Overcoming wound healing complications following radiotherapy in human breast dermal fibroblasts, through the influence of preadipocytes from the stromal vascular fraction

Abstract

Radiotherapy has major therapeutic benefits for cancer patients, but ionizing radiation causes damage of surrounding healthy tissues with poor wound healing a common side effect. Therefore, further oncoplastic, reconstructive surgery is challenging and often problematic. Current research models use normal human dermal fibroblasts irradiated in vitro to mimic radiation damage, but this is not comparable to ionising radiation and only measures acute changes. Since radiotherapy may induce epigenetic changes leading to alterations in dermal fibroblast phenotype, the first aim of this study was to compare fibroblasts cultured from irradiated skin with non-irradiated skin. As mesenchymal stem cells isolated from adipose tissue may offer beneficial effects in the regenerative capacity of irradiated tissue, the second part of this study was to compare those cultured from non-irradiated and irradiated breast tissue. Histological changes in the structural organisation of breast tissue in situ from donors exposed to radiotherapy was compared to untreated breast. Primary cultures of dermal fibroblasts from irradiated and non-irradiated breast skin were established and comparisons quantitated in proliferation (CyQuant), metabolism (Alamar Blue), migration (scratch-wound assay), collagen production (Sircol), levels of proteases and protease inhibitors (human protease/protease inhibitor array) and gene expression of COL1A1, COL3A1, MMP1, MMP2, TIMP1 and PPAR-γ mRNA (qPCR). Cells from the stromal vascular fraction (SVF) were cultured and characterised by immunocytochemistry and compared to human preadipocytes sourced commercially. The secretion of FGF, adiponectin and VEGF by the preadipocyte and the SVF mesenchymal cells was compared and the ability of their secretome to modulate dermal fibroblast proliferation, metabolism and migration was evaluated. Radiotherapy caused extensive disorganisation of the reticular dermis and flattening of the epidermal-dermal junction. Dermal fibroblasts cultured from irradiated skin had a pronounced spindle shaped morphology with longer thinner projections and took approximately twice as long to explant and grow. They had a lower proliferative and higher basal metabolic rate and did not respond to FGF-2. While they secreted similar amounts of total collagen they demonstrated distinct differences in proteolytic enzyme and protease inhibitor expression. This is the first report to culture cells from the SVF of irradiated breast tissue. The cells expressed the preadipocyte markers CD10, CD73 and CD105 and no CD45 (negative marker). SVF cells cultured displayed a typical ASC fibroblastoid morphology. Analysis of the secretome identified the presence of FGF, adiponectin and VEGF, while functional analysis demonstrated a stimulatory effect on normal dermal fibroblast migration, although irradiated dermal fibroblasts were unresponsive. Radiotherapy induces long term, detrimental changes in breast skin. This is the first quantitative characterisation of dermal fibroblasts and mesenchymal cells from the SVF, subjected to ionising radiation in situ. Changes in their phenotype that alter their function will impact on wound healing. Further characterisation of these cells may explain their dysfunctional behaviour, and lead to therapies to reverse or reduce this deleterious side-effect and significantly improve treatments facilitating wound healing following radiation injury.