Hypoxia or low oxygen concentration in tumor microenvironment has widespread effects ranging from altered angiogenesis and lymphangiogenesis, tumor metabolism, growth, and therapeutic resistance in different cancer types. A large number of these effects are mediated by the transcription factor hypoxia inducible factor 1⍺ (HIF-1⍺) which is activated by hypoxia. HIF1⍺ induces glycolytic genes and reduces mitochondrial respiration rate in hypoxic tumoral regions through modulation of various cells in tumor microenvironment like cancer-associated fibroblasts. Immune evasion driven by HIF-1⍺ further contributes to enhanced survival of cancer cells. By altering drug target expression, metabolic regulation, and oxygen consumption, hypoxia leads to enhanced growth and survival of cancer cells. Tumor cells in hypoxic conditions thus attain aggressive phenotypes and become resistant to chemo- and radio- therapies resulting in higher mortality. While a number of new therapeutic strategies have succeeded in targeting hypoxia, a significant improvement of these needs a more detailed understanding of the various effects and molecular mechanisms regulated by hypoxia and its effects on modulation of the tumor vasculature. This review focuses on the chief hypoxia-driven molecular mechanisms and their impact on therapeutic resistance in tumors that drive an aggressive phenotype. Impact statement Hypoxia contributes to tumor aggressiveness and promotes growth of many solid tumors that are often resistant to conventional therapies. In order to achieve successful therapeutic strategies targeting different cancer types, it is necessary to understand the molecular mechanisms and signaling pathways that are induced by hypoxia. Aberrant tumor vasculature and alterations in cellular metabolism and drug resistance due to hypoxia further confound this problem. This review focuses on the implications of hypoxia in an inflammatory TME and its impact on the signaling and metabolic pathways regulating growth and progression of cancer, along with changes in lymphangiogenic and angiogenic mechanisms. Finally, the overarching role of hypoxia in mediating therapeutic resistance in cancers is discussed.

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Lymphangiogenesis or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro 3D collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of Sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF), interleukin 8 (IL-8), and hepatocyte growth factor (HGF) were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion while synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expression of the integrin α5 and integrin β1 subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA mediated knockdown of the integrin β1 subunit suppressed LEC invasion. Further, exposing LECs to the inflammatory mediator, LPS inhibited sprouting. This optimized model for LEC invasion, includes S1P, VEGF-C and fibronectin within a 3D collagen matrix, along with VEGF-C, VEGF-A, bFGF and HGF in the culture medium, providing a useful tool to investigate the effect of various lymphangiogenic factors and inhibitors.

Cholangiocarcinoma (CCA), or cancer of bile duct epithelial cells, is a very aggressive malignancy characterized by early lymphangiogenesis in the tumor microenvironment (TME) and lymph node (LN) metastasis which correlate with adverse patient outcome. However, the specific roles of lymphatic endothelial cells (LECs) that promote LN metastasis remains unexplored. Here we aimed to identify the dynamic molecular crosstalk between LECs and CCA cells that activate tumor-promoting pathways and enhances lymphangiogenic mechanisms. Our studies show that inflamed LECs produced high levels of chemokine CXCL5 that signals through its receptor CXCR2 on CCA cells. The CXCR2-CXCL5 signaling axis in turn activates EMT (epithelial-mesenchymal transition) inducing MMP (matrix metalloproteinase) genes such as GLI, PTCHD, and MMP2 in CCA cells that promote CCA migration and invasion. Further, rate of mitochondrial respiration and glycolysis of CCA cells was significantly upregulated by inflamed LECs and CXCL5 activation, indicating metabolic reprogramming. CXCL5 also induced lactate production, glucose uptake, and mitoROS. CXCL5 also induced LEC tube formation and increased metabolic gene expression in LECs. In vivo studies using CCA orthotopic models confirmed several of these mechanisms. Our data points to a key finding that LECs upregulate critical tumor-promoting pathways in CCA via CXCR2-CXCL5 axis, which further augments CCA metastasis.