Angiogenesis and the Tumor Microenvironment

The Tumor Microenvironment

Several recent reviews underscore the contribution of the microenvironment to tumor development.12,15-18 While the normal cellular microenvironment can inhibit malignant cell growth, the modifications that occur in the tumor microenvironment synergistically support cell proliferation. Tumors shape their microenvironment and support the development of both tumor cells and non-malignant cells. An extensive review by Polyak, et al., concludes with a cautious summary, noting “although the importance of an altered microenvironment in tumorigenesis is no longer disputed, the nature of the molecular alterations underlying these changes remains unclear.” 15 Cancer treatments are now being considered that focus on the tumor microenvironment as a therapeutic target, as the non-malignant cells are more genetically stable and less likely to evolve into drug resistant phenotypes.
The tumor microenvironment affects angiogenesis by interfering with the signaling pathways required for cell recruitment and vascular construction. Endothelial progenitor cells (EPCs) that are recruited under hypoxic conditions for angiogenesis have been associated as well with metastasis.
The metabolic environment of the tumor is directly affected by lack of vasculature and subsequent oxygen starvation. Hypoxia in the microenvironment results in acidosis, as lactic acid builds up due to anaerobic glycolysis. The acidic environment inhibits the efficacy of more alkaline chemotherapeutic drugs. The absence of normal vasculature also places a physical constraint on the microenvironment. Because lymphatic fluid and waste products are inefficiently transported from the tumor microenvironment, the interstitial pressure in the proximity of the tumor is higher than for typical areas. In the tumor, the low pH of the microenvironment and the high interstitial pressure produce a hostile environment, making drug delivery difficult. An additional contributing factor to this hostile environment is the heterogeneous nature of the tumor and its environment. Not all areas of the tumor are equally hypoxic or acidic, since blood flow within tumors is inconsistent due to abnormal vascularization. The extracellular matrix is altered from normal environ­mental conditions. Also for solid tumors, the 3-D cellular structure can physically limit drug delivery. Hypoxic conditions also appear to promote tumor survival and growth by creating genomic instability and selecting for more aggressive phenotype cells.2,16
Stromal and other non-malignant cells create a supportive microenvironment for tumor growth, angiogenesis, and metastasis.15 The tumor recruits endothelial cells, fibroblasts, and inflammatory cells, pericytes, and these cells and the components of the extracellular matrix (ECM) secreted by them contribute to the microenvironment composition. Stromal cells generate both tumor enhancing and tumor suppressing signals. In a mouse model of pancreatic cancer, inhibition of the Hedgehog signaling pathway reduced the level of tumor-associated stroma and improved the vascular delivery of gemcitabine.19 However, while several theories have been proposed regarding the role of stroma in carcinogen induced tumors, the actual relationships are not yet proven. In addition, stromal cells may be a target for carcinogens, to either induce new cancers, or metastatic growths.15
Cancer-associated fibroblasts (CAF) and myofibroblasts are stromal cells that are abnormal, but not malignant, and promote angiogenesis and proliferation. These fibroblasts secrete growth factors and cytokines that produce oncogenic signals. In a comparative study which isolated both CAF and normal fibroblasts from six patients with invasive breast cancer, Ormio, et al., demonstrated that CAF were able to promote tumor growth more than normal fibroblasts.21 These activated fibroblasts were able to promote angiogenesis via expression of stromal cell-derived factor-1 (SDF-1 or CXCL12). SDF-1 produces an endocrine effect by recruiting circulating endothelial progenitor cells (EPC) to the tumor. Tumor cells also contain CXCR4, the receptor for SDF-1, so stromal SDF-1 is able to directly stimulate tumor growth through a paracrine effect. TThe EPC recruited to the tumor by the cancer-associated fibroblasts are capable of being differentiated into tumor-associated vascular enodothelial cells and used to construct new capillaries.21 Circulating EPC migrate to other tissues and form a “premetastatic niche” for the colonization of circulating tumor cells.20
SDF-1 is not the only factor that participates in recruiting cells to the tumor microenvironment. Growth factors secreted by the tumor also control the composition of the microenvironment. Transforming growth factor-β (TGF-β) recruits EPC to the microenvironment and is involved in activation of fibroblasts to CAF, while platelet derived growth factor (PDGF) is involved in recruiting fibroblasts and inducing their proliferation. VEGF does not directly recruit fibroblasts, but indirectly supports microenvironmental changes via creation of dysfunctional vascularization that allows plasma leakage, which attracts fibroblasts and other cells.20 The microenvironment may convert recruited stromal cells to cancer-associated cells through epigenetic changes, including DNA methylation and chromatin remodeling, as changes in histone modification and DNA methylation have been found in tumor cells.
Proteins secreted by the tumor modify the microenvironment by contributing growth factors and proteases that degrade the extracellular matrix, and affect cell motility and adhesion. Stromal cells secrete ECM proteins, cytokines, growth factors, proteases, protease inhibitors, and endoglycosidases such as heparanase. These proteins modify the ECM in what is thought to be a systematic manner. Osteopontin, galectin-3, transforming growth factor-β and matrix metalloproteinases (MMP) are important secreted proteins closely associated with cancer development.22 MMP are expressed at higher levels by tumor-associated epithelial cells than by normal epithelial cells. These MMP revise the composition of the EMC by degrading the basement membrane and other ECM proteins. This increase in proteolytic activity may also act to support tumor malignancy. Extreme shifts in the inhibition of protease activity by plasminogen activation inhibitor-1 (PAI-1), either by its absence or by excessive levels, demonstrate an anti-angiogenic effect in mouse cells, while physiological levels of PAI-1 support angiogenesis.23 Tissue inhibitors of metalloproteinases (TIMP) are endogenous inhibitors of MMP and may function to balance the protease activity of MMP to shift the balance from a pro-angiogenic to an inhibitory environment.