Construction and verification of matrix stiffness-related prognostic model of gastric cancer based on single-cell analysis and in vitro   experiments

Objective Although there is a significant correlation between tumour matrix sclerosis and the progression of gastric cancer, there is still a lack of a prognostic model based on Stiffness-related genes. For this reason, we have developed the matrix Stiffness-related risk score (MSRS) for prognostic prediction. In addition, this study integrates single-cell RNA sequencing technology to identify the cell source of characteristic genes and analyse the heterogeneity of cancer-related fibroblasts (CAF) lines. Methods Candidate stiffness-related genes were isolated by intersecting TCGA-STAD differentially expressed genes with four curated gene sets (e.g., Integrin, YAP). We constructed the MSRS via LASSO Cox regression in the GSE26253 training cohort (n = 432; endpoint: RFS) and validated it in the TCGA-STAD cohort (n = 386; endpoint: OS). Prognostic efficacy was gauged using Kaplan-Meier curves, ROC analyses, and nomograms, while ssGSEA assessed immune infiltration disparities. Furthermore, scRNA-seq data (GSE183904) was leveraged to dissect CAF heterogeneity, and core protein levels were ultimately corroborated by Western blotting. Results A 25-gene MSRS prognostic model was established. In the training cohort, high-risk patients exhibited significantly shorter recurrence-free survival (HR = 4.00, P  < 0.0001), with 3-, 5-, and 7-year AUCs of 0.770, 0.791, and 0.770, respectively. The validation cohort confirmed significantly reduced overall survival in the high-risk group ( P  = 0.0016; AUCs: 0.648, 0.772, 0.708). Multivariate analysis confirmed the risk score as an independent prognostic indicator (HR = 3.50, P  = 1.71×10⁻²¹). Three independent markers were identified: the oncogene MATN3 (HR = 1.559) and tumor suppressors CTSG (HR = 0.693)and MADCAM1 (HR = 0.813). Western blotting confirmed MATN3 upregulation and CTSG/MADCAM1 downregulation in tumor cells. High-risk patients showed increased monocyte infiltration but decreased activated B and NK cells. scRNA-seq revealed that stromal cells exhibited the highest stiffness scores. Specifically, inflammatory CAFs (iCAFs) displayed the highest score ( P  = 2.6×10⁻¹⁵) and were enriched in deep tumor regions.