Investigating the effects of hypoxia on brain tumour cell invasion using advanced in vitro and in vivo imaging

Glioblastoma (GBM) is the deadliest adult primary brain tumour. Limited treatment options for patients with GBM results in the dismal prognosis of 12–15 months, highlighting the unmet need for novel therapies for these patients. Tumour hypoxia is one reason for the highly aggressive and invasive nature of this tumour and is correlated with acquired therapy resistance, regulated by hypoxia inducible transcription factors (HIFs). Novel approaches to combat this disease has seen the development of therapies targeting alternative pathways such as tumour metabolism. Our aim was to firstly investigate the cellular mechanisms trigged by choline kinase alpha (ChoKα) inhibitor, JAS239 on four GBM cell lines (F98, 9L, U-87 MG and U-251 MG), and to assess the impact of hypoxia on JAS239 efficacy. Additionally, we also sought to determine the anti-tumour activity of JAS239 in vivo using orthotopic GBM rodent models and magnetic resonance frequency (MRS) for monitoring therapy response. Hypoxia and JAS239 treatment demonstrated significant alterations on cell metabolism across all cell lines. The ‘Warburg effect’ was the most commonly altered metabolic pathway in response to hypoxia and JAS239 treatment, along with glutamine/glutamate, branched chain amino acids and fatty acid metabolism. Furthermore, we observed that JAS239 induced significant reductions in cell proliferation and induced cell cycle arrest in all GBM cell lines. Interestingly, three out of the four cell lines (F98, U-87 MG and U-251 MG) were resistant to JA239 treatment under hypoxic conditions. To better explore the effect of the tumour microenvironment on the drug sensitivity, we cultured F98 and U-87 MG cells as 3D spheroids and tested the effects of JAS239 in these models. We found JAS239 induced antiinvasive responses, however these responses varied on parameters assessed and overall conclusive statements about the cell phenotypes based on these data were difficult to draw. We then used in vivo orthotopic F98 and 9L GBM models and observed a ~50% tumour growth arrest in JAS239 treated animals. In vivo MR spectra from the tumour regions of F98 and 9L tumours demonstrated a trend of reduction in total choline (tCho) metabolite levels. Similarly, we observed a significant reduction in tCho:NAA ratio in 9L tumours (*p