Dissecting the Transcriptional Variability of Breast Cancer-Associated Fibroblasts Using Different Culture Conditions

Marianna Talia; Francesca Cirillo; Domenica Scordamaglia; Azzurra Zicarelli; Marika Di Dio; Adelina Assunta Mondino; Francesca Silvestri; Chiara Meliti; Ernestina Marianna De Francesco; Roberta Malaguarnera; Anna Maria Miglietta; Marcello Maggiolini; Rosamaria Lappano

Abstract

The tumor microenvironment plays a critical role in breast cancer (BC) progression, with cancer-associated fibroblasts (CAFs) representing a highly plastic stromal population. Although primary CAFs are widely used to study tumor biology, the extent to which experimental conditions impact on their molecular identity and phenotype remains to be elucidated. Here, we investigated how culture conditions may influence the properties of CAFs isolated from female and male BC tissues. CAFs maintained under diverse culture conditions displayed marked differences in morphology and proliferation, indicating that in vitro environments may influence the cellular behavior. Accordingly, transcriptomic analysis revealed that culture conditions may represent a major determinant of CAFs identity, shaping distinct gene expression programs. Specifically, depending on medium composition, CAFs activated pathways associated with lipid metabolism or programs related to extracellular matrix organization. Collectively, our findings suggest that culture conditions may shape CAFs functional states, underscoring the need for careful standardization to ensure robust interpretation and enhance the translational relevance of studies using tumor tissue-derived cells.

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