Abstract 3852: The role of endothelial cell remodeling in driving immunotherapy resistance of hepatocellular carcinoma

Introduction: Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related deaths globally. Immune checkpoint blockade (ICB) therapy has shown promise but has yielded limited responses in HCC patients. Combining anti-vascular endothelial growth factor (VEGF) with ICB enhances therapeutic efficacy, however, a deeper understanding of endothelial cell (EC) remodeling is crucial for further improving ICB effectiveness. Our integrated analyses identified a transcription factor-4 (TCF4)-driven transcriptional program, coupled with elevated BRD4 expression, associated with EC remodeling in ICB-resistant HCC. Emerging studies have shown that TCF4 functions depend on BRD4 through recruitment to a specific enhancer region, and BRD4 inhibitors can reduce TCF4 expression. We aim to delineate the microenvironmental cue and transcriptional network that direct EC remodeling for tumor immunosuppression and their role in promoting ICB resistance. Methods: Single-cell transcriptomic profiling was performed on samples from a Phase II trial of pembrolizumab (anti-PD-1) in hepatitis B-related HCC patients (NCT03419481). An ICB-resistant HCC mouse model generated via iterative selection was used for validation. The BRD4 inhibitor AZD5153, administered in oral and nanoparticle formulations, was used to suppress BRD4 and TCF4 expression in systemic and endothelial cells. Quantitative immunofluorescence assessed ECs and detected TCF4 and BRD4 levels. Results: scRNA-seq analysis revealed that tumor vessels from ICB non-responders were enriched with disorganized macrovascular-like endothelial cell (MaVEC), which further increased upon ICB. RNA velocity analysis predicted that MaVEC in non-responders likely originate from liver sinusoidal endothelial cell (LEC), indicating LEC-to-MaVEC trans-differentiation during ICB resistance acquisition. TCF4 was identified as the top regulon in MaVEC, with increased activity along the LEC-to-MaVEC trajectory and association with BRD4. Using AZD5153, we observed that TCF4 can be suppressed in ECs, which caused a significant intra-tumoral reversal from MaVEC to LEC phenotypes, resulting in tumor cell apoptosis and regression. Additionally, nanoparticle-delivered EC-targeted AZD5153 achieved similar tumor-suppressive effects at a lower dosage. Conclusions: Our study reveals that the dynamic trans-differentiation from LEC to MaVEC is associated with ICB resistance in HCC, which can be reversed by BRD4 inhibition and possibly mediated by TCF4 transcriptional regulation, targeting their expression in ECs showed improved therapeutic outcomes. Keywords: Hepatocellular carcinoma, Immune-checkpoint blockade, Endothelial cells, Transcriptional network. Acknowledgement: Funded by General Research Fund (14118424) & AstraZeneca Pre-clinical Oncology Research Programme & Li Ka Shing Foundation. Citation Format: Baoyi Yin, Shufen Chen, Zhewen Xiong, Patrick Wong, Zhuo Yu, Stephan Chan, Alfred Sze-Lok Cheng. The role of endothelial cell remodeling in driving immunotherapy resistance of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3852.