(466f) Irradiation Promotes Breast Cancer Cell Proliferation and Invasion in Extracellular Matrix Hydrogels

While radiation therapy (RT) is an efficient treatment for patients with triple negative breast cancer (TNBC), patients continue to experience recurrence after RT. The alteration of extracellular matrix (ECM) of healthy breast tissue induced by radiation and its role on tumor locoregional recurrence are still unknown. We hypothesize that the irradiated ECM may influence pre-metastatic niche formation and lead to tumor cell recruitment and retention. The objectives of the present study were to develop irradiated ECM hydrogels and assess the effects of RT-induced ECM changes on breast cancer cell behavior. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

Scanning electron microscopy (SEM) was used to visualize irradiated breast tissue changes including the ECM deposition and lipid distribution. In addition, the Raman spectra of irradiated and non-irradiated healthy breast tissue were compared to confirm molecular ECM changes. To investigate the impact of irradiated ECM on tumor cell behavior, murine mammary fat pads (MFPs) were irradiated to a dose of 20 Gy ex vivo. Following incubation for 48h post-RT, the resulting control and irradiated MFPs were decellularized and digested to form ECM hydrogels for the encapsulation of murine 4T1 TNBC cells. Mechanical properties of the hydrogels were determined by rheology. The viability and proliferation of tumor cells were examined 48h after seeding by fluorescence microscopy, bioluminescence measurements, and viability measurements. We analyzed how cell-matrix interactions regulate tumor cell invasiveness and intracellular cytoskeletal dynamics by actin, cortactin, focal adhesion, and matrix metalloproteinase (MMP) staining.

SEM images and Raman spectra of irradiated MFPs indicated increased ECM deposition (p<0.05), which may provide a platform for cell migration, proliferation and infiltration. Stable hydrogel formation was demonstrated through the increased storage modulus compared to the loss modulus for all conditions. Enhanced tumor cell proliferation in irradiated hydrogels were proved by bioluminescence imaging and fluorescence microscopy (p<0.01). Viability staining suggested an increased live to dead cell ratio in RT hydrogels compared to the non-RT control hydrogels. Encapsulated cells were stained with F-Actin, and an increase in alignment and elongation was observed in irradiated ECM hydrogels (p<0.01). The invasion of breast cancer cells was confirmed by increased colocalization of actin and cortactin (p<0.01).

Our study establishes that the irradiated microenvironment influences tumor cell behavior. The developed ECM hydrogels are important for mimicking how the in vivo breast tissue environment responds to radiation damage. Our results suggest that the irradiated ECM promotes tumor cell proliferation and invasion. Future studies will utilize these ECM hydrogels to explore the interactions between tumor and immune cells that may contribute to recurrence following radiotherapy.