Reprogramming Liver Macrophages for Sex-based Hepatocellular Carcinoma Immunotherapy - PROJECT SUMMARY Hepatocellular carcinoma (HCC) is the third leading cause of global cancer mortality, with a higher prevalence in males. Although immunotherapy has lately broadened the landscape of liver cancer treatment, therapeutic resistance and a lack of accurate immune biomarkers remain a significant challenge due to HCC heterogeneity and sexual dimorphism. Owing to the current obesity endemic, metabolic dysfunction-associated steatohepatitis (MASH) has become the fastest-growing cause of HCC. Recently, we identified sphingosine kinase 2 (SphK2), which generates the bioactive sphingolipid sphingosine-1-phosphate (S1P), as a critical determinant of sexual dimorphism in MASH-driven HCC; SphK2 is tumor-protective in females and tumor-inducing in males. In addition, preliminary data showed that SphK2 deletion in male livers reduced the immuno-suppressive M2-like polarization of macrophages and promoted an antitumor phenotype. This led to the hypothesis that liver macrophage SphK2 mediates a pro-tumorigenic microenvironment in HCC. In Aim 1 (F99 phase), the sexually dimorphic role of SphK2 in liver macrophages will be examined in macrophage-specific SphK2 deleted male and female mice using our chronic Western diet model that recapitulates the progression and molecular hallmarks of MASH-HCC in humans. In-depth analyses will determine how SphK2 affects macrophage functions in response to Western diet, macrophage-hepatocyte cross-talk, and immune cell flux in the tumor microenvironment. Furthermore, the proposal will investigate the mechanism for SphK2-mediated regulation of liver macrophages to understand how targeting SphK2 could repolarize macrophages and enhance HCC anti-tumorigenicity. Aim 2 (K00 phase): Owing to the emerging role of immunometabolism as a central mechanism in immune-oncology, the K00 phase will focus on studying macrophage metabolic networks in the HCC tumor microenvironment to uncover novel immunotherapy targets. The study will consider different HCC models in males and females to emphasize on HCC tumor heterogeneity and sexual dimorphism. The F99 doctoral training will involve the study of diet-induced HCC model, primary cell isolation and culture, macrophage characterization, sphingolipid signaling, and epigenetic regulation. Building upon the F99 proposal, my post-doctoral training will involve the study of various HCC animal models, macrophage metabolomics, cell-signaling networks, and immune cell cross-talks to modulate the HCC tumor microenvironment. Collectively, the integrated research plan and training of my F99/K00 proposal will establish my career as an independent researcher and provide an opportunity to extend the boundaries of therapeutic resistance and sex-based cancer immunotherapy.