The oncometabolite D2HG reprograms macrophages to drive immunosuppression in IDH1-mutant cholangiocarcinoma

Background: Intrahepatic cholangiocarcinoma (CCA) is an aggressive primary liver cancer, with ~20% of cases driven by mutations in isocitrate dehydrogenase 1 (mIDH1). Compared with IDH1 wild-type tumors, mIDH1 CCA exhibits an immunosuppressive microenvironment enriched for M2-like macrophages and may derive reduced benefit from immune checkpoint therapy than the general CCA population, suggesting tumor-immune interactions contribute to therapeutic resistance. mIDH1 tumors secrete the oncometabolite D2HG, but its effects on macrophages within the tumor microenvironment (TME) remain incompletely understood. Methods: We exposed THP-1 monocytes, a human myeloid leukemia cell line commonly used to model macrophage differentiation, and PBMC-derived macrophages to D2HG. Flow cytometry, RT-qPCR, and Luminex were used to assess expression of immunosuppressive markers (CD206, CD163, IL-10) and cytokine secretion (IL-10, IL-4, IL-13). Lipid uptake and accumulation was quantified by BODIPY staining, and CD36 expression was measured by flow cytometry and RT-qPCR. Seahorse metabolic assays and fatty acid oxidation analyses evaluated metabolic activity, while phagocytosis assays with live-cell imaging assessed macrophage function. Results: Exposure to pathologically relevant levels of D2HG that mirror those in the mIDH1 TME led THP-1 and PBMC-derived macrophages to upregulate immunosuppressive markers including CD206, CD163, and IL-10, while also increasing secretion of IL-10, IL-4, and IL-13. Functionally, D2HG-treated macrophages showed a 51% reduction in phagocytic capacity compared to untreated cells. Mechanistic studies revealed that D2HG enhanced lipid uptake and strongly upregulated the lipid transporter CD36. Despite increased lipid accumulation, Seahorse analyses and fatty acid oxidation assays showed no rise in energy production, indicating lipids were not metabolically utilized. BODIPY staining confirmed neutral lipid accumulation, supporting the development of a lipid-laden macrophage phenotype. Metabolic lipidomic analyses of mIDH1 tumors revealed higher lipid content than IDH1 wild-type tumors, suggesting that the mIDH1 CCA TME is enriched in lipids. This tumor-derived lipid abundance, together with D2HG-induced upregulation of lipid uptake in macrophages via CD36, likely promotes a cooperative process in which macrophages take up excess lipids, reinforcing their lipid-laden, immunosuppressive phenotype. Conclusion: The oncometabolite D2HG reprograms macrophages into a lipid-rich, metabolically altered, immunosuppressive state. This mechanism reveals how mutant IDH1 tumors shape the immune microenvironment and highlights CD36 and lipid metabolism as potential therapeutic targets to restore antitumor macrophage function in mIDH1 CCA.