T UMOR ANGIOGENESIS OVERVIEW

1.1.1 Tumor growth is angiogenesis dependent.

Angiogenesis is defined as the formation of new blood vessels by proliferation of new capillaries from pre-existing microvessels. This process is distinct from vasculogenesis, which is defined as the formation of blood vessel de novo from angioblasts (Hanahan D and Folkman J 1996; Risau W 1997). Angiogenesis involves degradation of the basement membrane surrounding an existing capillary or venule, migration of endothelial cells through the basement membrane to create a sprout, proliferation of endothelial cells, formation of a lumen within the new sprout and joining of two sprouts to from a functional capillary loop, and vessel maturation (Bussolino F et al. 1997; Jendraschak E and Sage EH 1996). The idea that tumor growth is angiogenesis dependent was first proposed in 1971, allowing anti-angiogenic therapy to be used to treat cancer (Folkman J 1971). The development of a solid tumor progresses from a prevascular phase to a vascular phase. The prevascular tumor does not induce angiogenesis, is limited in size, and rarely metastasizes. The vascularized tumor induces host microvessels to undergo angiogenesis. The best characterized example is the hypoxia-dependent angiogenic switch in which host cell-derived endothelial cells invade into the tumor stroma to form new blood vessels (Carmeliet P and Jain RK 2000). One of the genetic proofs that tumor growth is angiogenesis-dependent is the induction of tumor angiogenesis by ras oncogene (Folkman J 1992). Ras induces the sequential activation of Myc, via a mechanism enables it to repress the expression of endogenous angiogenic inhibitors, e.g. thrombospondins (TSPs) (Watnick RS et al. 2003), and to activate angiogenic activators, e.g. vascular endothelial growth factor (VEGF) (Udagawa T et al. 2002). Thus, blocking angiogenesis can result in

tumor dormancy, tumors that can not expand beyond a microscopic size (Folkman J 2003b). Within the dormant tumors, the proliferating tumor cells are balanced by apoptotic tumor cells and few if any microvessels (Achilles EG et al. 2001).

1.1.2 Angiogenic activators and inhibitors are produced by tumor and host cells, respectively

In the early 1980’s, it became more clearly that tumor cells can produce specific angiogenesis activators that stimulate the proliferation of capillary endothelial cells (Shing Y et al. 1984). The growth of tumor vasculature is no longer regarded as an inflammatory reaction. Angiogenesis activators are stored in extracellular matrix (ECM) in a “stand-by mode” before they become activated by specific enzyme when angiogenesis is required, physiologically or pathologically (Folkman J 2003b).

Angiogenesis inhibitors can be expressed by tumor cells or by normal cells of the host and are endogenous molecular defense barriers in pathological hotspots of angiogenesis (Folkman J 2003b). Angiogenesis inhibitors are endogenous and from cryptic fragments of matrix protein, e.g. angiostatin from plasminogen (O'Reilly MS et al. 1994), endostatin from fragment of collagen XVIII (O'Reilly MS et al. 1997), and tumstatin from collage IV (Maeshima Y et al. 2002). The existence of naturally occurring angiogenic inhibitors which a tumor would have to overcome to induce angiogenesis also formed the basis for the subsequent concept of the

“angiogenic switch” (Bouck N 1990; Hanahan D and Folkman J 1996).

1.1.3 Angiogenic switch depends on the balance of angiogenic activators and inhibitors

Bouck et al. proposed that the onset of angiogenesis was the result of a shift in the balance of angiogenic activators and inhibitors, which were controlled by

oncogenes and tumor suppressor genes (Bouck N 1990). The balance of activators and inhibitors governs the angiogenic switch (Hanahan D and Folkman J 1996; Hawighorst T et al. 2001). The angiogenic switch is characterized by down-regulation of angiogenic inhibitors or up-regulation of angiogenic activators or both. When inhibitors are over activators, the switch is in the “off” position. When activators are over inhibitors, the switch is in the “on” position (Hanahan D and Folkman J 1996). Change in the relative balance of activators and inhibitors activate the angiogenic switch, before stabilizer molecules activate the maturation of nascent blood vessels (Bussolino F et al. 1997). This shift takes place between the angiogenic activators and inhibitors within the tumor cell itself; and between the tumor cell’s angiogenic proteins and the host’s anti-angiogenic proteins (Hanahan D and Folkman J 1996). For a tumor to switch to the angiogenic phenotype, it must overcome natural angiogenic inhibitors that might exist in the host’s circulation, extracellular matrix or within the tumor cells.

