Imaging tumor angiogenesis

Kristy Red-Horse, Napoleone Ferrara

J Clin Invest. 2006;116(10):2585-2587. https://doi.org/10.1172/JCI30058.

Commentary

Since the discovery of vascular-specific growth factors with angiogenic activity, there has been a significant effort to develop cancer drugs that restrict tumorigenesis by targeting the blood supply. In this issue of the JCI, Mancuso et al. use mouse models to better understand the plasticity of the tumor vasculature in the face of antiangiogenic therapy (see the related article beginning on page 2610). They describe a rapid regrowth of the tumor vasculature following withdrawal of VEGFR inhibitors, emphasizing the importance of fully understanding the function of these and similar treatments used in the clinic at the cellular and molecular level.

Find the latest version: https://jci.me/30058/pdf commentaries Imaging tumor angiogenesis Kristy Red-Horse and Napoleone Ferrara

Genentech Inc., South San Francisco, California, USA.

Since the discovery of vascular-specific growth factors with angiogenic activ- in 1 week (Figure 1). In accordance with ity, there has been a significant effort to develop cancer drugs that restrict previous findings (4, 6), posttreatment tumorigenesis by targeting the blood supply. In this issue of the JCI, Mancuso angiogenic growth correlated with the et al. use mouse models to better understand the plasticity of the tumor vas- disappearance of empty basement mem- culature in the face of antiangiogenic therapy (see the related article begin- brane sleeves, suggesting that regrowth ning on page 2610). They describe a rapid regrowth of the tumor vasculature occurred along these structures (Figure 1). following withdrawal of VEGFR inhibitors, emphasizing the importance of Attempts to prevent regrowth by block- fully understanding the function of these and similar treatments used in the ing MMP activity with the MMP inhibitor clinic at the cellular and molecular level. AG3340 or blocking a cryptic site within type IV collagen with the monoclonal Inhibiting tumor growth by limiting growth. However, not all vessels are elimi- antibody HUIV26 were unsuccessful. angiogenesis can, in combination with nated. A subset of VEGF-independent ves- However, this does not rule out the poten- chemotherapy, extend life expectancy in sels expressing lower levels of VEGFR2 (4) tial of regulating tumor angiogenesis by patients with certain malignancies and remain, resulting in a more “normalized” targeting these vascular relics. is being heavily pursued as a promising vascular bed, no longer exhibiting the tor- cancer therapy (1). There are several FDA- tuosity and dysfunction frequently asso- Role of basement membranes in the approved antiangiogenenic drugs current- ciated with tumor blood vessels. These context of normal angiogenesis ly used in the clinic to treat cancer and neo- remaining vessels could provide a source In the adult, the majority of angiogen- vascular age-related macular degeneration from which new vessels may bud follow- esis is associated with wound healing, the (bevacizumab, pegaptanib, ranibizumab, ing renewed VEGF activity. In addition, reproductive tract, and inflammation. At sunitinib, and sorafenib) and many others vascular regression leaves behind base- the cellular level, this process occurs by under development. These agents inhibit ment membrane sleeves — once associ- several different mechanisms, including a angiogenesis by targeting the signaling ated with the endothelial cell layer — that phenomenon termed sprouting (9), which pathways mediated by VEGF-A and poten- can persist for up to 21 days (5). In pre- is a major method by which tumors recruit tially other VEGF family members, such as vious work, McDonald and colleagues the preexisting vasculature. Sprouting is placental growth factor (PlGF), VEGF-B, established that, in normal vascular beds stimulated in response to local molecular VEGF-C, and VEGF-D. However, VEGF-A such as the trachea and thyroid, regrowth cues, such as hypoxia- or inflammation- is probably the major positive regulator of following drug withdrawal is rapid and induced VEGF-A production. Thought blood vessel formation. Given the success appears to be aided by the presence of to be a stepwise process, sprouting begins of anti-VEGF therapy in multiple mouse basement membrane sleeves, identifying with local increases in vascular perme- models of cancer and in clinical trials, it is these structures as potential antiangio- ability, followed by basement membrane important to fully understand the mech- genic targets (4, 6). and ECM degradation. Subsequently, anisms by which anti-VEGF treatment In their current report (2), the authors endothelial cells sometimes called tip cells affects tumor angiogenesis at the cellular use elegant microscopic imaging tech- send out projections and initiate migration and molecular level. This will be particu- niques to extend these findings to angio- along newly deposited ECM tracts. Finally, larly important when designing combina- genesis associated with 2 tumor models, lumen-containing vessels are formed and torial treatments aimed at circumventing a transgenic mouse model of insulinoma integrated into the circulation. Recent evi- drug resistance and tailoring patient- (RIP-Tag2 tumors) and mice implanted dence using real-time imaging techniques specific regimens. with Lewis lung carcinoma xenografts. in zebrafish demonstrates that the lat- In this issue of the JCI, Mancuso et al. Two different small molecule tyrosine ter steps involve, at least in some vessels, further our understanding of how anti- kinase inhibitors were used to target endothelial pinocytosis followed by coales- VEGF therapy affects blood vessels by VEGFR activity: AG-013736 (7), which cence of intracellular vacuoles (10). describing the dynamics of tumor vas- also blocks other receptor tyrosine kinas- At the molecular level, sprouting angio- culature regrowth after drug withdrawal es including PDGFR-β and c-kit, and genesis requires multiple factors, includ- (2). Work from multiple laboratories (3) AG-028262, which has been reported to ing MMPs such as MMP-2 (11) and has established that inhibiting the VEGF be a more selective inhibitor of VEGFR2 MMP-9 (12), which function in ECM pathway rapidly reduces the vascularity of (8). Similar results were obtained using remodeling and in the liberation and solid tumors, leading to a decline in tumor both drugs, suggesting that they function modification of angiogenic factors such primarily through VEGF inhibition. The as VEGF-A and FGF (13). Integrins are authors demonstrate that cessation of a also involved, as they bind sites within the Conflict of interest: The authors are employed by Genentech Inc., manufacturer of bevacizumab. 7-day therapy regimen is followed by rapid ECM and are critical for endothelial cell Citation for this article: J. Clin. Invest. 116:2585–2587 vascular regrowth, whereby the vessel den- proliferation, migration, and survival (14). (2006). doi:10.1172/JCI30058. sity returned to pretreatment levels with- In some cases, these 2 factors function in

