(23b) Radiation-Induced Changes in the Extracellular Matrix Alter the Invasiveness of Heterogeneous Tumors

Breast cancer metastasis remains a significant clinical problem linked to reduced patient survival. The role of the extracellular matrix (ECM) in metastasis, however, is not well understood. We hypothesize that the irradiated ECM influences tumor and immune cell behavior. In this study, we characterize the effects of radiation on heterogeneous tumor cell migration to evaluate how tumor-stromal interactions modulate metastasis after therapy. This work represents the first step toward elucidating how changes in the ECM after radiation contribute to tumor cell metastasis.

The effect of radiation on tumor-ECM changes was studied using an orthotopic breast cancer model. Nude mice were inoculated with heterogeneous mixtures of luciferase- and mCherry-labeled 4T1 or luciferase- and GFP-labeled 67NR murine breast cancer cells in the mammary fat pad (MFP) in ratios of 0:100 10:90, 50:50, 90:10, and 100:0. 4T1 cells exhibit a highly metastatic phenotype while 67NR cells are tumorigenic but not metastatic. Either primary tumors or contralateral MFPs were irradiated to a dose of 20 Gy with a 250 kVp cabinet x-ray machine when tumors were palpable. Cell migration was monitored with bioluminescence imaging 10 d after irradiation. In addition, lungs, livers, tumors, and MFPs were harvested for confocal imaging and flow cytometry to examine metastatic cell compositions. Flow cytometry was also performed on dissociated irradiated and control tissues to characterize immune cell populations. Of particular interest were CD11b+F4/80+ macrophages. Normal and irradiated MFPs were then decellularized and used as hydrogel scaffolds for studying cell-cell interactions. Scanning electron microscopy was performed to determine the structure of the control and irradiated MFPs.

The presence of 4T1 cells in the primary tumor directly enhanced the metastatic capacity of 67NR cells. With only 10% of 4T1 cells in the initial inoculated population, we observed 67NR metastases to the lung and liver. Different fluorophores allowed the visualization of metastatic cells as aggregates. Radiation of primary tumors enhanced circulating tumor cell levels, and irradiation increased tumor cell migration to normal tissues. Macrophage infiltration increased both in metastatic lesions and in tissues after irradiation. In decellularized MFP hydrogels, 67NR cells developed an invasive morphology when cultured with 4T1 cells, and this effect was enhanced in the irradiated MFP. The fibrous structure of the MFPs was also altered after irradiation.

This study establishes that tumor heterogeneity determines metastatic spread and underscores the importance of in vivo models that reflect the variation of naturally occurring tumors. We found that the ECM modulates tumor cell migration. The increase of macrophage infiltration in metastases as well as in irradiated tissues indicates a crucial role of the immune system in tumor progression and metastasis. Our work suggests that the ECM may facilitate tumor cell invasion and metastasis following radiotherapy. Future studies will utilize these results to engineer improved in vitro tumor microenvironment models to investigate the multifaceted physical, chemical, and biological cues that influence cancer metastasis.