Investigation of radiation-induced vascular abnormalities in glioblastoma tumour recurrences and the role of DOCK4 on pathological vessel development

Glioblastoma (GBM) is the most lethal form of brain tumour with an overall median survival of 12-15 months with current standard therapy of maximal safe surgery, radiotherapy and temozolomide chemotherapy. Abnormalities in the vascular niche are associated with defective vessel function and development of hypoxia, promoting disease progression and resistance to various anti-cancer therapies. Current anti-angiogenic therapies for GBM have not been promising in the clinic, contributing to no improvement in overall patient survival. Analysis of patient GBM tumours in our laboratory had shown augmentation of vascular abnormalities, including increase in vascularisation, larger lumen size and increase in mature blood vessels in recurrences compared to primary tumours. However, the precise nature of those vascular abnormalities and how they are affected by radiotherapy was not understood. This study explores the effects of radiotherapy on the vascularisation of regrown tumours with the use of an experimental CT2A murine glioma model. Similar to the vascular changes identified in the GBM patient recurrences, abnormal vascularisation was augmented in regrown tumours post radiotherapy in the experimental model. Larger lumen size, increase in vessel length and overall surface area covered by blood vessels were abnormities identified in CT2A tumours regrown post radiotherapy. Hypoxia has been shown previously to result in the formation of vessels with large lumens via the action of VEGF. Blood vessel enlargement in experimental recurrence tumours was associated with decreased vascular permeability and elevated hypoxia in the recurrent tumours post irradiation. While smaller and larger calibre blood vessels exhibited comparable levels of extravasation in unirradiated primary tumours, enlarged blood vessels showed increased extravasation and normoxia in their vicinity, suggesting that vessel enlargement is an adaptation response to vascular dysfunction and hypoxia in the regrown tumours post radiotherapy. DOCK4 (dedicator of cytokinesis 4) is a member of the DOCK180 family of guanine nucleotide exchange factors (GEFs) and GEF for the small GTPase Rac1, shown to operate downstream of VEGF signalling in organotypic co-cultures in vitro and to regulate blood vessel lumen size in in vivo. In CT2A tumours grown in global Dock4 heterozygous knockout mice to overcome homozygous Dock4 deletion embryonic lethality, blood vessel enlargement was reversed in tumours regrown post radiotherapy treatment, while DOCK4 played no role in the vascularisation of primary tumours. Additionally, regrown tumours post radiotherapy also exhibited higher number of blood vessels associated with a-SMA positive pericytes. This increase in blood vessel maturation that was unaffected by Dock4 deletion and had no impact on blood vessel permeability. Further in vivo experiments using CT2A tumours grown in the Dock4 endothelial conditional homozygous knockout mice showed that in primary tumours Dock4 may act as a blood vessel growth suppressor, however more experiments are necessary in the future to exclude the action of compensatory mechanisms in response to knocking out both of the Dock4 alleles in endothelial cells. The experiments were underpowered to confirm an endothelial cell autonomous role of Dock4 deletion in the reversal of blood vessel enlargement in regrown irradiated CT2A tumours. In summary, the study shows that in GBM tumours regrown post radiotherapy there is VEGF-driven augmentation of blood vessel calibre and growth to counteract radiation driven vasculopathy and elevated hypoxia in the regrown tumours. The resulting vasculature is also more mature with elevated pericyte coverage. Co-option of blood vessels situated in the peri-tumoural area treated with radiotherapy following surgical resection, and subsequent tumour-induced modification would provide an explanation for the abnormal function of the vasculature in the regrown tumours, and resistance to current anti-angiogenic therapies. The CT2A experimental model of tumour regrowth established here can be used to further elucidate the contribution of therapies on vascular abnormality. Elucidating the mechanisms of radiation-induced aberrant vascularisation may lead to the development of new blood vessel-targeting therapies and/ or provide rationale and alternative avenues for modifying existing therapies.