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Figure 1 The effects of VEGF inhibition on the tumor vasculature are reversible. (A) Tumor growth stimulates angiogenesis, producing an abnormal vas- cular bed with disorganized branching and increased permeability. In many cases, this is due to increased VEGF production. (B) Antiangiogenic therapy that inhibits VEGF activity decreases tumor vascularity. Vascular regression often leaves behind a pericyte layer and empty basement membrane sleeves that can persist for up to 21 days. The pericytes were, however, observed to have reduced immunoreactivity to α–smooth muscle actin. (C) Cessation of anti-VEGF therapy following a 7-day treatment regimen results in rapid vascular regrowth. Vessel density returns to pretreatment levels within 7 days. the same pathway where MMPs cleave the experimental models and clinical settings, efficacy of chemotherapeutics and radia- ECM to expose cryptic angiogenenic inte- treatment is given regularly for a sus- tion (18–20). The quantitative imaging grin-binding sites (15, 16). Thus, blocking tained period of time, somewhat longer techniques well established by the authors certain MMP and integrin family mem- than that used in the current study (2). In should be helpful in addressing these and bers can inhibit angiogenesis (12, 16). addition, antiangiogenic therapy is com- other issues by defining the tumor vascu- Mancuso et al. (2) similarly target MMPs bined with treatment with other cytotox- lature during critical stages of anti-VEGF and an integrin-binding site on type IV ic agents. Thus, to answer this question, treatment. Similarly, it may be informative collagen in their system, but there is no future work should focus on assessing to assess vasculature growth at the precise effect on tumor revascularization. The the dynamics of the tumor vasculature time when tumors begin to acquire resis- difference between their model and oth- following prolonged VEGF inhibition as tance to antiangiogenic drugs. Resistance ers may be the presence of preassembled well as after combination therapy. The is associated with reestablishment of the basement membrane sleeves that facilitate imaging analysis described in this manu- tumor vasculature due to compensatory sprouting. In support of this theory, the script could also be extended to human upregulation of other angiogenic factors authors localized VEGF to the basement tumor biopsy samples, perhaps leading to such as FGF (21). membrane and show microscopic images a method for monitoring patient respons- In summary, the work presented by Man- that capture new vessel sprouts enveloped es to anti-VEGF therapy. cuso et al. (2) expands our knowledge of within the type IV collagen matrix. In Despite it successes, antiangiogenic ther- how targeting the VEGF pathway influenc- addition, regrowth appears to occur along apy is continually evolving, with the goal es tumor angiogenesis. The rapid regrowth preexisting ECM tracts. Experiments of developing the most effective strategies of tumor-associated vessels following dere- analyzing regrowth in the presence and for different types of cancers and for each pression of VEGF signaling underscores the absence of these structures could address individual case. Current focuses are on powerful angiogenic activity of this growth the issue; for example, examining the rate establishing optimal dosages and combi- factor family. These and future studies of angiogenesis in comparable vascular nations as well as identifying mechanisms aimed at broadening our understanding beds before and after the basement mem- of drug resistance. As to the former, data of antiangiogenenic therapy are important brane has been degraded, which eventually from experimental models and clinical tri- steps in furthering the development of this occurs after longer treatment periods (5). als indicate that the optimal anti-VEGF class of cancer therapeutics. drug dosage may be critical to getting the Clinical implications maximum synergistic effects between it Address correspondence to: Napoleone of tumor revascularization and chemotherapy or radiation (17). It Ferrara, Genentech Inc., 1 DNA Way, South What is the potential significance of rapid has been suggested that inhibiting VEGF San Francisco, California 94080, USA. vascular regrowth to patients undergo- signaling “normalizes” the tumor vascu- Phone: (650) 225-2968; Fax: (650) 225- ing antiangiogenic therapy? Typically, in lature, resulting in improved delivery and 6327; E-mail: [email protected].

