Fibroblast-associated migrasome programs define TSPAN4-driven progression and clinical risk stratification in gastric cancer

Gastric cancer (GC) is a highly aggressive malignancy with poor prognosis, characterized by complex tumor microenvironment (TME) interactions. Migrasomes have been implicated in cancer progression, but their specific roles and spatial correlation in GC remain unclear. Using 10x Genomics scRNA-seq (14 GC specimens), CT radiomics (46 patients), bulk RNA-seq (385 TCGA training and 357 GSE84433 validation samples), and spatial transcriptomics, we performed integrative analyses. Machine learning (101 algorithm combinations) was applied to construct prognostic models, while shRNA knockdown/overexpression, xenograft models, and molecular docking validated candidate genes. Single-cell analysis identified nine transcriptionally distinct clusters that were annotated into eight major cellular compartments. Fibroblasts exhibited the highest migrasome scores and the highest Augur AUC (0.805), and 11 fibroblast-specific migrasome-associated genes (FSMAGs) were identified. Pseudotime analysis revealed five distinct fibroblast differentiation states, with CXCL12 and TSPAN4 expression increasing along the differentiation trajectory, whereas TSPAN9 remained relatively stable across states. CellChat analysis predicted stronger communication between high-migrasome-score fibroblasts and macrophages/dendritic cells, with the MIF-CD74/CXCR4 axis emerging as a candidate signaling route for fibroblast-dendritic-cell communication. The Radiomics-Derived Migrasome-associated Gene Signature (Rad_MGsig), integrating five genes and nine radiomic features, showed high prognostic accuracy, with 1-/3-/5-year AUCs of 0.96/0.90/0.88 in the training cohort and 0.97/0.90/0.90 in the validation cohort. Uprosertib inhibited GC cell growth in a dose-dependent manner, with IC50 values of 0.368 µM in HGC-27 cells and 0.460 µM in AGS cells, and attenuated TSPAN4-overexpression-induced proliferation in AGS cells. Molecular docking predicted the strongest binding affinity between Uprosertib and TSPAN4 (-9.0 kcal/mol). Multiplex immunofluorescence showed that TSPAN4-positive signals were observed in close proximity to Vimentin-positive fibroblast regions at the tumor-stroma interface. The fibroblast-associated migrasome signature is closely associated with GC progression and prognosis, and Rad_MGsig serves as a robust prognostic signature. Tumor cell-intrinsic TSPAN4 exerts pro-proliferative effects via activating the PI3K/AKT pathway, representing a potential druggable target for gastric cancer. These findings offer insights for biomarker-guided precision therapy in GC.