. (a) Tumor growth in the dorsal window chamber over a 20-day period without (top) and with (bottom) hormone ablation therapy. In the treated animal, the tumor regresses but then eventually escapes the therapy to regrow (*). (b) Tumor vessels which are abnormally tortuous with chaotic branching patterns can pass through a phase of “normalization” during anti-angiogenic therapy. This can help in delivering drugs more efficiently to the tumor cells. If the blood supply becomes inadequate, drug delivery will be impaired. (Reprinted with permission from Nature Reviews Cancer, copyright 2002 Macmillan Magazines Ltd.1)
strated short-term as well as long-term tumor control in combination with radiation.24,25 Hormone-dependent tumors often relapse following hormone ablation therapy, and patients may benefit from the combination of antihor-
monal and anti-VEGF therapy. Androgen withdrawal in prostate cancer inhibits VEGF expression and angiogenesis and induces endothelial cell apoptosis.26 We examined the role of androgens in angiogenesis using the Shionogi murine tumor, an
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androgen-dependent male mammary carcinoma. Castration of tumor-bearing mice was associated with tumor regression, a decrease in VEGF expression, and apoptosis of tumoral endothelial cells, as well as neoplastic cells, as shown by intravital microscopy. Tumoral endothelial cells underwent apoptosis before the neoplastic cells, however.26 Two weeks after castration, an increase in VEGF expression was observed, accompanied by an increase in angiogenesis and tumor growth, supporting the need for addition of anti-VEGF therapy. In breast cancer, overexpression of HER2, a receptor tyrosine kinase in the endothelial growth factor receptor family, is associated with poor prognosis and is thought to confer a growth advantage to neoplastic cells and to enhance their metastatic potential. HER2 signaling is known to increase VEGF expression in breast cancer cells, possibly through increasing the synthesis of HIF-1␣.27,28 Our laboratory showed that therapy with the monoclonal antibody trastuzumab (Herceptin; Genentech, Inc, South San Francisco, CA), which targets HER2, inhibits angiogenesis by modulating at least five different angiogenic pathways.29 Using intravital microscopy, we observed that trastuzumab “normalized” the vasculature of a HER2/neu-overexpressing human tumor xenograft, potentially allowing increased access to the target tumor cells. We also discovered that trastuzumab-induced downregulation of VEGF in cancer cells is accompanied by compensatory upregulation of VEGF in the host cells. Thus, it will be of great clinical interest to evaluate the potential of combining trastuzumab with bevacizumab.29 CONCLUSIONS
Antiangiogenic therapy targeting the VEGF signaling pathway is a promising new strategy for the management of solid tumors, primarily as a component of multiagent and multimodality treatments. By decreasing vascular permeability and IFP, judicious application of these agents may allow more effective delivery of other antitumor agents, including macromolecules. Prolonged therapy with these agents has been shown to reduce vascularization and inhibit tumor growth and metastases. One of the key goals for the future will be to determine the optimal doses and schedules of these agents to improve the delivery of other anticancer therapies, and the doses required to in-
hibit tumor angiogenesis. The actual doses are likely to vary with different tumors, corresponding to differences in composition, location, and VEGF expression. The development of dosing strategies and combination therapies will be an important clinical challenge for the future.30 REFERENCES 1. Jain RK, Munn LL, Fukumura D: Dissecting tumour pathophysiology using intravital microscopy. Nat Rev Cancer 2:266-276, 2002 2. Carmeliet P, Jain RK: Angiogenesis in cancer and other diseases. Nature 407: 249-257, 2000 3. Stohrer M, Boucher Y, Stangassinger M, et al: Oncotic pressure in solid tumors is elevated. Cancer Res 60:4251-4255, 2002 4. Netti PA, Hamberg LM, Babich JW, et al: Enhancement of fluid filtration across tumor vessels: Implication for delivery of macromolecules. Proc Natl Acad Sci U S A 96:3137-3142, 1999 5. Pluen A, Boucher Y, Ramanujan S, et al: Role of tumorhost interactions in interstitial diffusion of macromolecules: Cranial vs. subcutaneous tumors. Proc Natl Acad Sci U S A 98:4628-4633, 2001 6. Jain RK: Barriers to drug delivery in solid tumors. Sci Am 271:58-65, 1994 7. Padera TP, Kadambi