Integrative analysis unveils ECM signatures and pathways driving hepatocellular carcinoma progression: A multi-omics approach and prognostic model development

Abstract

Liver hepatocellular carcinoma (LIHC) is a highly lethal form of cancer that is among the deadliest cancer types globally. In terms of cancer-related mortality rates, liver cancer ranks among the top three, underscoring the severity of this disease. Insufficient analysis has been conducted to fully understand the potential value of the extracellular matrix (ECM) in immune infiltration and the prognostic stratification of LIHC, despite its recognised importance in the development of this disease. The scRNA-seq data of GSE149614 was used to conduct single-cell analysis on 10 LIHC samples. CellChat scores were calculated for seven cell populations in the descending cohort to investigate cellular communication, while PROGENy scores were calculated to determine tumour-associated pathway scores in different cell populations. The pathway analysis using GO and KEGG revealed the enrichment of ECM-associated genes in the pathway, highlighting the potential role of the ECM in LIHC development. By utilizing the TCGA-LIHC cohort, an ECM-based prognostic model for LIHC was developed using Lasso regression. Immune infiltration scores were calculated using two methods, and the performance of the ECM-related risk score was evaluated using an independent cohort from the CheckMate study. To determine the precise expression of ECM-associated risk genes in LIHC, we evaluated hepatocellular carcinoma cell lines using a range of assays, including Western blotting, invasion assays and Transwell assays. Using single-cell transcriptome analysis, we annotated the spatially-specific distribution of major immune cell types in single-cell samples of LIHC. The main cell types identified and annotated included hepatocytes, T cells, myeloid cells, epithelial cells, fibroblasts, endothelial cells and B cells. The utilisation of cellchat and PROGENy analyses enabled the investigation and unveiling of signalling interactions, protein functionalities and the prominent influential pathways facilitated by the primary immune cell types within the LIHC. Numerous tumour pathways, including PI2K, EGFR and TGFb, demonstrated a close correlation with the involvement of ECM in LIHC. Moreover, an evaluation was conducted to assess the primary ECM-related functional changes and biological pathway enrichment in LIHC. Differential genes associated with ECM were identified and utilised to create prognostic models. The prognostic stratification value of these models for LIHC patients was confirmed through validation in multiple databases. Furthermore, through immune infiltration analysis, it was discovered that ECM might be linked to the irregular expression and regulation of numerous immune cells. Additionally, histone acetylation was mapped against gene mutation frequencies and differential expression profiles. The prognostic stratification efficacy of the ECM prediction model constructed in the context of PD-1 inhibitor therapy was also examined, and it exhibited strong stratification performance. Cellular experiments, including Western blotting, invasion and Transwell assays, revealed that ECM-associated risk genes have a promoting effect on the development of LIHC. The creation of biomarkers for LIHC using ECM-related genes unveiled substantial correlations with immune microenvironmental infiltration and functional mutations in various tumour pathways. This enlightens us to the possibility that the influence of ECM on tumours may extend beyond simply promoting the fibrotic process and the stromal composition of tumours.

Keywords: drug selection; extracellular matrix; immune infiltration; immunotherapy; liver hepatocellular carcinoma; single‐cell sequencing.