Effect of Meflin overexpression on the fibroblast transcriptome in a three-dimensional culture using cell accumulation technique

We previously reported that Meflin, encoded by the immunoglobulin superfamily containing leucine-rich repeat (ISLR) gene, is a marker for tumor-restraining cancer-associated fibroblasts. However, the functional roles of Meflin in fibroblasts remain unclear. To investigate the functional impact of Meflin expression in fibroblasts, RNA sequencing was performed on Meflin-overexpressing NHDF-Ad cells cultured under conditions that better mimic the in vivo tumor microenvironment, using a cell accumulation technique that accurately recapitulates the fibrotic stroma observed in aggressive human cancers. Meflin overexpression resulted in the downregulation of PDPN, a well-established marker of tumor-promoting CAFs, and upregulation of DPT and COL15A1, which are markers of universal fibroblasts that resemble normal fibroblasts and are considered a potential source of diverse fibroblast subtypes.These findings suggest that high-level expression of Meflin is crucial for skewing CAFs toward a more universal fibroblast–like state. Overall design: RNA-seq profiling of Meflin-overexpressing NHDF-Ad cells and control NHDF-Ad cells cultured either alone or co-cultured with MDA-MB-468, a triple-negative breast cancer cell line, in a three-dimensional culture system using the cell accumulation technique.