(582e) Characterizing the Irradiated Microenvironment with Normal Tissue Mammary Organoids

Authors: 
Hacker, B. C., Vanderbilt University
Gomez, J. D., Vanderbilt University
Silvera Batista, C., Vanderbilt University
Rafat, M., Vanderbilt University
Despite receiving chemotherapy, surgical intervention, and ionizing radiation treatment, a significant percentage of triple negative breast cancer (TNBC) patients experience cancer recurrence at the primary tumor site. The role of the normal tissue microenvironment in recurrence, specifically interactions between immune, stromal, and tumor cells, is not well-understood. Pre-clinical studies have shown that tumor cell recruitment to the irradiated microenvironment is preceded by infiltrating macrophages, and this effect is enhanced in the absence of CD8+ T cells. Essential in vitro models to evaluate how radiation of normal cells influences tumor and immune cell infiltration, however, have not been established. In this study, we developed and characterized a robust mammary organoid model to analyze the effects of radiation on cell behavior.

To obtain irradiated epithelial mammary organoids, murine mammary glands of 8-12 week old female Nu/Nu mice were resected, irradiated ex vivo to 20 Gy, and enzymatically digested in a collagenase solution. Growth comparisons were done in low adhesion plates and Matrigel, and they were cultured for 7 to 14 days. Expression of epithelial and cytoskeletal markers on irradiated and control organoids were evaluated using immunofluorescence staining. Organoids were co-cultured with dTomato expressing RAW 264.7 macrophages, and cellular dynamics were monitored up to 48 hours with live cell fluorescence and phase contrast imaging.

Organoids recapitulated expression and localization of relevant epithelial markers, including E-cadherin, tight junction protein 1, and cytokeratin 14. An increase in F-actin expression was observed in irradiated organoids, which suggests that radiation induced reorganization of the cytoskeleton. Finally, macrophage migration toward irradiated organoids was significantly enhanced (p < 0.001), recapitulating pre-clinical observations.

This work describes a model that can be used to generate organ-like structures to study cell recruitment following cancer therapy. The importance of recreating tissue architecture is highlighted via observations of cytoskeletal reorganization. Future studies will include the addition of pertinent growth factors, microfluidic channels to study extravasation, and further co-culture with tumor cells, fibroblasts, and adipocytes. This model will provide insights into the mechanisms behind radiation-induced tumor cell recruitment, which will have significant implications for TNBC patients.