Developing an in vitro to in vivo pipeline of mammary gland exposure-response relationships to per- and poly-fluoroalkyl substances (PFAS)

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Project Abstract Per- and polyfluoroalkyl substances (PFAS) are a family of over 5000 man-made chemicals that are ubiquitous in the environment, due to their chemical stability and bioaccumulative properties. Many of these “forever chemicals” have been linked with health concerns, including strong evidence of developmental health and harm to hormone-sensitive tissues. Manufacturers continue to substitute new PFAS for which exposure- based health risks are unknown. There is an urgent public health need to determine the effects of PFAS in use on both mammary gland development and increased breast cancer incidence. Current exposure studies use rodent models that require cumbersome end-point analyses as well as large monetary and time investments. Our proposal is aimed at developing an in vitro to in vivo extrapolation (IVIVE) pipeline of mammary gland development and maintenance to identify and prioritize potentially toxic PFAS, to ultimately mitigate number of animals needed for environmental exposure studies. Our approach is to develop in vitro models of the mammary gland of increasing complexity but decreasing throughput, identifying links between high-throughput and high- complexity model endpoint readouts to best prioritize large chemical libraries. A key technology to establish links across multiple in vitro culture platforms is optical coherence tomography-based structural-functional imaging (OCT-SFI), developed by MPI Oldenburg, which non-invasively visualizes label-free cells, their intracellular motility, and morphology of formed spheroids, within optically turbid tissue models. Our first specific aim advances a high-throughput paper-based culture system, developed by MPI Lockett, to study mammary epithelial cell invasion in physiologically relevant tissue microenvironments. The platform will evaluate 96 different exposure conditions in parallel. Our second specific aim employs 3D co-culture models that include fibroblasts to model stromal signaling known to affect mammary gland development. OCT-SFI will provide cellular motility and morphology of the organotypic spheroids that form in these cultures. Finally, our third aim will screen a library of 40 PFAS, with a particular focus on the perfluoroethercarboxylic acids (PFECAs) currently used in industrial coatings. In addition, 12 PFAS will be screened for which there is existing in vivo rodent model data available, and comparisons between in vitro assay outputs and in vivo gland remodeling will be used to refine the assay models and establish initial thresholds for screening. The models developed as part of this proposal will thus be predictive of biology, enabling the high-throughput capability needed for future screening of all PFAS as well as other emerging endocrine disruptors. The project’s risk is balanced by the known imaging capabilities of OCT-SFI to probe responses in 3D spheroid and paper- based co-cultures. The high-throughput nature of this IVIVE pipeline makes it ideal for screening libraries of potential toxicants, providing information-rich datasets of spatially and temporally resolved morphological and molecular changes across the tissue-like structures.