(604b) Radiation-Enhanced Proliferation and Invasion of Breast Cancer Cell in Extracellular Matrix Hydrogels

The poor overall survival in triple negative breast cancer (TNBC) patients is associated with a high incidence of local recurrence following radiation therapy (RT), and previous work suggests a link between radiation damage and TNBC recurrence under immunocompromised conditions. How radiation impacts the microenvironment, especially the extracellular matrix (ECM) and its role in locoregional recurrence is unknown. We hypothesize that the radiation-damaged ECM facilitates pre-metastatic niche formation and leads to tumor cell recruitment and retention. In this study, the structure, composition, and mechanical properties of irradiated breast tissue are evaluated. ECM hydrogels were also developed to characterize the influence of RT on breast cancer cell behavior. This work represents a crucial step toward illustrating how modulation of the ECM after RT contributes to breast cancer recurrence.

We used scanning electron microscopy (SEM) and Raman spectroscopy to investigate murine tissue changes post-RT, including ECM deposition and lipid distribution. In addition, the stiffness of healthy and irradiated breast tissue was determined by atomic force microscopy (AFM). To characterize the effects of the irradiated ECM on breast cancer cell behavior, we irradiated mammary fat pads (MFPs) ex vivo to a dose of 20 Gy and decellularized the resulting treated and control MFPs after 48h of incubation. ECM hydrogels were formed after pepsin digestion, and AFM was utilized to measure their mechanical properties. GFP- and luciferase-labeled murine 4T1 TNBC cells were encapsulated within these hydrogels, and cell proliferation was evaluated by fluorescence microscopy and bioluminescence measurements after 48h. Cytoskeletal organization and invasion of the encapsulated tumor cells were analyzed by phalloidin staining of F-actin and cortactin immunofluorescence staining.

SEM and Raman spectra revealed increased ECM deposition in irradiated tissues. Additionally, ECM fibers were thinner and more dense post-RT, which may allow for tumor cell adhesion and retention. The stiffness of MFPs increased significantly following RT, which may influence tumor progression and metastasis. Bioluminescence imaging and fluorescence microscopy 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. 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.

Our study establishes that RT-induced alterations in the ECM impact breast cancer cell behavior. The ECM hydrogels developed in this work recapitulate the radiation-damaged microenvironment. Our work suggests that the irradiated ECM promotes tumor cell proliferation and invasion, which may lead to TNBC recurrence following RT. Future studies will utilize these ECM hydrogels to explore the role of immune cells on tumor cell behavior in the post-treatment microenvironment.