Stromal responses to fractionated radiotherapy

Purpose: Recently, several landmark randomized trials were published that justify the use of alternative fractionation schemes, e.g., hypofractionation, in adjuvant applications of whole-breast radiotherapy following breast-conserving surgery. We are studying effects of fractionated photon radiotherapy on stromal cell biology to understand how fractionation parameters influence the cellular microenvironment. Methods and materials: Three-dimensional (3-D) collagen matrices, fibroblasts, and transforming growth factor beta 1 (TGF-β1) were combined to model microenvironmental components of mammary stroma. We explored the effects of fractionation schemes on collagen matrix stiffness and fibroblast activation using this culture model. Samples were exposed to 6 MV X-rays from a linear accelerator in daily fraction sizes of 90, 180 and 360 cGy over three days in a manner consistent with irradiation exposure during radiotherapy. Results: Fibroblast-cell activation and collagen sample stiffness both increased over time for all samples, but marked changes were noted when samples were irradiated and/or stimulated with growth factors in relation to the magnitude of the stimulus. We found a significant reduction in fibroblast proliferation and activation with fraction size but a modest and irreversible increase in matrix stiffness as the dose increased. Overall, larger fraction sizes reduced conditions leading to the formation of a reactive stroma. Conclusion: There is a significant reduction in fibroblast activation and a modest increase in matrix stiffness with increasing fraction size over a 72-hour observation time in 3-D cultures modeling mammary stroma. However, expanded in vitro studies with more mammary components are needed to evaluate the net effects of stromal reactivity to radiotherapy. Our results suggest that the stromal cell microenvironment is an important consideration when optimizing fractionation schedules.