Sivakumar, N.; Fritz, G.

The anticancer efficacy of radiotherapy (RT) is limited by acquired radioresistance (RR). Here, we aim to characterize prolonged responses of breast carcinoma cells to hypofractionated irradiation (hFI). To this end, murine mammary 4T1 tumor cells (4T1WT) were subjected to a clinically oriented hFI protocol (56 Gy cumulative dose) to select radioresistant cells in vitro (4T1RS). Furthermore, hFI of subcutaneously growing 4T1CTR tumors (hFI; 24 Gy cumulative dose) was performed to radioselected 4T1IR cells in vivo. Following single irradiation in vitro, radioselected 4T1RS cells revealed increased proliferation, attenuated G2/M arrest and reduced apoptosis as compared to parental 4T1WT cells. Moreover, 4T1RS cells showed increased expression of DNA-damage response (DDR)-related proteins (pKAP1, pCHK2, gH2AX) and improved DSB repair efficiency as demonstrated by nuclear gH2AX foci analyses. The mRNA expression of factors regulating cell cycle progression, DDR, apoptosis and oxidative stress was substantially different between both cell variants in vitro. Ten days after hFI of in vivo growing tumors, residual DNA damage and apoptosis were increased in the radioselected 4T1IR tumors, whereas proliferation was reduced as compared to non-irradiated 4T1CTR control tumors. Both irradiated and non-irradiated tumors revealed complex differences in the mRNA expression profile of susceptibility- and metastasis related genes, including GADD45a, DUSP1, CDKN1a and NQO1 as well as CD44 and Rho-related factors, respectively. Moreover, hFI stimulated the infiltration of MPO-positive immune cells into tumor tissue while the presence of CD3-positive cells was reduced in the tumor area. In addition, hFI in vivo resulted in a dysregulated mRNA expression of various immune cell markers, Rho-regulatory factors, tissue remodeling molecules and cell adhesion factors. Summarizing, we identified long-lasting adaptive changes following hFI in vitro and in vivo that are associated with DNA replication, DNA repair, senescence and apoptosis as well as immune cell infiltration and tissue remodeling.