(460c) Radiation Therapy Promotes Breast Cancer Cell Proliferation and Invasion in Extracellular Matrix Hydrogels

Patients with triple negative breast cancer (TNBC) often experience local recurrence even after undergoing radiation therapy (RT), and existing research has indicated a correlation between radiation-induced damage and TNBC recurrence in cases of immunocompromised patients. However, the impact of radiation on the microenvironment, particularly the extracellular matrix (ECM) and its role in locoregional recurrence, remains unclear. We propose that the ECM, which is damaged by radiation, could potentially facilitate pre-metastatic niche formation and lead to the recruitment and retention of tumor cells. We developed extracellular matrix (ECM) hydrogels to test this hypothesis and to study the effects of RT on breast cancer cell behavior.

To determine how the irradiated extracellular matrix (ECM) affects breast cancer cells, we created an ECM hydrogel model that mimics in vivo conditions. To achieve this, we irradiated murine mammary fat pads (MFPs) ex vivo to a dose of 20 Gy, and then decellularized and digested them to form ECM hydrogels. We used scanning electron microscopy (SEM) and atomic force microscopy (AFM) to examine the structure and stiffness of the ECM hydrogels. Luciferase-labeled murine 4T1 TNBC cells were encapsulated within these hydrogels, and cell proliferation was evaluated by bioluminescence measurements. We also analyzed cytoskeletal organization and invasion of the encapsulated tumor cells through phalloidin staining of F-actin, cortactin immunofluorescence staining, and invasion assays. In addition, we collected conditioned media from 4T1 cells cultured in the ECM hydrogels and used a Luminex multiplex immunoassay to analyze cytokine secretion. Finally, we co-cultured GFP-labeled 4T1 cells with bone marrow derived macrophages (BMDMs) stained with CellTrace Far Red to investigate the interactions between tumor and immune cells in the irradiated ECM hydrogels.

SEM analysis revealed thinner and denser ECM following RT, which may allow for tumor cell adhesion and retention. Irradiated ECM hydrogels also exhibited elevated stiffness compared to unirradiated controls consistent with increased ECM deposition. Bioluminescence imaging confirmed enhanced tumor cell proliferation in irradiated ECM hydrogels (p<0.01). We further demonstrated that irradiated ECM hydrogels promote a higher invasive capacity in tumor cells through quantifying cell elongation using F-actin staining and invasion assay. Additionally, tumor cell invadopodia, as determined by the colocalization of F-actin and cortactin, increased in irradiated hydrogels, suggesting that tumor cell invasion is enhanced in the radiation-damaged microenvironment. The secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) increased after encapsulating 4T1 tumor cells in irradiated hydrogels, implying that macrophages contribute to recurrence after radiation damage. Moreover, co-culturing 4T1 cells BMDMs resulted in a 3-fold enhanced proliferation compared to 4T1 cells cultured in ECM hydrogels alone, emphasizing the role of immune cells in promoting cancer cell proliferation in the irradiated microenvironment.

Our research investigates the effects of radiation on the extracellular matrix (ECM) and highlights the potential link between irradiation and cancer recurrence. This work represents a significant step toward understanding how post-RT modulation of the ECM contributes to breast cancer recurrence. We demonstrate the importance of using ECM hydrogels derived from mammary tissue to understand how the breast tissue environment responds to radiation damage. Our findings indicate that the radiation-damaged ECM facilitates tumor cell proliferation and invasion and also alters the interactions between tumor and immune cells. Further studies will focus on developing ECM hydrogels from in vivo irradiated mouse models to further evaluate the role of the ECM in TNBC recurrence following RT.