Angiogenesis and Apoptosis

Seminars in Cancer Biology 13 (2003) 159–167 Angiogenesis and apoptosis Judah Folkman∗ Department of Surgery, Children’s Hospital and Harvard Medical School, Hunnewell 103 300 Longwood Avenue, Boston, MA 02115, USA Abstract This review assembles the laboratory and clinical evidence that cytotoxic chemotherapy and antiangiogenic therapy are each dependent on endothelial cell apoptosis. During cytotoxic chemotherapy, apoptosis of endothelial cells in the vascular bed of tumors precedes apoptosis of tumor cells, even when the tumor has been made drug resistant. Administration of an angiogenesis inhibitor which is not directly cytotoxic to tumor cells can increase tumor cell apoptosis and inhibit tumor growth by inhibiting endothelial proliferation and migration and/or by inducing endothelial apoptosis. Furthermore, oncogene expression and loss of tumor suppres- sor gene activity can at once protect tumor cells against apoptosis and increase their angiogenic output. Both of these survival ad- vantages conferred on the tumor can be overcome by antiangiogenic therapy. They can also be overcome by cytotoxic chemotherapy administered on a low dose ‘antiangiogenic schedule’ which continuously exposes endothelial cells in the tumor bed to the drug. As a result, endothelial apoptosis can be demonstrated to precede tumor cell apoptosis, and tumors regress or are inhibited, whether or not the tumor cells are resistant to the drug, and with little or no host toxicity. In contrast, cytotoxic chemotherapy administered on a ‘conventional schedule’ of maximal tolerated dose followed by an off-therapy interval, becomes ineffective after drug resistance is acquired. On the basis of these experimental findings, chemotherapy of cancer may possibly be improved—i.e. decreased drug resistance and decreased toxic side-effects—by changing dose and schedule to maximize apoptosis of endothelial cells in the vascular bed of tumors. Further improvement may be achieved by combining angiogenesis inhibitors with ‘antiangiogenic chemotherapy’. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Apoptosis; Antiangiogenic chemotherapy 1. Introduction 2. The angiogenic switch in a tumor is followed by decreased apoptosis Angiogenesis, the growth of new capillary blood vessels, is critical for development, reproduction and repair and Tumor growth is angiogenesis-dependent (for experimen- dominates many pathological conditions. The hypothesis tal evidence see reviews [2,3]). In the absence of angiogen- that tumor growth is angiogenesis-dependent was first pro- esis, tumor growth is restricted to a microscopic size; tumor posed in 1971 [1,2] and has subsequently been proven by a cells do not shed into the circulation. In non-angiogenic in variety of experiments including genetic methods [3]. The situ tumors [4] or in dormant micro-metastases [5,6] tumor ability to culture endothelial cells in vitro, the development cell proliferation continues (e.g. up to 30% BrdU labeling in of bioassays for angiogenesis, the discovery of molecules experimental tumors) balanced by high rates of tumor cell which stimulate angiogenesis as well as those which inhibit apoptosis (up to 8–10% apoptotic cells identified by frag- this process, have led to the emergence of an active field mented DNA with the TdT staining technique [5]). High of angiogenesis research now pursued worldwide. A fun- rates of human tumor cell apoptosis can persist for as long damental question of how angiogenesis is linked to apop- as a tumor remains non-angiogenic. Non-angiogenic clones tosis of endothelial cells and of tumor cells is addressed of tumor cells were isolated from human osteosarcomas and here. transplanted from one SCID mouse to another after 8 months residence in each mouse as a <0.5 mm dormant subcuta- neous tumor [7]. After 3 years, a small percentage of these dormant tumors spontaneously became angiogenic. In con- ∗ Tel.: +1-617-355-7661; fax: +1-617-355-7662. trast, non-angiogenic dormant human liposarcomas became E-mail address: [email protected] (J. Folkman). angiogenic within 2–4 months of transplantation in SCID 1044-579X/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. PII: S1044-579X(02)00133-5 160 J. Folkman/ Seminars in Cancer Biology 13 (2003) 159–167 mice [4]. Other human tumors, such as breast cancer spon- non-angiogenic and angiogenic, are mixed at different ra- taneously switched to the angiogenic phenotype over a pe- tios before implantation into SCID immunodeficient mice riod of 6 months. Therefore, an in situ dormant tumor may reveal that as few as 1% angiogenic cells will lead to a neo- not be harmful to its host until it has recruited microvascular vascularized tumor [7]. However, the latent period before a endothelial cells. detectable tumor appears is longer with a lower percentage The angiogenic switch can be induced at a predictable of angiogenic cells (e.g. months with 1% angiogenic cells time and usually more rapidly, by introduction of oncogenes versus weeks with 50% angiogenic cells). into the tumors. When the human non-angiogenic osteosar- While pre-existing microvessels, are