Response to ionising radiation of glioblastoma stem-like cells

Introduction: Glioblastoma (GBM) is characterised by local recurrence following surgery, radiotherapy and chemotherapy. GBM has a poor prognosis and novel approaches are required. Recently, a hierarchical organisation of tumour cells in GBM has been proposed. This hypothesis suggests only a subset of cancer cells, termed ‘cancer stem-like cells’ (CSCs) drive tumour growth and possess properties of self renewal and unlimited proliferative capacity. CSCs have been described as radioresistant, implicating CSCs as a determinant of tumour recurrence following therapy. Therefore improved patient outcomes could potentially be achieved by targeting GBM CSCs. Nevertheless, reports of GBM CSC radioresistance have been conflicting, with some authors demonstrating CSC radiosensitivity. Furthermore, investigations of GBM CSC radioresponse have lacked robust radiobiological quantification and this aspect of the CSC phenotype remains controversial. Aims: To investigate the radioresponse of GBM CSCs in comparison to non CSCs, characterise the DNA damage response (DDR) in GBM CSCs to radiation and investigate effects of inhibition of DNA damage response (DDR) in GBM CSCs. Methods: Primary GBM cells were cultured in CSC enriching conditions and differentiating (‘tumour bulk’) conditions. The radioresponse of CSC and tumour bulk cultures derived from single parental tumours were thus compared by clonogenic survival assay. DDR was analysed in CSC and tumour bulk cells via Western blotting for DDR phosphoproteins and flow cytometric quantification of mitotic cells. DNA double strand break (DSB) repair was quantified by analysis of gamma H2AX foci. CSCs and tumour bulk response to irradiation in combination with inhibition of key DDR elements (ataxia telangiectasia mutated, (ATM); ataxia telangiectasia and Rad3 related, (ATR); and poly (ADP-ribose) polymerase, (PARP) by small molecule inhibitor agents was characterised. Results: CSC cultures were tumourigenic or recapitulated pathological features of parental tumours in orthotopic mouse models, whereas differentiated tumour bulk cultures did not. CSC cultures exhibited upregulation of putative CSC markers relative to tumour bulk. CSC cultures were radioresistant, demonstrated upregulated DDR and more efficient activation of the G2/M checkpoint compared to tumour bulk. CSC cultures repaired DNA DSBs more efficiently at 24 hours following irradiation. Inhibition of ATM in CSCs led to abrogation of the G2/M checkpoint response, reduced efficiency of DNA DSB repair and potent radiosensitisation. Inhibition of PARP in CSCs produced an increase in unresolved DNA DSBs in GBM CSCs at 24 hours post irradiation in G2 phase cells and modest levels of radiosensitisation. Inhibition of ATR in CSCs abrogated the G2/M checkpoint in CSCs efficiently and was associated with modest radiosensitisation. Dual ATR and PARP inhibition provided highly potent radiosensitisation of GBM CSCs. Conclusions: GBM CSCs were shown to be radioresistant relative to tumour bulk cells due to upregulated DDR, in support of the hypothesis that CSCs contribute to local recurrence, implying a need for CSC targeted therapies. The inhibition of G2/M checkpoint activation and DNA DSB repair via ATM inhibition or combined ATR/PARP inhibition potently radiosensitised GBM CSCs suggesting targeting both checkpoint and DNA DSB repair is important for optimal radiosensitisation of GBM CSCs. This study has demonstrated that DDR is a potential therapeutic target for radiosensitisation of GBM CSCs.