In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type–specific associations with age and prognosis

Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer–predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals e...