PFHxA and PFHxS promote breast cancer progression in 3D culture: MEX3C-associated immune infiltration revealed by bioinformatics and machine learning

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with widespread use and bioaccumulative potential. Short-chain PFAS such as perfluorohexanoic acid (PFHxA) and perfluorohexane sulfonate (PFHxS) have been introduced as safer alternatives to long-chain PFAS, yet their toxicological impacts remain poorly defined. In this study, we employed a 3D Gelatin methacryloyl (GelMA) hydrogel model to mimic the tumor microenvironment and investigated the effects of PFHxA and PFHxS on triple-negative breast cancer (TNBC) progression. At environmentally relevant concentrations (0.1–10 μM), both compounds significantly enhanced proliferation, migration, and invasion of MDA-MB-231 cells. Transcriptomic and machine learning analyses identified MEX3C as a key gene upregulated by PFAS exposure. Gene set enrichment analysis (GSEA) revealed activation of the PI3K-AKT-mTOR signaling pathway, which was further supported by siRNA-mediated knockdown of MEX3C, leading to a marked reduction in the expression levels of phosphorylated PI3K, AKT, and mTOR proteins. Furthermore, immune cell co-culture experiments showed that MDA-MB-231 cells with high MEX3C expression promoted M2 macrophage polarization, suppressed M1 polarization, and enhanced macrophage chemotactic activity, the immunomodulatory effects were significantly attenuated upon MEX3C knockdown. These findings establish MEX3C as a central mediator of PFAS-induced tumor progression and immune remodeling. This study provides mechanistic insight into the carcinogenic potential of emerging short-chain PFAS and underscores the need for stricter regulation to safeguard public health.