Abstract
BACKGROUND: Cancer-associated fibroblasts (CAFs) are the predominant cell type in the stroma of many solid organ malignancies, including non-small cell lung cancer (NSCLC). They exhibit considerable phenotypic and functional heterogeneity and are widely used in functional assays and co-culture models. CAF research frequently involves the in vitro expansion and maintenance of CAFs to facilitate functional assays and co-culture studies. However, less is known about how in vitro culture temporally affects CAF phenotype. METHODS: We characterised the phenotype of CAFs from NSCLC patients compared to non-cancerous lung fibroblasts using conventional in vitro conditions by tracking changes in CAF subset marker expression levels by flow cytometry. Additional transcriptomic and functional analyses were performed to determine differences between CAFs and non-cancerous fibroblasts. RESULTS: We demonstrate that CAFs from NSCLC undergo phenotypic drift in culture, and that there is a convergence to a subset phenotype predominantly upregulated in non-cancerous lung. Additionally, we demonstrate the phenotype, transcriptome and function of fibroblasts converge between CAFs and fibroblasts from non-cancerous lung by the third culture passage, suggesting that in vitro conditions promote this phenotype. CONCLUSION: We highlight the need to understand and monitor the culture phenotype during functional studies with CAFs, as the heterogeneity found in the tumour microenvironment is rapidly lost in cultured cells.