Adaptation of breast cancer cells to serum-free defined medium reveals changes in proliferation, adhesion, and lipid metabolism

Cancer cell metabolic dependencies are strongly influenced by nutrient availability, yet conventional culture conditions contain non-physiological nutrient concentrations and poorly defined serum components that may obscure metabolic vulnerabilities. Here, we investigated the adaptation of three breast cancer cell lines to physiologically relevant, serum-free defined medium (DM). Using a stepwise reduction in fetal bovine serum (FBS), we evaluated proliferation, adhesion, morphology, and transcriptional changes in HER2-positive SK-BR-3, triple-negative MDA-MB-231, and ER-positive/HER2-positive BT-474 cells. The three cell lines differed substantially in their ability to adapt to serum-free conditions. MDA-MB-231 cells adapted readily, maintaining proliferation and adhesion without exogenous substrate coatings. SK-BR-3 cells also achieved sustained proliferation in serum-free DM, but required a serum-derived coating to maintain adhesion. In contrast, BT-474 cells failed to sustain adhesion and proliferation following complete serum withdrawal, although they remained proliferative in medium containing 1% FBS. Adapted cells retained characteristic HER2 or vimentin expression despite changes in morphology and proliferation. RNA sequencing revealed extensive transcriptional remodeling, including reduced expression of cell-cycle-associated programs in SK-BR-3 cells and enrichment of cholesterol homeostasis gene sets in SK-BR-3 and MDA-MB-231 cells. Expression of cholesterol regulatory genes, including SREBF2, HMGCR, and LDLR, increased across all three lines. Together, these findings highlight cell-line-specific adhesion requirements and shared metabolic responses to serum withdrawal, establishing a framework for developing defined culture systems to investigate cancer metabolic dependencies through genetic screening.