Abstract
Introduction: PANoptosis is a newly identified form of programmed cell death that integrates elements of pyroptosis, apoptosis, and necroptosis. It plays a pivotal role in shaping the tumor immune microenvironment. Despite its significance, the specific functions and mechanisms of PANoptosis within the tumor microenvironment (TME) of hepatocellular carcinoma (HCC) remain unclear. This study aims to investigate these mechanisms using single-cell RNA sequencing data. Methods: Single-cell RNA sequencing data from HCC patients were obtained from the GEO database. The AUCell algorithm was used to quantify PANoptosis activity across various cell types in the TME. Cell populations with high PANoptosis scores were further analyzed using CytoTRACE and scMetabolism to assess their differentiation states and metabolic profiles. Associations between these high-score cell subsets and patient prognosis, tumor stage, and response to immunotherapy were examined. Cell-cell communication analysis was performed to explore how PANoptosis-related APO+ endothelial cells (ECs) may influence HCC progression. Immunofluorescence staining was used to assess the spatial distribution of APO+ ECs in tumor and adjacent tissues. Finally, a CCK8 assay was conducted to evaluate the effect of APOH+ HUVECs on HCC cell proliferation. Results: A total of 16 HCC patient samples with single-cell RNA sequencing data were included in the study. By calculating the PANoptosis scores of different cell types, we found that ECs, macrophages, hepatocytes, and fibroblasts exhibited higher PANoptosis scores. The PANoptosis scores, differentiation trajectories, intercellular communication, and metabolic characteristics of these four cell subpopulations with high PANoptosis scores were visualized. Among all subpopulations, APO+ ECs demonstrated the most significant clinical relevance, showing a positive correlation with better clinical staging, prognosis, and response to immunotherapy in HCC patients. Cellular communication analysis further revealed that APO+ ECs might regulate the expression of HLA molecules, thereby influencing T cell proliferation and differentiation, potentially contributing to improved prognosis in HCC patients. Immunofluorescence staining results indicated that APO+ ECs were primarily located in the adjacent tissues of HCC patients, with lower expression in tumor tissues. The results of cellular experiments showed that APOH+ HUVECs significantly inhibited the proliferation of HCC cells. Conclusions: This study systematically mapped the cellular landscape of the TME in HCC patients and explored the differences in differentiation trajectories, metabolic pathways, and other aspects of subpopulations with high PANoptosis scores. Additionally, the study elucidated the potential molecular mechanisms through which APO+ ECs inhibit HCC cell proliferation and improve prognosis and immunotherapeutic efficacy in HCC patients. This research provides new insights for clinical prognosis evaluation and immunotherapy strategies in HCC.