Wangxia Tong ¹ Tao Lu ² Han Lin ¹ Bolun Chen ³ Chen Jibing ² Ning Luo ^2,3*

• ¹ Department of Hepatology, Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning, Guangxi Zhuang Region, China
• ² Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning, China
• ³ Department of Neurology, Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning, Guangxi Zhuang Region, China

Background Hepatocellular carcinoma (HCC) patients exhibiting portal vein tumor thrombosis (PVTT) face a high risk of rapid malignant progression and poor outcomes, with this issue being compounded by a lack of effective treatment options. The integration of bulk RNA-sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) datasets focused on samples from HCC patients with PVTT has the potential to yield unprecedented insight into the dynamic changes in the tumor microenvironment (TME) and associated immunological characteristics in these patients, providing an invaluable tool for the reliable prediction of disease progression and treatment responses.Methods scRNA-seq data from both primary tumor (PT) and PVTT cells were downloaded from the Gene Expression Omnibus (GEO) database, while the International Cancer Genome Consortium (ICGC) and Cancer Genome Atlas (TCGA) databases were used to access bulk RNA-seq datasets.scRNA-seq, clustering, GSVA enrichment, mutational profiling, and predictive immunotherapeutic treatment analyses were conducted using these data with the goal of systematically assessing the heterogeneity of PT and PVTT cells and establishing a model capable of predicting immunotherapeutic and prognostic outcomes in patients with HCC. Results These analyses revealed that PVTT cells exhibited patterns of tumor proliferation, stromal activation, and low levels of immune cell infiltration, presenting with immune desert and immune rejection-like phenotypes.PT cells, in contrast, were found to exhibit a pattern of immunoinflammatory activity. Core PVTTassociated genes were clustered into three patterns consistent with the tumor immune rejection and immune desert phenotypes. An established clustering model was capable of predicting tumor inflammatory stage, subtype, TME stromal activity, and patient outcomes. PVTT signature genes were further used to establish a risk model, with the risk scores derived from this model providing a tool to evaluate patient clinicopathological features including clinical stage, tumor differentiation, histological subtype, microsatellite instability status, and tumor mutational burden. These risk scores were also able to serve as an independent predictor of patient survival outcomes, responses to adjuvant chemotherapy, and responses to immunotherapy. These results offer new insight into the biological and immunological landscape of PVTT in HCC patients, By utilizing individual patient risk scores, providing an opportunity to guide more effective immunotherapeutic interventional efforts.