Tumor Angiogenesis

Normal Vascular Development

Development of the vascular system is one of the first events in embryonic organogenesis. Mesoderm-derived vascular endothelial cells (ECs) generate lumen-containing tubular structures that form the basic functional unit of blood vessels. Initially, vascular networks form independently in the yolk sac and the embryo, and then connect to generate a closed circulatory system. In a process known as vasculogenesis , endothelial cell progenitors ( angioblasts ) and their derivative ECs aggregate de novo in the yolk sac to form a primitive vascular network or plexus of approximately uniform dimensions ( Figure 17-1 ). Subsequent angiogenesis occurs through vessel sprouting , in which ECs from existing vessels respond to angiogenic signals by degrading their basement membrane, loosening their association with support cells, altering their morphology, and proliferating. These ECs migrate in response to chemotactic signals and coalesce to form new vessels that connect to the existing vasculature. The coordinated recruitment of supporting mural cells, including pericytes and smooth muscle cells , results in vessel maturation. In a parallel mechanism termed intussusception , columns of endothelial cells create a barrier in the lumen of a preexisting vessel, thus partitioning it into multiple independent vessels ( Figure 17-2 ). This complex series of events produces a closed, highly arborized system of larger and smaller vessels including arteries, veins, and capillaries.

In contrast to the yolk sac, angioblasts in the embryo migrate along specific pathways and aggregate directly to form the dorsal aorta and posterior cardinal vein, without passing through an intermediate plexus phase. These vessels undergo subsequent remodeling and ultimately connect to the extra-embryonic yolk sac vessels to form a mature vascular system. Interestingly, vascular development is also intimately associated with the development of hematopoietic cell lineages, as hematopoietic stem cells (HSCs) have been shown to arise from the hemogenic endothelium , which comprises Runx-1 expressing ECs restricted to the ventral portion of the developing dorsal aorta (see Figure 17-1 ).

The vessels of the parallel lymphatic system collect and return interstitial fluids, particulates, and extravasated cells to the venous circulation. Lymphatic vessels differ from blood vessels in that lymphatic capillaries have internal membranous valves that prevent fluid backflow, and they are generally not surrounded by support cells. Lymphatic ECs are derived from primitive veins and express and respond to a different spectrum of receptors and signaling molecules than ECs in blood vessels (Ref. , and see later discussion). The ability of cancer cells to invade lymphatics and collect in lymph nodes, complex organs involved in local immune surveillance, is an important indicator of tumor metastasis. It is likely that the lymphatic vessels at the periphery of a solid tumor are most directly involved in metastasis, as interstitial pressure within the tumor often leads to vessel collapse. Recent evidence supports the idea that lymphatic ECs may secrete chemokines that attract tumor cells and may therefore participate more actively in metastasis than was previously recognized.

In adult humans and mice, there is little regular angiogenic activity, with the notable exception of the female reproductive system. Localized angiogenesis is, however, an important aspect of normal wound healing, and inflammatory cells including macrophages, neutrophils, and mast cells, as well as activated resident fibroblasts, are an important source of angiogenic modulators during wound repair. Recently, it was shown that macrophages directly bind angiogenic ECs and promote the formation of anastomoses between them. On remodeling and fusion with the surrounding vasculature, these new vessels restore normal blood supply to the wounded area. These infiltrating stromal cells also represent an important component of many solid tumors, where they can produce angiogenic factors as part of what may be considered an aberrant wound healing response, leading to the idea that tumors represent “wounds that never heal.” Genetic experiments using multiple murine cancer models have established that tumor-associated macrophages play a critical role in driving tumor angiogenesis and metastasis.

Rapid growth of any tissue (embryos, neoplasias, adipose tissue, regenerating liver, etc.) invariably requires a supply of oxygen, nutrients, and hormones and is typically accompanied by active angiogenesis. Consequently, angiogenesis can be seen as a genetically programmed, dynamic process that can be activated locally in response to stimulatory signals. The fact that most blood vessels in the adult body are quiescent has been proposed as an advantage of anti-angiogenic strategies, which typically target actively dividing ECs, as these drugs may be less generally toxic to quiescent ECs lining established vessels.

Tumor Vasculature

The blood vessels found in solid tumors are notable for being highly disorganized compared to those of normal organs and are characterized by tortuous and misshapen vessels that sometimes terminate in open-ended blood lakes ( Figure 17-3 ). These aberrant vessels are thought to result from dysregulated angiogenic signaling in the tumor bed, as a result of oncogene activation and tumor suppressor loss. Microscopic analysis of tumor vessels reveals disrupted junctions between tumor ECs and reduced or inconsistent coverage by pericytes, which helps explain the increased permeability characteristic of tumor vessels. The origin of some tumor ECs is also controversial: In addition to ECs recruited through sprouting of preexisting vessels, growing evidence supports a role of circulating endothelial progenitor cells (EPCs) that either differentiate into endothelial-like cells or promote expansion of bona fide ECs (see Figure 17-2 ). The precise cellular origins and complexity of these cells remain controversial, and the degree to which murine EPCs actually contribute directly to the lining of new tumor vessels varies considerably, depending on the model used, genetic background, and other factors. In addition, bone marrow–derived myeloid cells contribute to tumor angiogenesis; these cells have been reported to express a variety of cell surface markers, including those common to endothelial cells (Tie-2) and myeloid cells (CD11b, Gr-1), and may function by providing paracrine angiogenic signals. It is interesting to note that genetic ablation of bone marrow–derived Tie-2 expressing monocytes (TEMs), in particular, has profound effects on tumor angiogenesis in mice (see Refs. , and references therein).

Tumors often display sluggish, uneven, and highly variable patterns of blood flow, as well as direct arteriole-venule shunts. Tumor vessels also differ from normal vasculature in being exposed to an acidic microenvironment characterized by oxygen and nutrient deprivation. In rapidly growing tumors, aberrant angiogenic regulation and high interstitial pressure can produce regions of localized anoxia and/or ischemia. This typically results in pockets of necrosis surrounded by a penumbra of hypoxic but living cells. Severely hypoxic conditions are thought to protect tumor cells from radiation therapy, which depends on the generation of reactive oxygen intermediates to kill tumor cells. Moreover, hypoxic regions in tumors appear to select for highly malignant cancer cells. In particular, hypoxia directly promotes angiogenic signaling in tumors, as discussed in more detail later.

The degree to which tumors generate vascular beds is often expressed as microvessel, or mean vessel , density (MVD) , which can vary widely within a given tumor and between tumors of similar or different tissues. MVD is traditionally determined by staining tumor sections with antibodies raised against proteins expressed on ECs, including CD31 (PECAM), CD34, and von Willebrand factor. Clinical studies have demonstrated that MVD is a useful prognostic indicator for a wide array of cancers, including breast, prostate, non–small-cell lung, gastrointestinal, and even hematological tumors. It is important to note, however, that not all tumor vessels are functional and that MVD may greatly exceed the basic metabolic requirements of a growing tumor. The striking functional heterogeneity of vessels within a tumor, and the ability of many cancer cells to withstand severe hypoxia, glucose deprivation, and tissue acidity, makes it difficult to assess the effects of angiogenesis-based therapies based solely on MVD.

Pro-angiogenic Factors