Single-cell multi-omics reveal epigenomic reprogramming and multicellular ecosystem shaping venous tumor thrombus in hepatocellular carcinoma

ABSTRACT Portal vein tumor thrombus (PVTT) in hepatocellular carcinoma (HCC) is a major contributor to metastasis and poor prognosis, yet its underlying molecular mechanisms remain poorly understood. Here, we comprehensively delineated the epigenomic reprogramming and dynamic cellular crosstalks within the PVTT microenvironment by integrating single-cell transcriptomic, chromatin accessibility, and spatial transcriptomic analyses. Our analysis revealed non-coding cis -regulatory enhancers (CREs) as orchestrators of transcriptional dysregulation in both cancer and stromal cells, driving metastatic phenotypes. FAP-positive fibroblasts (FAP + Fib) and GJA5-expressing arterial endothelial cells (GJA5 + EC) as central stromal hubs within PVTT. These components collaborate with hypoxia-induced epithelial-mesenchymal transition (hypoxia-EMT) cancer cells to establish a spatially co-localized niche fostering pro-arterialization and pro-EMT programs. Furthermore, in vitro co-culture experiments validated that the SLIT2/ROBO1 axis mediates cytoskeletal remodeling of HCC cells and facilitates metastatic progression, highlighting its potential as a therapeutic target in HCC patients with PVTT. In summary, our study highlights the importance of non-coding regulatory mechanisms in metastatic progression and underscores the crucial role of stromal cells in remodeling of the tumor microenvironment (TME) in PVTT, offering new insights into precision medicine strategies against this aggressive complication.