Brain metastasis‐associated fibroblasts shape the tumour microenvironment to enhance cancer cell invasion

Abstract

Abstract Brain metastases (BrM) are a frequent and life‐threatening complication of solid tumours, with lung cancer representing their most common source. While cancer‐associated fibroblasts (CAFs) are well‐established contributors to tumour progression in many extracranial malignancies, their presence and function within the brain tumour microenvironment, where fibroblasts have long been considered scarce or absent, remain poorly understood. Here, we isolated and characterised fibroblast‐like cells from 13 human BrMs of diverse origins. These BrM‐associated CAFs (BrM‐CAFs) expressed canonical CAF markers and showed reduced proliferation and increased senescence compared to normal fibroblasts. Their transcriptome was enriched for extracellular matrix (ECM)‐related genes, including multiple collagens, fibronectin, and matrix‐remodelling enzymes. In vitro, BrM‐CAFs produced a fibrillar ECM, and in BrM tissues, their abundance was associated with collagen I and fibronectin deposition. Transcriptomic, proteomic, and secretome analyses further revealed that BrM‐CAFs produce multiple cytokines, chemokines, and growth factors that promote cell motility. BrM‐CAF conditioned medium promoted both monocyte migration and the migration of cancer cells, including established cell lines and patient‐derived lung cancer BrM cells; monocyte migration was partially reduced by inhibition of CCL2/CCR2 signalling, whereas blocking CXCL12, CXCL16, or CX3CL1 attenuated BrM‐CAF‐induced cancer cell migration. Beyond these effects on migration, exposure to BrM‐CAFs increased cancer cell invasion in transwell and heterotypic 3D spheroid assays. In contrast, their effects on cancer cell proliferation were limited and did not indicate a growth‐promoting role. Exposure to BrM‐CAFs was also associated with increased expression of interferon‐stimulated genes in cancer cells. Together, our findings support a role for BrM‐CAFs in shaping the brain metastatic microenvironment through ECM remodelling and the secretion of pro‐migratory mediators, promoting monocyte and cancer cell migration and enhancing cancer cell invasion. These data identify BrM‐CAFs as active stromal participants in BrM biology and support further investigation of their biological, diagnostic, and therapeutic relevance.