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1. Ferrara, N., and Kerbel, R.S. 2005. Angiogenesis as Assoc. Cancer Res. 43:A5357. esis. Am. J. Pathol. 161:1429–1437. a therapeutic target. Nature. 438:967–974. 8. Zou, H.Y., et al. 2004. AG-028262, a novel selective 16. Xu, J., et al. 2001. Proteolytic exposure of a cryp- 2. Mancuso, M.R., et al. 2006. Rapid vascular regrowth VEGFR tyrosine kinase antagonist that potently tic site within collagen type IV is required for in tumors after reversal of VEGF inhibition. J. Clin. inhibits KDR signaling and angiogenesis in vitro angiogenesis and tumor growth in vivo. J. Cell Biol. Invest. 116:2610–2621. doi:10.1172/JCI24612. and in vivo. Proc. Am. Assoc. Cancer Res. 45:A2578. 154:1069–1079. 3. Gerber, H.P., and Ferrara, N. 2005. Pharmacology 9. Conway, E.M., Collen, D., and Carmeliet, P. 2001. 17. Bergsland, E., and Dickler, M.N. 2004. Maximizing and pharmacodynamics of bevacizumab as mono- Molecular mechanisms of blood vessel growth. Car- the potential of bevacizumab in cancer treatment. therapy or in combination with cytotoxic therapy diovasc Res. 49:507–521. Oncologist. 9(Suppl. 1):36–42. in preclinical studies. Cancer Res. 65:671–680. 10. Kamei, M., et al. 2006. Endothelial tubes assem- 18. Winkler, F., et al. 2004. Kinetics of vascular normal- 4. Kamba, T., et al. 2006. VEGF-dependent plastic- ble from intracellular vacuoles in vivo. Nature. ization by VEGFR2 blockade governs brain tumor ity of fenestrated capillaries in the normal adult 442:453–456. response to radiation: role of oxygenation, angio- microvasculature. Am. J. Physiol. Heart Circ. Physiol. 11. Itoh, T., et al. 1998. Reduced angiogenesis and poietin-1, and matrix metalloproteinases. Cancer 290:H560–H576. tumor progression in gelatinase A-deficient mice. Cell. 6:553–563. 5. Inai, T., et al. 2004. Inhibition of vascular Cancer Res. 58:1048–1051. 19. Tong, R.T., et al. 2004. Vascular normalization endothelial growth factor (VEGF) signaling in can- 12. Bergers, G., et al. 2000. Matrix metalloproteinase-9 by vascular endothelial growth factor receptor 2 cer causes loss of endothelial fenestrations, regres- triggers the angiogenic switch during carcinogen- blockade induces a pressure gradient across the sion of tumor vessels, and appearance of basement esis. Nat. Cell Biol. 2:737–744. vasculature and improves drug penetration in membrane ghosts. Am. J. Pathol. 165:35–52. 13. Mott, J.D., and Werb, Z. 2004. Regulation of matrix tumors. Cancer Res. 64:3731–3736. 6. Baluk, P., et al. 2004. Regulated angiogenesis and biology by matrix metalloproteinases. Curr. Opin. 20. Jain, R.K. 2005. Normalization of tumor vascula- vascular regression in mice overexpressing vascular Cell Biol. 16:558–564. ture: an emerging concept in antiangiogenic thera- endothelial growth factor in airways. Am. J. Pathol. 14. Stupack, D.G., and Cheresh, D.A. 2004. Integrins py. Science. 307:58–62. 165:1071–1085. and angiogenesis. Curr. Top. Dev. Biol. 64:207–238. 21. Casanovas, O., Hicklin, D.J., Bergers, G., and Hana- 7. Hu-Lowe, D., et al. 2002. Characterization of 15. Hangai, M., et al. 2002. Matrix metalloproteinase-9- han, D. 2005. Drug resistance by evasion of antian- potency and activity of the VEGF/PDGF recep- dependent exposure of a cryptic migratory control giogenic targeting of VEGF signaling in late-stage tor tyrosine kinase inhibitor AG013736. Proc. Am. site in collagen is required before retinal angiogen- pancreatic islet tumors. Cancer Cell. 8:299–309.

