Inserm U1242 - OSS - SPECIAL SEMINAR - Dr Isabel FABREGAT - Bellvitge Biomedical Research Institute - IDIBELL - BARCELONA, SPAIN

Targeting the TGF-beta pathway to improve immunotherapy in hepatocellular carcinoma ----------------------------------------------------------------------------------- ### Dr Isabel FABREGAT ### Bellvitge Biomedical Research Institute - IDIBELL - BARCELONA SPAIN **Introduction** Liver cancer incidence has risen in recent years, with high mortality and limited treatment response. Transforming Growth Factor-beta (TGF-β) is emerging as a promising therapeutic target, especially in combination with immune checkpoint inhibitors. However, TGF-β is a pleiotropic cytokine that can act as both a tumor suppressor and promoter, depending on the tumor context and microenvironment. Its pro-tumorigenic roles in fibroblast activation, inflammation, and immunosuppression remain poorly understood in liver cancer. **Objectives** This study aims to develop novel in vitro and in vivo models to better understand the role of the TGF-β pathway in liver tumor and stromal cells, and to evaluate whether TGF-β inhibitors could serve as an effective treatment for liver cancer. **Methods** We established a new liver cancer cell line (AL1099) from tumors induced by hydrodynamic tail vein injection of a transposon vector (MYC-LucOS; CTNNB1; SB13). A syngeneic orthotopic model was generated by injecting tumor cells into the liver parenchyma of C57BL/6J mice. We tested galunisertib (a TGF-β receptor inhibitor, 150 mg/kg orally), anti-PD-L1 (5 mg/kg), and their combination to assess their effects on tumor progression. **Results** AL1099 cells displayed an epithelial phenotype, responded to TGF-β with growth inhibition and apoptosis, and underwent epithelial-mesenchymal transition (EMT). To model resistance to TGF-β’s tumor-suppressive effects, AL1099 cells were chronically treated with TGF-β (2 ng/mL) for four weeks, generating a resistant variant (AL1099-R). These two lines mimic possible clinical scenarios regarding immunotherapy response. Upon orthotopic injection, AL1099-R tumors progressed more aggressively than AL1099 tumors. Treatment with galunisertib alone had no effect or worsened tumor progression in both models. Immunotherapy (anti-PD-L1 treatment) was effective in AL1099 tumors but less so in AL1099-R. Interestingly, combining galunisertib with anti-PD-L1 improved outcomes in both models, reducing tumor burden. Inhibition of cell cycle and induction of apoptosis, stromal remodelling and immune reactivation were found as central mechanisms of efficacy in driving tumor regression under TGF-β and PD-L1 blockade. **Conclusion** These models provide valuable tools for studying the dual roles of TGF-β in liver cancer. Our findings suggest that TGF-β inhibition alone may be ineffective or even detrimental. However, its combination with immunotherapy could offer therapeutic benefits in hepatocellular carcinoma (HCC), particularly in tumors where TGF-β acts as a tumor suppressor. **Funding** This research was supported by the Spanish Association for Cancer Research (AECC), grant #PRYGN211279FABR.