surrounded by nor- coma was transfected with the ras oncogene, neovascular- mal cells, under some conditions such as in brain metastasis, ized tumors appeared within 2 weeks [7]. In transgenic mice tumor cells can displace normal cells around a microvessel, in which the large T-antigen was expressed in all ␤-cells a process of vessel cooption [16]. However, new tissue mass of the pancreatic islets, carcinomas appeared at approxi- requires the recruitment of new microvessels. This principle mately 4 weeks of life and the angiogenic switch occurred applies to tumors as well as to normal tissue (such as fat) at approximately 7 weeks in 4–10% of islets [8,9]. The [17]. Neoplastic tissue usually exceeds the oxygen diffusion angiogenic switch occurred slightly later in fibrosarcomas limit when tumor cell layers accumulate to a thickness of ap- arising in transgenic mice carrying the bovine papilloma proximately 150–200 ␮m from a nearest open microvessel. virus oncogenes [10], or when squamous cell carcinomas Tumor cells beyond this limit undergo apoptosis (Fig. 1). arose in mice carrying the human papillovirus type 16 Therefore, almost any tumor that has reached a diameter of oncogenes targeted to basal cells of the epidermis [11]. The >10–100 mm, is probably already neovascularized. spontaneous angiogenic switch [9] in human tumors can be After the angiogenic switch, new microvessels converge driven by: (1) angiogenic oncogenes [12] which up-regulate on the dormant in situ tumor and tumor cells cluster around expression of pro-angiogenic proteins (i.e. VEGF, bFGF each microvessel in a cylindrical configuration. The radius etc.), and/or down-regulate expression of angiogenesis in- of the microcylinder of tumor cells is limited by the oxygen hibitors, such as thrombospondin); (2) tumor-associated hy- diffusion requirements for that particular tumor, but it would poxic conditions which activate hypoxia-inducible factor-1 be rare to find layers of viable tumor cells beyond 200 ␮m (HIF-1) [13], which itself up-regulates angiogenic proteins; (Fig. 1) [18]. The angiogenic switch is associated with a (3) fibroblasts in the tumor bed which can be induced by marked decrease (3- to 4-fold) in overall tumor cell apoptosis tumor cells to elaborate pro-angiogenic proteins [14]; and [5]. In contrast to apoptosis, the proliferation rate of tumor (4) bone marrow derived progenitor endothelial cells which cells may remain at the same level and in many angiogenic traffic to tumors [15]. Of interest is that not all cells in a tumors appears to be relatively independent of the onset of non-angiogenic dormant human tumor must switch to the angiogenesis or its intensity [19]. angiogenic phenotype for a tumor itself to become neovas- The mechanisms of increased tumor cell survival and de- cularized. Experiments in which human cancer cells both creased tumor cell apoptosis after the onset of angiogenesis, Fig. 1. Neovascularized transplanted tumors growing in SCID immunodeficient mice to show supported viable tumor cells forming perivascular cuffs (indicated by black dashed ovals). Perivascular cuff size is roughly indicative of the metabolic burden of the tumor cells and their ability to survive under hypoxic conditions. Human melanoma cells (a) within approximately 80 ␮m of the vasculature are viable. Beyond this radius, at the limits oxygen and nutrient diffusion from the microvessel, an abrupt shift to tumor cell necrosis is observed. Prostate carcinoma cells (Dunning rat), (b) cannot exceed an oxygen/nutrient limit of 110 ␮m. Sections in both panels (a) and (b) were stained with hematoxylin for DNA, highlighting areas of necrosis, and with an antibody to CD31 in (b) showing the endothelial lining of blood vessels. (a) From Nava Almog in the Folkman laboratory, unpublished. (b) From Hlatky et al. [18] with permission from the publisher. J. Folkman/ Seminars in Cancer Biology 13 (2003) 159–167 161 depend not only on delivery of oxygen and nutrients and the hibitors endostatin or TNP-470 (Shay Soker, unpublished removal of catabolites by new microvessels, but also on a data). paracrine release of anti-apoptotic factors from the endothe- Several clinical studies indicate that despite an initial lial cells in these new vessels. Microvascular endothelial increase in tumor blood flow during the early phase of an- cells recruited into a tumor bed supply at least 20 mitogens, tiangiogenic therapy, chronic antiangiogenic therapy causes anti-apoptotic factors, and survival factors (including among total tumor blood flow to reach a steady state or to gradu- others, bFGF, HB-EGF, IL-6, G-CSF, IGF-1 and PDGF) for ally decrease. When endostatin was continued for weeks or those tumor cells apposed to the new microvessels [20–22]. months in cancer patients, PET scans revealed a gradual, It is unclear whether angiogenesis inhibitors also directly dose-dependent reduction in total tumor blood flow [28]. decrease endothelial cell production of paracrine factors, but This could be caused by a dropout of individual microves- this could be tested in vitro.

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