(549d) Co-Cultures of Mammary Organoids and Macrophages Provide Insight into Breast Cancer Recurrence

Although breast cancer patients receive chemotherapy, surgery, and radiation treatment, up to a quarter of triple negative breast cancer (TNBC) patients experience locoregional recurrence. Patient data and pre-clinical studies have established that radiotherapy, immune status, and macrophage infiltration are associated with TNBC recurrence. However, there is much about the role of the normal tissue microenvironment in recurrence that remains unknown. Although macrophages have been implicated in facilitating tumor cell recruitment to treated sites, characterization of macrophage polarization following radiation damage has not been undertaken. Determining recurrence associated immune cell infiltration in vivo and pairing these studies with biologically relevant in vitro models are therefore necessary to elucidate further mechanisms of tumor cell recruitment. In this study, we examined the radiation response of immune cells in vivo and applied these observations to an in vitro mammary organoid model to analyze the effects of radiation on cell behavior.

To evaluate immune cell recruitment, we irradiated inguinal murine mammary fat pads (MFPs) of 8-12 week old female Nu/Nu and BALB/c mice in vivo to a dose of 20 Gy. MFPs were resected 10 days post-irradiation. Tissues were either processed for immunohistochemical characterization or into a single cell suspension for flow cytometry. Cells were stained with F4/80 in addition to CD86+ or CD206+ to determine M1 and M2 macrophage polarization, respectively. To obtain mammary organoids, MFPs were resected, irradiated ex vivo to 20 Gy, and digested with collagenase to isolate epithelial cells before self-assembly in low adhesion plates. Control unirradiated and irradiated organoids were then cultured with biologically relevant ratios of M1/M2-polarized RAW 264.7 macrophages. Co-culture experiments were monitored up to 48 hours with live cell imaging. Conditioned media from the co-culture was used for in vitro invasion assays with murine TNBC 4T1 cells.

We identified an increase in F4/80+ macrophage infiltration following in vivo irradiation, recapitulating observations from previous studies. We also quantified enhanced recruitment of tumor associated M2 macrophages in irradiated sites (p < 0.05), potentially implicating the role of wound healing macrophages in tumor cell recruitment. These results were confirmed in vitro as tumor cell invasiveness was associated with irradiation and higher M2/M1 macrophage ratios in the co-culture model.

This work describes the interplay between an in vivo and in vitro model that may elucidate the role of radiation in immune-facilitated recurrence in a biologically relevant environment. This model will provide insight into the mechanisms behind radiation-induced tumor and immune cell recruitment, which will have significant implications for patients suffering from TNBC recurrence.