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Cancer stem cells (CSC) are a major cause for the failure of tumor therapy. This is mainly attributed to increased DNA repair capacity and immune escape. Recent studies showed that functional DNA repair via Homologous recombination (HR) avoids radiation-induced accumulation of DNA in the cytoplasm, thus inhibiting the intracellular immune response. Yet, it is unclear whether CSC suppress radiation-induced cytosolic dsDNA formation and thus suppress an innate immune response.

Investigations were performed in four breast cancer cell lines, their respective radioresistant clones (RR clones) which were produced by repeated irradiation (10x4Gy) and ALDH1 positive CSC, which were isolated from the RR clones. Functionality of HR was determined (plasmid reporter assay, RAD51 foci), general markers for DNA-repair (53BP1-foci) and DNA replication stress (yH2AX/RPA foci, DNA-Fiber) were analyzed. Cytosolic dsDNA formation was investigated by PicoGreen™-assay, the expression of PD-L1, PD-L2 and the activation of cGAS/STING/IRF3 via Western Blot. Radiosensitization was investigated by inhibition of ATR with the small-molecule inhibitor VE-821 in colony assays.

A significantly increased activity of ALDH1 was observed in all RR clones and their isolated, ALDH1-positive CSC. After irradiation, survival in the RR clones was significantly increased and the number of residual 53BP1 foci was significantly decreased. This was especially apparent after irradiation in S-Phase (p<0.0001), indicating improved DNA repair by HR. This was confirmed by an increased HR capacity and replication fork stability after irradiation and resulted in significantly decreased yH2AX- and RPA foci after irradiation (p<0.001). The avoidance of radiation-induced replication stress resulted in a significantly lower accumulation of dsDNA in the cytoplasm and a low cGAS/STING/IRF3 activation. Indeed, the proportion of ALDH1-positive CSC correlated significantly with the amount of cytosolic dsDNA after irradiation (p<0.001). Strikingly, the CSC showed an endogenously increased expression of PD-L1 and PD-L2, which was increased ~3 fold after fractionated irradiation (5x5.2 Gy). The inhibition of ATR led to a distinct radiosensitization of the radioresistant CSC (EF=3) and significantly reduced PD-L1 expression. Furthermore, ATR-inhibition to a significant increase of nuclear IRF3 after irradiation, thus an increased activation of the intracellular immune response (p<0.001).

The results show that CSC express more PD-L1 and PD-L2 and minimize the formation of cytosolic DNA after irradiation through enhanced DSB repair and protection of replication forks by HR. Disruption of the ATR-CHK1 signaling pathway by ATR inhibition leads to radiation sensitization, reduced PD-L1 expression and increased activation of the cGas-STING pathway. Therefore, we hypothesize that inhibitors to inactivate the S-phase DNA damage response, such as ATR, may be used to further develop existing therapies in the future.