Stromal-epithelial responses to fractionated radiotherapy in a breast cancer microenvironment

Muqeem A. Qayyum, Jin Tae Kwak, Michael F. Insana

The stromal-epithelial-cell interactions that are responsible for directing normal breast-tissue development and maintenance play a central role in the progression of breast cancer. In the present study, we continued our development of three-dimensional (3-D) cell co-cultures used to study cancerous mammary cell responses to fractionated radiotherapy. In particular, we focused on the role of the reactive stroma in determining the therapeutic ratio for post-surgical treatment. Cancerous human mammary epithelial cells (MRC-7) were cultured in a 3-D collagen matrix with human fibroblasts (MRC-5) stimulated by various concentrations of transforming growth factor beta 1 (TGF-β1). These culture samples were designed to model the post-lumpectomy mammary stroma in the presence of residual cancer cells. We tracked over time the changes in medium stiffness, fibroblast-cell activation (MRC-5 converted to cancer activated fibroblasts (CAFs)), and proliferation of both cell types under a variety of fractionated radiotherapy protocols. Samples were exposed to 6 MV X-rays from a linear accelerator in daily fraction sizes of 90, 180 and 360 cGy over five days in a manner consistent with irradiation exposure during radiotherapy. We found in fractionation studies with MRC-5 fibroblasts and CAFs that higher doses per fraction may be more effective early on in deactivating cancer-harboring cellular environments. Higher-dose fraction schemes inhibit contractility in CAFs and prevent differentiation of fibroblasts, thereby metabolically uncoupling tumor cells from their surrounding stroma. However, higher dose fraction appears to increase ECM stiffening. The findings suggest that dose escalation to the region with residual disease can deactivate the reactive stroma, thus minimizing the cancer promoting features of the cellular environment. Large-fraction irradiation may be used to sterilize residual tumor cells and inhibit activation of intracellular transduction pathways that are promoted during the post-surgical wound-healing period. The higher dose fractions may slow wound healing and increase ECM stiffening that could stimulate proliferation of surviving cancer cells.