A, di Tomaso E, et al: Lymphatic metastasis in the absence of functional intratumor lymphatics. Science 296:1883-1886, 2002 8. Netti PA, Berk DA, Swartz MA, et al: Role of extracellular matrix assembly in interstitial transport in solid tumors. Cancer Res 60:2497-2503, 2000 9. Jain RK: Normalizing tumor vasculature with anti-angiogenic therapy: A new paradigm for combination therapy. Nat Med 7:987-989, 2001 10. Ferrara N: VEGF: An update on biological and therapeutic aspects. Curr Opin Biotechnol 11:617-624, 2000 11. Dvorak HF, Nagy JA, Feng D, et al: Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr Top Microbiol Immunol 237:97-132, 1999 12. Melder RJ, Koenig GC, Witwer BP, et al: During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat Med 2:992-997, 1996 13. Compernolle V, Brusselmans K, Acker T, et al: Loss of HIF-2␣ and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med 8:702-710, 2002 14. Presta LG, Chen H, O’Connor SJ, et al: Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57:4593-4599, 1997 15. Yuan F, Chen Y, Dellian M, et al: Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad Sci U S A 93:14765-14770, 1996
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16. Lee CG, Heijn M, di Tomaso E, et al: Anti-vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. Cancer Res 60:5565-5570, 2000 17. Brown EB, Campbell RB, Tsuzuki Y, et al: In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med 7:864-868, 2001 18. Kadambi A, Moutra Carreira C, Yun CO, et al: Vascular endothelial growth factor (VEGF)-C differentially affects tumor vascular function and leukocyte recruitment: role of VEGF-receptor 2 and host VEGF-A. Cancer Res 61:24042408, 2001 19. Fukumura D, Xavier R, Sugiura T, et al: Tumor induction of VEGF promoter activity in stromal cells. Cell 94:715725, 1998 20. Tsuzuki Y, Fukumura D, Oosthuyse B, et al: Vascular endothelial growth factor (VEGF) modulation by targeting hypoxia-inducible factor-1␣ 3 hypoxia response element 3 VEGF cascade differentially regulates vascular response and growth rate in tumors. Cancer Res 60:6248-6252, 2000 21. Carmeliet P, Dor Y, Herbert JM, et al: Role of HIF1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394:485-490, 1998 22. Hansen-Algenstaedt N, Stoll BR, Padera TP, et al: Tumor oxygenation in hormone dependent tumors during vascular endothelial growth factor receptor-2 blockade, hormone ablation, and chemotherapy. Cancer Res 60:4556-4560, 2000 23. Gorski DH, Bechett MA, Jaskowiak NT, et al: Blockage of the vascular endothelial growth factor stress response in-
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creases the antitumor effects of ionizing radiation. Cancer Res 59:3374-3378, 1999 24. Kozin SV, Boucher Y, Hicklin DJ, et al: Vascular endothelial growth factor receptor-2-blocking antibody potentiates radiation-induced long-term control of human tumor xenografts. Cancer Res 61:39-44, 2001 25. Geng L, Donnelly E, McMahon G, et al: Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy. Cancer Res 61: 2413-2419, 2001 26. Jain RK, Safabakehsh N, Sckell A, et al: Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: Role of vascular endothelial growth factor. Proc Natl Acad Sci U S A 95:1082010825, 1998 27. Laughner E, Taghavi P, Chiles K, et al: HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: Novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 21:3995-4004, 2001 28. Yen L, You XL, Al Moustafa AE, et al: Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis. Oncogene 19:3460-3469, 2000 29. Izumi Y, Xu L, di Tomaso E, et al: Tumor biology: Herceptin acts as an anti-angiogenic cocktail. Nature 416:279280, 2002 30. Jain RK, Carmeliet PF: Vessels of death or life. Sci Am 285:38-45, 2001
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OLID TUMORS are multicompartmentalized structures, consisting of three major components: cancer and stromal cells, the extracellular matrix (ECM), and the vasculature.1 The volumes of each of these components vary depending on the origin and size of the tumor and the organ in which the primary tumor develops.1 Tumors require vasculature to gain access to oxygen and other nutrients, allowing growth and metastasis.2 While most anticancer therapies target neoplastic cells, novel approaches that target other tumor compartments may be potentially useful additions to the armamentarium of antitumor agents. Specifically, the development of new blood vessels (angiogenesis) is essential for tumor growth and metastasis, and several unique features of tumor Seminars in Oncology, Vol 29, No 6, Suppl 16 (December), 2002: pp 3-9