The discoveries of endogenous angiogenesis inhibitors are associated with neovascularized tumors, suggest a new paradigm of tumorigenesis (Folkman J 1995a). The extent to which the inhibitors are decreased during this switch dictates whether a primary tumor grows rapidly or slowly and whether metastases grow at all (Folkman J 1995a). Rastinejad et al. demonstrated that tumor cells did not become angiogenic until they had significantly reduced their own production of TSP (Rastinejad F et al. 1989). In certain tumors, the angiogenic switch also involves down-regulation of endogenous angiogenic inhibitors, in addition to increased expression of angiogenic activator. For example, ras transfection decreases expression of TSP and increases VEGF expression (Kerbel R and Folkman J 2002).

1.1.4 The angiogenic process relies on a complex tumor-host interaction The mechanisms of angiogenesis involve angiogenic activity arising from at least two sources: tumor cells that mediate the release of angiogenic activators and host cells (e.g. macrophages) recruited by the tumor from surrounding host ECM (Folkman J 1992). During angiogenic processes, there exist a complex interaction among cell components such as tumor, stromal, endothelial and inflammatory cells, growth factors, and the ECM, which are regulated by angiogenesis activators and inhibitors (Rice A and Quinn CM 2002).

Tissue mass, whether it is neoplastic or normal, may be regulated by microvascular endothelial cells (Folkman J 2003b). Both tumors and organs may limit their own growth by increasing the production of angiogenic inhibitors with size, until a critical size is reached where further growth is actively self-inhibited (Folkman J et al. 2000). Tumor mass, as well as normal organ size, is under the tight control of microvascular endothelium (Franck-Lissbrant I et al. 1998). If normal cells are similarly dependent upon endothelial-derived paracrine factors, the ratio of endothelial cells to normal parenchymal cells is likely to be lower than for tumor cells. Nevertheless, the regulation of tissue mass or organ size by vascular endothelial cells may be based upon mechanisms which also operate in tumors (Folkman J 1998). Cancer growth requires the proliferation of endothelial cells, in addition to malignant cells (Folkman J 1996). Without the proliferation of endothelial cells, a tumor cannot grow beyond the size of a colony. Folkman proposed the “two compartment model” explain the interaction of endothelial cells and tumor cells, which constitute two important compartments of a tumor. Within a tumor, these two cell compartments can stimulate each other’s growth via a perfusion effect or paracrine effect (Folkman J 1996). Coordinated tumor/vascular growth exploits an ultimate limitation to tumor size under angiogenic control, where

opposing angiogenic stimuli come into dynamic balance (Hahnfeldt P et al. 1999).

During tumor angiogenesis, there is continued recruitment of endothelia and continued expansion of the tumor mass. Ultimately, each newly recruited endothelial cell can support a large population of tumor cells. This leverage may be exploited to cancer therapeutic advantage (Folkman J et al. 2000). Administration of an angiogenic inhibitor, which is not directly cytotoxic to tumor cells, can increase tumor cell apoptosis and inhibit tumor growth by inhibiting endothelial proliferation and migration, or by inducing apoptosis of the endothelial cells (Folkman J 2003a). By administering angiogenic inhibitors to swift the stimulatory climate in the tumor back to inhibition, recruited endothelial cells are removed, followed by the subsequent loss of the relatively abundant amount of supported tumor cells (Folkman J et al. 2000; Hahnfeldt P et al. 1999).