The role of lipid-laden Kupffer cells in immunosuppression and immunotherapy response in MASLD-related hepatocellular carcinoma.

2568 Background: Hepatocellular carcinoma (HCC) related to metabolic dysfunction-associated steatotic liver disease (MASLD) is a rising global health burden. Despite systemic immunotherapy being the first-line treatment for advanced HCC, clinical observation shows that immune checkpoint inhibitors (ICI) offer lower benefit to patients with non-viral HCC, including MASLD-HCC. Macrophages, especially Kupffer cells (KCs), are the major PD-L1+ liver cells. With previous studies showing that more KCs are associated with poorer survival, we hypothesise that KCs in MASLD-related HCC show an immunosuppressive phenotype secondary to lipid accumulation, contributing to the poorer ICI responses in patients. Methods: As proof-of-concept, we characterised macrophage phenotypes and lipid accumulation in matched tumour and non-tumour tissues from HBV+ and non-viral HCC patients ( n = 6 / group). We next assessed the functional consequences of lipid loading using induced pluripotent stem cell (iPSC)-derived KCs in vitro, followed by validation in KC-containing iPSC- and patient-derived HCC organoids. We evaluated lipid accumulation, paracrine signalling, transcriptomic changes, and cell-cell interactions in these organoids. Results: In patient samples, macrophage lipid accumulation is associated with a PD-L1 high, TREM2 high, KC-like phenotype in both non-tumour and tumour (Cohen’s d > 1.0, power > 99%). In non-viral HCC samples, lipid-laden macrophages, with an M2-KC phenotype, are enriched twofold in non-tumour (Cohen’s d > 1.0, power = 72%) and tumour (Cohen’s d = 0.3, power = 6%). Light-sheet microscopy confirmed colocalization of lipids and PD-L1+ immune cells. Exposing iPSC-KCs to free fatty acids and IL-6 suppressed antigen presentation, while enhancing phagocytosis and T-cell exhaustion ( p < 0.05) – effects not reversed by ICI alone but alleviated when combined with tocilizumab or a CD36 inhibitor. These changes are likely independent of lipophagy or FASN-mediated fatty acid synthesis. Our HCC organoid models preserve KCs and recapitulate their immunosuppressive phenotype. scRNA-seq and CellPhoneDB analysis of HCC organoids highlighted significant KC-hepatocyte crosstalk, mediated by IL-6, SPP1, and other cytokines, corroborated by Luminex assay. Conclusions: These findings suggest that MASLD-associated lipid loading promotes a distinctly immunosuppressive KC phenotype, contributing to diminished ICI responsiveness in HCC. Targeting the lipid-KC axis, for instance with IL-6 blockade or CD36 inhibitors, may help restore immune competence and improve ICI-based treatment outcomes. Future studies, especially with a larger patient cohort and with our organoid platform, should determine whether lipid-laden KCs can serve as a biomarker for ICI responsiveness and further delineate the pathways driving their immunosuppressive behaviour.