(72d) Irradiated Extracellular Matrix Hydrogels Enhance Tumor Cell Proliferation and Invasion

Authors: 
Zhu, T., Vanderbilt
Alves, S. M., Vanderbilt University
Shostak, A., Vanderbilt University
Rossen, N., Stanford University
Rafat, M., Vanderbilt University
Triple negative breast cancer (TNBC) is an aggressive and difficult to treat subtype of breast cancer, and over 13% TNBC patients experience recurrence after radiation therapy (RT). 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. We hypothesize that the irradiated ECM may influence pre-metastatic niche formation that could lead to tumor cell recruitment and retention. In this study, we develop irradiated ECM hydrogels to characterize the effects of RT-induced ECM changes on proliferation and cytoskeleton organization of breast cancer cells. We evaluate how tumor-ECM interactions modulate tumor behavior at sites of damage. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer progression.

The impact of irradiated ECM on tumor cell behavior was studied using decellularized, irradiated normal tissues as ECM hydrogels. We irradiated murine mammary fat pads to a dose of 20 Gy ex vivo, and the resulting control and irradiated tissues were decellularized following incubation for 2 days post-RT. Hydrogels were formed following pepsin digestion, and 4T1 cells, a murine TNBC cell line, were encapsulated within the hydrogels. Rheology of the irradiated and control hydrogels was performed to determine mechanical properties. Cell proliferation was examined by fluorescence microscopy, bioluminescence measurements, and viability staining 48 h after encapsulation. Phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells.

The increased storage modulus compared to the loss modulus for all conditions demonstrated stable hydrogel formation. Fluorescence microscopy and bioluminescence imaging confirmed enhanced tumor cell proliferation in irradiated hydrogels. Viability staining indicated an increased live to dead cell ratio in irradiated hydrogels compared to the unirradiated control. An increase in actin alignment and tumor cell elongation was observed in irradiated ECM hydrogels, which suggests that the irradiated ECM environment promotes tumor cell adhesion and invasion.

Our study establishes that the irradiated microenvironment alters tumor cell behavior. The developed ECM hydrogels mimic the in vivo breast tissue environment and its response to radiation damage. The tumor cell proliferation and cytoskeleton changes suggest that the irradiated ECM facilitates tumor cell retention and invasion in irradiated normal tissues. Future studies will utilize these ECM hydrogels to study tumor-immune interactions to provide insight into the mechanisms influencing TNBC recurrence.