(604d) Primary Murine Mammary Spheroids and Macrophage Co-Cultures Provide Insight into Triple Negative 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, mechanisms of macrophage mediated recurrence following radiation damage have not been studied in-depth in vitro. 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 spheroid model to analyze the effects of radiation on cell behavior.

First, to determine physiologically relevant proportions of macrophage infiltrate post irradiation in vivo, we inoculated 8-12 week old Nu/Nu and BALB/c mice with 4T1 tumors in the left inguinal mammary fat pad (MFP). Once tumors reached a size of 100 mm³, we irradiated the contralateral mammary gland to a dose of 20 Gy to induce macrophage and tumor cell migration, modeling radiation induced cancer recurrence. We characterized macrophage populations via flow cytometry. CD45⁺CD11b⁺F4/80⁺ macrophages were further characterized by phenotype with iNOS (M1) and CD206 (M2) expression. Next, we constructed an in vitro model of irradiated normal mammary tissue by first isolating primary cells from the stromal vascular fraction (SVF). Cell populations within the SVF were characterized by immunofluorescence, including markers for fibroblasts (PDGFRA) and epithelium (Pan-Cytokeratin). We treated SVF cells with clinically relevant doses of irradiation up to 20 Gy. To form spheroids, SVF cells were plated with primary bone marrow derived macrophages (BMDMs) in low adhesion U bottom plates. Spheroids were plated with physiologically relevant concentrations of M0, M1, and M2 BMDMs, determined from the previous in vivo studies of macrophage migration. Conditioned media was collected 2 and 7 days after irradiation of spheroids. Additionally, tumor cell infiltration and invasion assays were conducted using GFP expressing 4T1 (4T1^GFP) cells. Kinetics of 4T1^GFP cell infiltration and invasion were characterized via live cell imaging.

We identified an increase in CD45⁺CD11b⁺F4/80⁺ macrophage infiltration following in vivo irradiation, recapitulating observations from previous studies. We also observed enhanced recruitment of tumor associated M2 macrophages in irradiated sites, potentially implicating the role of wound healing macrophages in tumor cell recruitment. In our in vitro model, primary SVF spheroids displayed expression of multiple cell types biologically relevant to the mammary gland, including fibroblasts and epithelial cells. Co-cultures of SVF cells and primary macrophages consistently self-assembled into spherical constructs, and we were able to culture primary macrophages in irradiated spheroids for over 7 days.

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.