Myeloid suppressor cells regulate the adaptive immune response to cancer Alan B. Frey

Department of Cell Biology, New York University School of Medicine, New York, New York, USA.

Inflammation resultant from tumor growth, infection with certain patho- including activation of Tregs (2), cancer gens, or in some cases, trauma, can result in systemic release of cytokines, “immunoediting” (3), direct tumor down- especially GM-CSF, that in turn stimulate the abundant production and regulation of the effector phase (4, 5), and activation of a population of immature myeloid cells, termed myeloid sup- activation of non–T cell suppressor cells pressor cells (MSCs), that have potent immunosuppressive functions. In (6). Myelopoiesis is often dramatically this issue of the JCI, Gallina and colleagues have illuminated some complex stimulated during tumor growth, resulting issues concerning the development, activation, and function of MSCs (see in accumulation (in secondary lymphoid the related article beginning on page 2777). They show that activation of organs, blood, and tumor tissue) and acti- MSCs is initiated in response to IFN-γ, presumably produced in situ by anti- vation of a population of myeloid suppres- tumor T cells in the tumor microenvironment. After this triggering event, sor cells (MSCs). In mice, removal of the MSCs express 2 enzymes involved in l-arginine metabolism, Arginase I and primary tumor results in normalization of iNOS, whose metabolic products include diffusible and highly reactive per- the number of systemic MSCs, revealing a oxynitrites, the ultimate biochemical mediators of T cell immune suppres- causal role for tumor growth in their aber- sion. The multifaceted regulation of this complex suppressive effector sys- rant accrual (7). Purified MSCs have been tem provides several potential therapeutic targets. shown to inhibit both CD4+ and CD8+ T cell responses in vitro, and indirect but Immune response to tumor growth recognition of tumor antigens, coincident compelling studies imply that these cells As shown elegantly by North, priming of with or immediately subsequent to T cell can downregulate T cell functions in vivo. the adaptive immune response occurs dur- priming, the antitumor immune response ing the early stage of tumor growth and is inadequate to eliminate the tumor and MSCs in cancer results in development of CD8+ T cells is eventually dampened, thereby leading Recent research has focused on decipher- reactive to tumors (1). Despite evident host to tumor escape. Understanding how can- ing the role played by MSCs in the subver- cer growth affects the antitumor immune sion, inhibition, or downregulation of the response and discovering how escape from immune response to cancer (reviewed in Nonstandard abbreviations used: IL-4Rα, IL-4 recep- antitumor immunity can be reversed are ref. 6). MSCs are uniformly CD11b+TCR– tor α; l-Arg, l-arginine; MSC, myeloid suppressor cell. Conflict of interest: The author has declared that no major goals in tumor immunology. but express surface markers indicative of a conflict of interest exists. Several non–mutually exclusive phenom- mix of partially differentiated monocytes Citation for this article: J. Clin. Invest. 116:2587–2590 ena are likely to contribute to inhibition and DCs. The exact differentiation status (2006). doi:10.1172/JCI29906. of effective antitumor T cell immunity, of MSCs probably reflects the mechanism

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