Characterisation of the glioblastoma perivascular niche and the role of DOCK4 in blood vessels and tumour invasion

Blood vessels play a vital role in cancer development and progression as tumours cannot grow beyond a small diameter in their absence. Blood vessels are abundant and intrinsically abnormal in glioblastoma multiforme (GBM), while current antiangiogenic therapies are disappointing as these tumours are either unresponsive or gradually develop resistance. Hence, characterisation of aberrant blood vessels in primary and recurrent GBM, and following radiotherapy could open new therapeutic avenues for GBM in the future. This study identified different blood vessel morphologies in patientderived glioblastoma samples. Patients presented with varying degrees of blood vessel abnormality which increased in recurrent tumours following surgery, radiotherapy and chemotherapy. High nestin positivity and large lumen size were key characteristics, particularly in recurrent tumours. Nestin positive cells were closely associated with the blood vessel endothelial lining but distinct to CD31 positive endothelial cells. Nestin positive cells were negative for the glioblastoma stem-like cell (GSC) markers OLIG2 and SOX2, and astrocyte or immune cell markers, but positive for the pericyte marker PDGFRb and smooth muscle cell (SMC) marker alpha smooth muscle actin (a-SMA), suggesting they are distinct from GSCs. Nestin and SMA positive blood vessels were found in close proximity to tumour necrotic regions, but were associated with low hypoxia levels suggesting functionality. The effects of radiotherapy on blood vessel abundance, morphology and function are poorly characterised, as are the molecular mechanisms of glioblastoma blood vessel formation. DOCK4 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1, which is highly expressed in neuronal and endothelial cells, and regulates cellular protrusions, cell migration and blood vessel lumen size. Irradiation experiments were performed using the CT2A model of glioblastoma grown intracranially in syngeneic mice. While a decrease in vascularisation and lumen size were observed early after irradiation, the abundance and size of lumenised blood vessels increased at later stages. Using Dock4 heterozygous knockout mice to overcome early embryonic lethality of homozygous Dock4 deletion, it was shown that reduction of DOCK4 levels reverses the increase in blood vessel size in response to irradiation, however without observed concurrent changes in tumour growth, or survival. Invasion of cells into the normal brain parenchyma limits the efficacy of surgery and radiotherapy thus contributing to recurrence and poor prognosis of glioblastoma. Glioblastoma invasion is known to take place along white matter tracts and alongside blood vessels. The role of DOCK4 in glioblastoma invasion was investigated in spheroid assays in collagen, using both established glioblastoma cell lines and patient-derived glioblastoma stem-like cells (GSCs). Lentiviral shRNA-mediated knockdown of DOCK4 reduced invasion into the surrounding matrix in spheroid assays. However, DOCK4 knockdown did not alter invasive capacity when glioblastoma cells were co-cultured with endothelial cells. On the other hand, co-culture with endothelial cells increased the sensitivity of some glioblastoma cell lines to irradiation. In summary, this study shows that aberrant pericyte/SMC blood vessel coverage and larger blood vessel size are prevalent in GBM and increase with recurrence. These aberrant blood vessels appear functional and may play an important role in glioblastoma resistance to chemo- and radioradiotherapy, and facilitate tumour recurrence. Radiotherapy promotes aberrant blood vessel morphology and size in experimental tumours. DOCK4 inhibition may normalise the glioblastoma vasculature, and concomitantly inhibit invasion of a subpopulation of glioblastoma cancer stem cells.