Patient derived cancer-associated fibroblasts from non-small cell lung cancer undergo phenotypic drift in culture

Nanostring nCounter targeted RNA Seq. data of fibroblast cells isolated from human non-cancerous lung tissue and non-small cell lung cancer tumour tissue as well as fibroblasts maintained for three passages in culture from both origins as shown in Mathieson et al. (submission) "Patient derived cancer-associated fibroblasts from non-small cell lung cancer undergo phenotypic drift in culture". 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 are widely studied and represent a diverse population of cells presenting with different phenotypes and functions. CAF research often relies on propagation and culture in vitro for functional assay and co-culture experimentation. In this study we investigated the phenotype of CAFs from NSCLC patients compared to non-cancerous lung fibroblasts by tracking changes in CAF subset marker expression levels by flow cytometry. We demonstrate that CAFs from NSCLC undergo phenotypic drift in culture, and that of the previously defined subsets there is a convergence to a phenotype predominantly upregulated in non-cancerous lung when CAFs are maintained in standard culture conditions. Together we show the phenotype, transcriptome and function of fibroblasts converge between CAFs and fibroblasts from non-cancerous lung, suggesting that standard culture conditions promote this phenotype. 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.