Gastrointestinal cancers represent a heterogeneous group of diseases including gastric, pancreatic and colorectal cancer and hepatocellular carcinoma. In this complex scenario of different etiology and clinical management, oxygen limitation is one common condition that fosters cancer malignancy through a metabolic rewire, stimulating glycolysis and inhibiting mitochondrial activity. In particular, hypoxia is involved in fatty acids (FA) and lipid de novo synthesis and import, which in turn play an important role in cancer cell migration, invasion and angiogenesis regulation. Indeed, the hypoxic microenvironment is usually generated by scarce or aberrant vascularization. In this scenario, FA are reported to be capable on one hand of stimulating the expression of FA-binding proteins which are positive regulators of angiogenesis and on the other, drugs blocking FA oxidation decrease endothelial cell proliferation.

Nine different cancer cell lines (gastric, pancreas and colon cancer) were subjected to hypoxia (0.5% O2) for 24 or 72 hours as acute or chronic stimuli in a gas-tight manipulator and their mitochondrial mass and membrane potential assayed through MitoTracker and TMRM via flow cytometry, respectively. Their metabolic profile and the capacity to β-oxidate FA were evaluated via the Seahorse platform, while the hypoxic gradient was monitored continuously thanks to the Hypoxia Green dye. FA were measured in cells and conditioned media (CM) via Nile Red staining. The vessel formation capacity was assessed through 2D and 3D angiogenic models.

A chronic hypoxic stimulus was capable, with respect to an acute one or normoxic oxygen tension, to induce an increased FA storage, as intracellular neutral lipids. The secretion of FA produced by cancer cells in lipid-depleted media was measured and human endothelial cells were subjected to the collected CM in standard conditions. We observed that the CM could regulate vessel formation in an inversely proportional manner to the amount of FA in the media. We validated this negative tendency by using drugs blocking the FA oxidation (Etomoxir), the FA synthase (Denifanstat) or the FA uptake (Sulfosuccinimidyl oleate) and by administering increasing doses of palmitic acid.

Our findings suggest that chronic hypoxia induces a metabolic shift in gastrointestinal cancer cells, leading to increased FA storage and secretion, which in turn modulates endothelial cell behavior and angiogenesis. The inverse correlation between secreted FA levels and vessel formation highlights a potential mechanism for aberrant tumor vascularization. Interestingly, gastric cancer cells appear to follow a distinct metabolic pattern, warranting further investigation. Overall, these results provide new insights into the interplay between hypoxia, FA metabolism, and angiogenesis, offering potential therapeutic targets for modulating tumor vascularization.