Short-chain fatty acids suppress invasion and clonogenic survival in triple-negative breast cancer cells

Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers. Defined by the absence of estrogen receptors, progesterone receptors, and HER2 expression, this subtype is highly aggressive and has limited therapeutic options, resulting in a five-year survival rate of 78% compared to over 90% for other subtypes. Previous data from our laboratory demonstrated that short-chain fatty acids (SCFAs), microbiota derived metabolites, reduce TNBC cell viability, proliferation, and migration, with butyrate showing the strongest inhibitory effects. However, the impact of SCFAs on invasion and long-term survival remains unclear. Objective: This study aimed to investigate the effects of SCFAs on invasion and clonogenic potential in a TNBC cell line (MDA-MB-231) as indicators of metastatic behavior and sustained proliferative capacity. Methods: MDA-MB-231 cells were treated with SCFAs butyrate, acetate, and propionate at 5 mM and invasion was assessed using Boyden chambers coated with Matrigel. Cells were seeded in the upper chamber in serum-free medium, while the lower chamber contained complete medium as a chemoattractant. After 24 hours, cells that passed through the Matrigel layer were fixed, stained, and quantified using ImageJ. To evaluate long-term proliferative potential, clonogenic assays were performed with butyrate-treated cells, chosen for its strong inhibitory effects on TNBC growth and migration. MDA-MB-231 cells were seeded at a density of 1,000 cells per well of a six-well plate and treated with butyrate at 1 mM and 5 mM for 9-10 days. Colonies were fixed, stained, photographed, and quantified using ImageJ. Results: Butyrate and propionate treatment significantly suppressed invasion compared to control (p<0.0001 and p=0.0031, respectively), while acetate had no significant effect in invasion (p=0.4084). Clonogenic assays showed complete inhibition of colony formation in cells treated with 1 mM and 5 mM butyrate, while control cells formed a mean of 429 colonies per well. Morphologically, treated cells displayed loss of adherence and rounded shapes consistent with cytotoxicity. Conclusion: Butyrate and propionate significantly inhibited invasion of MDA-MB-231 cells, while butyrate also completely suppressed clonogenic survival. These findings align with our previous transcriptomic data indicating SCFA modulation of pathways involved in oncogenic processes such as migration and cell adhesion. SCFAs demonstrate potent inhibitory effects on metastatic behavior and long-term proliferation, supporting further investigation into their therapeutic potential.