Dysregulated Complement Pathway in Macrophage Reprogramming and Progression of HCC in MASH - ABSTRACT Hepatocellular carcinoma (HCC), a deadly form of liver cancer, is becoming increasingly common in patients with metabolic dysfunction-associated steatohepatitis (MASH), a rapidly growing cause of chronic liver disease. Macrophages are believed to play a key role in promoting HCC in MASH. Interestingly, the complement pathway, which regulates macrophage function, seems to worsen MASH instead of resolving it. This paradoxical behavior of the complement pathway might be a critical link between pro-tumor macrophage reprogramming in MASH and the development of HCC. Our prior research demonstrates that pro-tumor macrophage reprogramming fosters an immune-evasive microenvironment in HCC. Building on this, we conducted spatial analysis of human MASH-HCC, uncovering three critical findings: a unique immune-evasive spatial organization characterized by pro-tumor-macrophage neighborhoods and CD8T cell exhaustion, the activation of the complement pathway through the C3-C3AR1 axis, and the downregulation of SERPING1, a crucial complement pathway inhibitor, in MASH-HCC. Based on these findings, we hypothesize that the loss of SERPING1 leads to complement-mediated pro-tumor macrophage reprogramming in MASH, thereby promoting HCC. We are using three innovative methods to test this hypothesis. First, we will manipulate SERPING1 in patient-derived 3D tumor models composed of cancer cells and macrophages, using a microfluidics system. Second, we will target C3-mediated complement activation in immunocompetent mouse models of MASH-HCC which recapitulate major components of MASH, including metabolic syndrome, steatohepatitis, fibrosis, and HCC. Third, we will employ CODEX, a 53-plex single-cell spatial technology, to visualize and quantify complement pathway activation in specific tumor and macrophage compartments in human HCC. To test our central hypothesis, we have conceived three interconnected, yet independent, specific aims. In aim 1 we will investigate if SERPING1 is necessary for pro-tumor macrophage reprogramming in MASH-HCC. We will perform in vitro and in vivo studies using 3D tumoroids and orthotopic implantation of HCC cells with varying SERPING1 levels in diet-induced MASH models, and examine tumor progression and macrophage reprogramming. In aim 2 we will assess the efficacy of C3ar1 inhibition, alone or combined with anti-Pdl1, in a mouse model of MASH-HCC, using MRI and mass cytometry for tumor and immune response analysis. In aim 3 we will quantify complement pathway expression in HCC tissue and peripheral blood, using a retrospective-prospective study design to correlate complement levels with disease progression and clinical outcomes in patients with MASH and HCC. Overall, this research targets a pivotal gap in understanding immune-evasion of HCC arising in MASH, aiming to identify novel treatment strategies. Successful completion of this study will lead to novel use of therapies targeting the complement pathway in MASH-HCC, with the goal to improve long-term survival of patients with this deadly cancer.
