Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

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Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. The alteration of extracellular matrix (ECM) of healthy breast tissue induced by radiation and its role on tumor recurrence are still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. Alterations in irradiated ECM components including collagen I, IV, VI, and fibronectin were observed. TNBC cell proliferation was enhanced in irradiated hydrogels. Encapsulated TNBC cell morphology analysis indicated an increase in invasive capacity within irradiated ECM hydrogels. In addition, TNBC cells co-cultured with macrophages in irradiated ECM hydrogels exhibited further increases in cell proliferation. Our study establishes that the ECM in the irradiated microenvironment promotes TNBC invasion and proliferation that is enhanced in the presence of macrophages. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

Triple negative breast cancer (TNBC) recurrence rates remain high despite aggressive therapeutic intervention, including surgery, chemotherapy, and radiotherapy (RT). Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence rather than persistent tumor cells in the irradiated surgical bed. However, the role of the microenvironment in recurrence is not well understood. An emerging risk factor for breast cancer patients is lymphopenia or abnormally low systemic lymphocyte counts following therapy. In this study, we examined whether lymphopenia following therapy correlates to local recurrence. To investigate the relationship between prolonged low lymphocyte count and recurrence, we evaluated radiation effects on tumor and immune cell recruitment to normal tissues using an orthotopic breast cancer model of lymphopenia. We tested the hypothesis that local recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. Local cytokine secretion as well as protein expression levels were analyzed to evaluate how tumor-stromal interactions modulate tumor cell recruitment to sites of damage. Recurrence free survival at 5 years for TNBC patients with sustained low ALC 1-5 months after RT (n=37) was 62.5% compared with 97% for patients with normal ALC (n=46; p<0.0001). Ex vivo BLI analysis revealed that normal tissue irradiation attracted tumor cells to the mammary fat pad (MFP) and surrounding tissues in BALB/c mice with depleted CD8+ T cells. Macrophage infiltration was greatly enhanced in BALB/c mice with depleted CD8+ T cells (p<0.0001) and necessary for tumor cell recruitment. Luminex analysis of MFPs in mice lacking CD8+ T cells after RT showed enhanced cytokine secretion of factors that regulate the inflammatory microenvironment and influence tumor cell proliferation and invasion. RPPA analysis of irradiated MFPs in the absence of CD8+ T cells revealed factors that promote epithelial-mesenchymal transition. Taken together, these results demonstrate that the response of the tissue microenvironment to therapy is dependent on the immune milieu and may encourage local recurrence. Our study establishes the importance of considering tumor subtype and immune function when assessing failure and outcome risks in breast cancer. We show that normal tissue radiation response may play a role in modulating tumor and immune cell migration. These results suggest that the stroma facilitates local recurrence in breast cancer through influencing immune and tumor cell behavior following RT. Citation Format: Benjamin C. Hacker, Steven M. Alves, Dadi Jiang, Albert C. Koong, Amato J. Giaccia, Edward E. Graves, Marjan Rafat. The irradiated tissue microenvironment and its role in breast cancer recurrence: Enhanced macrophage infiltration promotes tumor cell recruitment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3931.

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