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

Background : Hepatocellular carcinoma (HCC) has shown limited durable responses to immune checkpoint blockade (ICB) therapies due to tumor microenvironment (TME)heterogeneity. Combining anti-vascular endothelial growth factor (VEGF) with ICB has enhanced therapeutic efficacy, but the limited benefits suggest that understanding endothelial cell (EC) remodeling beyond angiogenesis is essential for enhancing ICB effectiveness. Integrated analyses identified a novel transcription factor(TF) transcriptional program, coupled with elevated bromodomain and extra terminal domain (BET) protein BRD4 expression, that was associated with EC remodeling in ICB-resistant HCC. Emerging studies have shown that this TF functions depend on BRD4 through its recruitment to a specific enhancer region, and inhibiting BRD4 with BET inhibitors can reduce its expression. Hence, we aim to delineate the microenvironmental cue 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 clinical trial of pembrolizumab in HCC patients (NCT03419481). An ICB-resistant HCC mouse model generated via iterative selection was utilized for validation. BET inhibitor AZD5153, administered in nanoparticle formulations, was used to suppress BRD4 and TF expression selectively in ECs. Quantitative immunofluorescence and flow cytometry was employed for EC assessment and intratumoral immune microenvironment. Results: scRNA-seq analysis revealed that tumor vessels in ICB non-responders were enriched with disorganized macrovascular-like endothelial cells (MaVEC), which likely originate from liver sinusoidal endothelial cells (LEC). Suggesting a dynamic trans-differentiation process from LEC to MaVEC during the development of ICB resistance. Notably, we identified a novel TF as the top regulon in MaVEC, exhibiting increased transcription factor activity along the LEC-to-MaVEC trajectory, and closely associated with BRD4 in MaVEC. Treatment with the EC-targeted nanoparticle-delivered AZD5153 can suppress this TF expression, caused remarkable intratumoral MaVEC-to-LEC reversion and reshaped the intratumoral immune microenvironment, leading to an augmented anti-PD-L1 efficacy. Conclusions : Our study reveals that the dynamic trans-differentiation from LEC to MaVEC is related with ICB resistance in HCC. Blocking this EC transformation shifts the TME from immunosuppressive to stimulatory, re-sensitizing tumors to anti-PD-L1 therapy. This project may yield new strategies to counter adaptive immune resistance in HCC. Keywords: Hepatocellular carcinoma, Immune-checkpoint blockade, Endothelial cells remodeling. Acknowledgement: The work is funded by General Research Fund (14118424) & AstraZeneca Pre-clinical Oncology Research Programme & Li Ka Shing Foundation.