vasculature offer attractive targets for anticancer therapy. VASCULAR AND EXTRAVASCULAR BARRIERS IN TUMORS
Unlike normal angiogenesis, which is an ordered and highly regulated process that occurs during fetal and early postnatal development and during wound repair in the adult, the development of tumor vasculature is chaotic and irregular.2 Tumor blood vessels are functionally abnormal; their diameter is variable, they are excessively branched and twisted, and they are often dilated. Additionally, the walls of tumor vessels have many openings (endothelial fenestrae and transcellular holes), widened interendothelial junctions, and a defective basement membrane. The structural and functional abnormalities of tumor vasculature result in variable blood flow in the tumor, which in turn leads to hypoxic and acidic regions in tumors.1,2 The abnormal and chaotic architecture of tumor vasculature is one of several physiologic barriers that impede the effective delivery of therapeutic agents to target cells.3,4 To reach the cancer cells, any systemically administered therapeutic agent must enter the tumor via the vascular compartment, cross the vessel wall, and move through the interstitial compartment. In addition to abnormal features of tumor vasculature, the ECM also impedes movement of therapeutic agents toward target cells. The total barrier strength varies not only across different tumor types but also for the same tumor implanted in different body locations.5
From the Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Harvard Medical School and Massachusetts General Hospital, Boston, MA. Supported in part by an unrestricted educational grant from Genentech BioOncology and by grants from the National Cancer Institute. Address reprint requests to Rakesh K. Jain, PhD, Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Harvard Medical School and Massachusetts General Hospital, 100 Blossom St – Box 7, Boston MA 02114. Copyright 2002, Elsevier Science (USA). All rights reserved. 0093-7754/02/2906-1602$35.00/0 doi:10.1053/sonc.2002.37265 3
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Elevated interstitial fluid pressure (IFP) is a key barrier that prevents effective transport of therapeutic agents into tumors and thus reduces the efficacy of those agents. This is of particular concern with high–molecular-weight anticancer agents, such as monoclonal antibodies, because of their low diffusibility. The elevation of tumor IFP to the level seen in microvasculature is thought to be the result of the high vascular permeability of tumor blood vessels and the absence of a functional lymphatic circulation system.6,7 Our studies show that the oncotic pressure within human tumor xenografts is equivalent to that of plasma.3 In contrast, under normal conditions, IFP is lower than the pressure of the surrounding vasculature, allowing convective movement of macromolecules across the vessel wall and into the interstitial space. The composition of the ECM also influences the penetration of a drug into a solid tumor. Both tumor and stromal cells secrete components of the ECM, such as collagens and proteoglycans. Studies with fluorescently labeled macromolecules showed a direct correlation between ECM composition and macromolecular transport resistance. Specifically, greater mechanical stiffness of the tumor, a property dependent on fibrillar collagen networks and their interactions with proteoglycans, was associated with increased resistance to macromolecular penetration. Collagen contributes to ECM stiffness, and our studies showed that interstitial diffusion rates were increased by treating penetration-resistant tumors with collagenase.8 ANTIANGIOGENIC THERAPY
Antiangiogenic therapy offers several potential advantages over conventional key cytotoxic therapies, most notably with respect to physical accessibility and genetic stability of target cells. Unlike antitumor agents, whose target cells are often inaccessible and genetically unstable, antiangiogenic drugs target vascular endothelial cells, which are easily accessible and genetically stable. Furthermore, because most vasculature in adults is relatively quiescent, the potential for adverse effects with antiangiogenic therapy is reduced. Antiangiogenic therapy may be particularly effective in combination with other treatment modalities, such as conventional chemotherapy, antihor-
monal therapy, immunotherapy, and radiation therapy.9 VASCULAR ENDOTHELIAL GROWTH FACTOR AND ANGIOGENESIS
