An analytical screening platform to differentiate acute and prolonged exposures of per- and polyfluoroalkyl substances on invasive cellular phenotypes

Per- and polyfluoroalkyl substances (PFAS) are "forever chemicals" and pervasive environmental contaminants associated with cancer. Epidemiological studies found that an increased incidence of hormone-sensitive breast cancer is correlated with PFAS exposure. Cell-based assays provide a well-controlled experimental platform to quantify cellular responses as a function of exposure. Given the nearly 15,000 known PFAS on the Environmental Protection Agency's toxicity database (DSSTox), in vitro models are the only feasible approach to screen this large molecular library. One of the hallmarks of cancer is increased migration and invasion, processes that are the gateway to metastasis. Using a paper-based invasion assay developed in our lab, we compared the invasion of the MCF7 and M231 cell lines after acute and prolonged exposures to 2 legacy PFAS compounds, individually and in an equimolar mixture: perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS). The acute exposures quantified cellular movement after a 24-h period in the presence of the molecule of interest. The prolonged exposures in this work exposed 5 consecutive cell passages to the PFAS. We hypothesized that prolonged PFAS exposures would select for invasive subpopulations. These prolonged exposures increased the invasion of MCF7 and M231 cells compared to acute exposures of the same PFAS concentration (10 µM). The prolonged exposures to PFOA and PFOS at environmentally relevant concentrations (10 nM) did not increase invasion. Our results highlight the need to assess different exposure durations in vitro and that the paper-based invasion assay is a reasonable screening tool.