Single-Nucleus and Spatial Transcriptome Profiling Delineates the Multicellular Ecosystem in Hepatocellular Carcinoma...

Hepatic arterial infusion chemotherapy (HAIC) has emerged as a promising treatment strategy for hepatocellular carcinoma (HCC), but a detailed understanding of the multicellular ecosystem after HAIC treatment is lacking. Here, we collected tumor samples from treatment-naïve primary and post-HAIC HCC, and integrated single-nucleus RNA sequencing with spatial transcriptomics to characterize the tumor ecosystem in the post-HAIC HCC. Increased fractions and enhanced cellular communication of CD4⁺ T, CD20⁺ B, and dendritic cell subtypes were identified in post-HAIC tumors. Moreover, it is substantiated that HAIC promoted tertiary lymphoid structures (TLS) formation, and addressed the roles of TLSs as spatial niches of cellular communication. Specifically, intermediate exhausted CD8⁺ T cells expressing Granzyme-K and PD-1 (PD-1⁺CD8⁺ Tex-int) expanded following HAIC and exhibited a functionally antitumor phenotype. PD-1⁺CD8⁺ Tex-int accumulated in the TLS vicinity and disseminated throughout the tumor microenvironment, demonstrating potential as an effective biomarker for HAIC-based treatment in HCC. This study provides valuable resources and biological insights in the cellular underpinnings of HAIC treatment.