The focus of our studies is the vascular endothelial growth factor (VEGF, also known as vascular permeability factor [VPF], or VEGF-A), a critical and central regulator of angiogenesis. The other members of the VEGF family (VEGF-B, VEGF-C, VEGF-D, and PlGF) also play a role in angiogenesis. Vascular endothelial growth factor is both a vascular growth factor and a vascular permeability factor. Its expression is upregulated in many tumors,10,11 and it can upregulate expression of adhesion molecules on the vascular endothelium.12 It is required to initiate the formation of blood vessels during development, as demonstrated by the fact that disruption of one copy of the VEGF gene in mice leads to embryonic lethality because of severe vascular abnormalities. It is also required for normal vascular development for the first few weeks postnatally, while its inactivation in older (adult) animals affects only those structures that undergo vascular remodeling, such as the corpus luteum and bone growth plates.2,10 Chronic anti-VEGF downregulation during embryonic development may also lead to neurologic and lung impairment.2,13 ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN TUMOR ANGIOGENESIS
Bevacizumab (Avastin, rhuMAb-VEGF; Genentech, South San Francisco, CA) is a humanized mouse monoclonal antibody that neutralizes human, but not mouse, VEGF. Bevacizumab is derived from A4.6.1, the mouse monoclonal antibody directed against human VEGF (VEGF-A), and has the same high affinity and biological properties as A4.6.1.14 Both bevacizumab and A4.6.1 recognize all spliced variants of VEGF, but no other members of the VEGF family of proteins.14 Preclinical studies showed that inhibition of VEGF with A4.6.1 could alter and ultimately decrease tumoral blood supply. When human cancers (glioblastoma, melanoma, and colon adenocarcinoma) were transplanted into immunodeficient mice, intravenous treatment with A4.6.1 produced a rapid decrease in tumor vessel permeability
TUMOR ANGIOGENESIS AND ACCESSIBILITY
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Fig 1. Four requirements for intravital microscopy. (a) An appropriate animal model. Three different models are shown: a chronic transparent window in which a tumor grown on the inner surface of the skin can be viewed through a coverslip; an in situ preparation in which an optical tracer is injected into the interstitial space of the tail; and an acute liver model in which the animal is surgically prepared to reveal a tumor in the internal organ. (b) A molecular probe (usually fluorescent) that can be imaged. Two examples are shown: green fluorescent protein (GFP) that is expressed in genetically engineered cells/animals and controlled by the promoter of interest; and an optical probe that is activated by a specific enzyme or microenvironment. (c) A microscope equipped with an excitation source and a detection system (such as photomultiplier tube (PMT) or charged-coupled device/silicon-intensified target (SIT) camera), which passes the images to a computer acquisition and analysis workstation. (d) Image processing and analysis algorithms are then applied to extract quantitative data. (Reprinted with permission from Nature Reviews Cancer, copyright 2002 Macmillan Magazines Ltd.1)
(within 6 hours) and led to vascular regression.15 These effects persisted for 72 hours following a single intravenous dose of A4.6.1. Intraperitoneal administration of a single dose of A4.6.1 produced a decrease in vascular permeability in glioma after 6 days, whereas multiple doses over an 11-day period were associated with the regression of preformed vessels.15 This study also showed the specificity of A4.6.1 for human cancers, as the vascularization and vascular permeability of a transplanted murine breast cancer cell line that produces murine VEGF was unaffected by treatment with A4.6.1. In another study, treatment with A4.6.1 significantly reduced tumor IFP, leading to an increase in oxygen levels in some tumors.16 The antiangiogenic effect of bevacizumab on human tumor xenografts was further confirmed by multiphoton laser scanning intravital microscopy.17 This technique allows tumor images to be continuously captured by a video camera attached to a microscope that visualizes images through a
