Analysis of collagen architecture in brain metastasis and paired primary tumors in Non-small cell lung cancer supports the recruitment of fibroblasts originating outside the brain

Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer (NSCLC) and frequently metastasizes to the brain. Lung brain metastases arise in a microenvironment that is structurally and mechanically distinct from the collagen-rich, highly desmoplastic stroma of primary LUAD tumors, which is driven by the chronic activation of cancer-associated fibroblasts (CAFs). In contrast, the normal brain lacks fibroblasts and is virtually devoid of fibrillar collagens. Although recent work suggests that disseminated tumor cells may co-travel with fibroblasts to recreate a primary tumor-like stroma at metastatic sites, the extent to which LUAD brain metastases recapitulate the fibrotic landscape of the primary tumor remains poorly defined. We retrospectively analyzed collagen architecture and common markers of immunosuppressive CAF phenotypes in LUAD patient samples, including primary tumors, paired brain metastases (n=17), and normal brain tissue from epilepsy patients (n=5). Collagen organization was assessed by picrosirius red staining, polarized light microscopy, and quantitative extraction of collagen fiber descriptors using CT-FIRE (Almici et al., Mod Pathol 2023). As expected, collagen fibers were virtually absent in normal brain. In contrast, LUAD brain metastases displayed prominent fibrillar collagen deposition. However, collagen architecture differed substantially from that of primary tumors, with brain metastases exhibiting fewer, shorter, and less aligned fibers. To explore whether EMT contributes to collagen production, LUAD cells and normal pulmonary fibroblasts were stimulated with TGF-beta1. Although TGF-beta1 increased vimentin and fibrillar collagens in cancer cells, their levels remained substantially lower than in activated fibroblasts. Consistently, brain metastases contained numerous elongated, fibroblast-like stromal cells expressing alpha-SMA but not markers of activated astrocytes and pericytes, supporting a fibroblast origin. Our findings demonstrate for the first time that LUAD brain metastases develop a fibrotic stroma characterized by de novo collagen deposition driven by fibroblast-lineage cells originating outside the brain, rather than by cancer cells undergoing EMT. These results indicate that collagen-rich fibrosis is a defining feature of the LUAD metastatic niche in the brain and may influence immune cell localization, tumor progression, and therapeutic response. Ongoing studies are examining CAF heterogeneity and functional differences between primary tumors and brain metastases.