surgically implanted glass window near the tumor locus. Using a high-powered multiphoton microscope and computer-assisted analysis of captured images, multiple parameters of the tumor vasculature (vessel morphology, hemodynamics, pH, pO2, vascular permeability, leukocyte adhesion, and molecular distribution patterns) can be monitored (Fig 1).1,17 In addition, fluorescent dyes or live fluorescent reporters, such as green fluorescent protein, can be used to visualize vasculature or gene expression patterns. In intravital microscopy studies, A4.6.1 and an anti-VEGF receptor-2 antibody, DC101 (ImClone, New York, NY) inhibited neovascularization and growth of implanted tumors.15,18 Intravital microscopy was also used to determine the relative contribution of host stromal cells to tumoral VEGF levels. Transgenic mice expressing green fluorescent protein under the control of the VEGF promoter show fluorescence around the healing margins and throughout the granulation tissue of superficial wounds, a finding consistent
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with the role of VEGF in wound healing.19 Implantation of a non– green fluorescent protein-producing tumor cell line in dorsal skin chambers of the transgenic mice induced VEGF promoter activity and led to an accumulation of VEGF promoter-directed fluorescence. With time, the fluorescent cells (predominantly fibroblasts) invaded the tumor and could be seen throughout the tumor mass, indicating that normal stromal cells actively participate in tumor vascularization.17,19 We further examined the tumoral VEGF contribution of normal cells by transplantation of VEGF-/- tumors into VEGF⫹/⫹ mice. VEGF-/- tumors contained approximately 50% of VEGF levels found in wild-type (VEGF⫹/⫹) tumors, showing that host stromal cells contribute significantly to the VEGF levels found in tumors.19,20 Tumor cells have previously been shown to have elevated VEGF levels.10,11 HYPOXIA AND ANGIOGENESIS
Physiologic adaptation to hypoxia is a necessity for organisms having an oxygen-based metabolism. In mammals, these adjustments include vasodilatation, angiogenesis, upregulation of glucose transport and activation of glycolysis, and apoptosis (programmed cell death). By limiting tumor blood flow and oxygen supply, antiangiogenic therapy may select tumor cells that have acquired an unusual degree of tolerance to chronic hypoxia. To determine if tumors could acquire tolerance to chronic hypoxia, we examined the effect of inactivation of a key factor in the hypoxia-induced signaling cascade. Hypoxia inducible factor-1 alpha (HIF-1␣) is a transcription factor that mediates hypoxia-induced responses, including apoptosis, and VEGF gene regulation.21 We evaluated angiogenesis, tumor growth, and apoptosis in tumors that lacked HIF-1␣ (HIF-1␣-/-) and in wild type tumors. Whereas, the growth of the two tumor types was similar, HIF-1␣-/-tumors had reduced angiogenesis (and VEGF levels)20 and also decreased apoptosis.21 Thus, the decreased apoptosis in HIF-1␣-/- tumors effectively compensates for reduced vascularization and allows normal tumor growth. In other words, HIF-1␣-/- tumors show tolerance to chronic hypoxia, and their growth is not substantially affected by oxygen deprivation. Moreover, analysis of heterogeneous tumors established from mixtures of HIF-1␣⫹/⫹ and HIF-1␣-/-
viable cells showed that the proportion of cells expressing HIF-1␣ was increased in perivascular areas and decreased in distal tumor regions, indicating increased tolerance to hypoxia by HIF-1␣-/cells. In other words, HIF-1␣⫹/⫹ cells were highly dependent on proximity to blood vessels for their growth and survival in vivo, whereas HIF-1␣-/cells were not.17 This finding confirms the potential of antiangiogenic therapy to select tumor cells tolerant to hypoxia, which may have important implications for the long-term efficacy of antiangiogenic therapy as a single treatment modality. COMBINATION OF ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR APPROACHES WITH OTHER THERAPEUTIC MODALITIES
Although A4.6.1 and DC101 suppress tumor vascularization and growth, they do not appear to be curative. Therefore, it is important to evaluate their role in combination with other treatment modalities such as radiation, antihormonal, antibody, and chemotherapies. Both A4.6.1 and DC101 reduce tumor vascular permeability and IFP15,16,18 and thus “normalize” the abnormal tumor vasculature (Fig 2).9 This normalization may translate into enhanced intratumoral delivery of other drugs (eg, antitumor antibodies, cytotoxic agents). Additionally, this normalization may increase local oxygen level in some tumors, which may result in enhanced efficacy of antitumor radiation.9,16,22 The effect of anti-VEGF or VEGF receptor-2 antibody in combination with radiation therapy on the growth of human tumor xenografts was evaluated under normal and hypoxic conditions. Radiation treatment showed a dose-dependent increase in tumor growth delay, a finding that was sensitive to hypoxia (less tumor growth delay under hypoxic conditions). Treatment with A4.6.1 alone also increased tumor growth delay under both normoxic and hypoxic conditions. Treatment with A4.6.1 augmented radiation-induced tumor growth delay under both normal and hypoxic conditions.16 A similarly enhanced therapeutic effect was reported with the combined use of anti-(murine)VEGF monoclonal antibody and radiation therapy in murine tumor models, such as the Lewis lung carcinoma.23 Other inhibitors of the VEGF signaling pathway also have demon-
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Fig 2. Imaging therapeutic responses. (a) Tumor growth in the dorsal window chamber over a 20-day period without (top) and with (bottom) hormone ablation therapy. In the treated animal, the tumor regresses but then eventually escapes the therapy to regrow (*). (b) Tumor vessels which are abnormally tortuous with chaotic branching patterns can pass through a phase of “normalization” during anti-angiogenic therapy. This can help in delivering drugs more efficiently to the tumor cells. If the blood supply becomes inadequate, drug delivery will be impaired. (Reprinted with permission from Nature Reviews Cancer, copyright 2002 Macmillan Magazines Ltd.1)
strated short-term as well as long-term tumor control in combination with radiation.24,25 Hormone-dependent tumors often relapse following hormone ablation therapy, and patients may benefit from the combination of antihor-
monal and anti-VEGF therapy. Androgen withdrawal in prostate cancer inhibits VEGF expression and angiogenesis and induces endothelial cell apoptosis.26 We examined the role of androgens in angiogenesis using the Shionogi murine tumor, an
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androgen-dependent male mammary carcinoma. Castration of tumor-bearing mice was associated with tumor regression, a decrease in VEGF expression, and apoptosis of tumoral endothelial cells, as well as neoplastic cells, as shown by intravital microscopy. Tumoral endothelial cells underwent apoptosis before the neoplastic cells, however.26 Two weeks after castration, an increase in VEGF expression was observed, accompanied by an increase in angiogenesis and tumor growth, supporting the need for addition of anti-VEGF therapy. In breast cancer, overexpression of HER2, a receptor tyrosine kinase in the endothelial growth factor receptor family, is associated with poor prognosis and is thought to confer a growth advantage to neoplastic cells and to enhance their metastatic potential. HER2 signaling is known to increase VEGF expression in breast cancer cells, possibly through increasing the synthesis of HIF-1␣.27,28 Our laboratory showed that therapy with the monoclonal antibody trastuzumab (Herceptin; Genentech, Inc, South San Francisco, CA), which targets HER2, inhibits angiogenesis by modulating at least five different angiogenic pathways.29 Using intravital microscopy, we observed that trastuzumab “normalized” the vasculature of a HER2/neu-overexpressing human tumor xenograft, potentially allowing increased access to the target tumor cells. We also discovered that trastuzumab-induced downregulation of VEGF in cancer cells is accompanied by compensatory upregulation of VEGF in the host cells. Thus, it will be of great clinical interest to evaluate the potential of combining trastuzumab with bevacizumab.29 CONCLUSIONS
Antiangiogenic therapy targeting the VEGF signaling pathway is a promising new strategy for the management of solid tumors, primarily as a component of multiagent and multimodality treatments. By decreasing vascular permeability and IFP, judicious application of these agents may allow more effective delivery of other antitumor agents, including macromolecules. Prolonged therapy with these agents has been shown to reduce vascularization and inhibit tumor growth and metastases. One of the key goals for the future will be to determine the optimal doses and schedules of these agents to improve the delivery of other anticancer therapies, and the doses required to in-
hibit tumor angiogenesis. The actual doses are likely to vary with different tumors, corresponding to differences in composition, location, and VEGF expression. The development of dosing strategies and combination therapies will be an important clinical challenge for the future.30 REFERENCES 1. Jain RK, Munn LL, Fukumura D: Dissecting tumour pathophysiology using intravital microscopy. Nat Rev Cancer 2:266-276, 2002 2. Carmeliet P, Jain RK: Angiogenesis in cancer and other diseases. Nature 407: 249-257, 2000 3. Stohrer M, Boucher Y, Stangassinger M, et al: Oncotic pressure in solid tumors is elevated. Cancer Res 60:4251-4255, 2002 4. Netti PA, Hamberg LM, Babich JW, et al: Enhancement of fluid filtration across tumor vessels: Implication for delivery of macromolecules. Proc Natl Acad Sci U S A 96:3137-3142, 1999 5. Pluen A, Boucher Y, Ramanujan S, et al: Role of tumorhost interactions in interstitial diffusion of macromolecules: Cranial vs. subcutaneous tumors. Proc Natl Acad Sci U S A 98:4628-4633, 2001 6. Jain RK: Barriers to drug delivery in solid tumors. Sci Am 271:58-65, 1994 7. Padera TP, Kadambi A, di Tomaso E, et al: Lymphatic metastasis in the absence of functional intratumor lymphatics. Science 296:1883-1886, 2002 8. Netti PA, Berk DA, Swartz MA, et al: Role of extracellular matrix assembly in interstitial transport in solid tumors. Cancer Res 60:2497-2503, 2000 9. Jain RK: Normalizing tumor vasculature with anti-angiogenic therapy: A new paradigm for combination therapy. Nat Med 7:987-989, 2001 10. Ferrara N: VEGF: An update on biological and therapeutic aspects. Curr Opin Biotechnol 11:617-624, 2000 11. Dvorak HF, Nagy JA, Feng D, et al: Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr Top Microbiol Immunol 237:97-132, 1999 12. Melder RJ, Koenig GC, Witwer BP, et al: During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat Med 2:992-997, 1996 13. Compernolle V, Brusselmans K, Acker T, et al: Loss of HIF-2␣ and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med 8:702-710, 2002 14. Presta LG, Chen H, O’Connor SJ, et al: Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57:4593-4599, 1997 15. Yuan F, Chen Y, Dellian M, et al: Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad Sci U S A 93:14765-14770, 1996
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16. Lee CG, Heijn M, di Tomaso E, et al: Anti-vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. Cancer Res 60:5565-5570, 2000 17. Brown EB, Campbell RB, Tsuzuki Y, et al: In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med 7:864-868, 2001 18. Kadambi A, Moutra Carreira C, Yun CO, et al: Vascular endothelial growth factor (VEGF)-C differentially affects tumor vascular function and leukocyte recruitment: role of VEGF-receptor 2 and host VEGF-A. Cancer Res 61:24042408, 2001 19. Fukumura D, Xavier R, Sugiura T, et al: Tumor induction of VEGF promoter activity in stromal cells. Cell 94:715725, 1998 20. Tsuzuki Y, Fukumura D, Oosthuyse B, et al: Vascular endothelial growth factor (VEGF) modulation by targeting hypoxia-inducible factor-1␣ 3 hypoxia response element 3 VEGF cascade differentially regulates vascular response and growth rate in tumors. Cancer Res 60:6248-6252, 2000 21. Carmeliet P, Dor Y, Herbert JM, et al: Role of HIF1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394:485-490, 1998 22. Hansen-Algenstaedt N, Stoll BR, Padera TP, et al: Tumor oxygenation in hormone dependent tumors during vascular endothelial growth factor receptor-2 blockade, hormone ablation, and chemotherapy. Cancer Res 60:4556-4560, 2000 23. Gorski DH, Bechett MA, Jaskowiak NT, et al: Blockage of the vascular endothelial growth factor stress response in-
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creases the antitumor effects of ionizing radiation. Cancer Res 59:3374-3378, 1999 24. Kozin SV, Boucher Y, Hicklin DJ, et al: Vascular endothelial growth factor receptor-2-blocking antibody potentiates radiation-induced long-term control of human tumor xenografts. Cancer Res 61:39-44, 2001 25. Geng L, Donnelly E, McMahon G, et al: Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy. Cancer Res 61: 2413-2419, 2001 26. Jain RK, Safabakehsh N, Sckell A, et al: Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: Role of vascular endothelial growth factor. Proc Natl Acad Sci U S A 95:1082010825, 1998 27. Laughner E, Taghavi P, Chiles K, et al: HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: Novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 21:3995-4004, 2001 28. Yen L, You XL, Al Moustafa AE, et al: Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis. Oncogene 19:3460-3469, 2000 29. Izumi Y, Xu L, di Tomaso E, et al: Tumor biology: Herceptin acts as an anti-angiogenic cocktail. Nature 416:279280, 2002 30. Jain RK, Carmeliet PF: Vessels of death or life. Sci Am 285:38-45, 2001