Metabolism based isolation of invasive glioblastoma cells with specific gene signatures and tumorigenic potential

Smith, Stuart James; Rowlinson, Jonathan; Estevez-Cebrero, Maria; Onion, David; Ritchie, Alison; Clarke, Phil; Wood, Katie; Diksin, Mohammed; Lourdusamy, Anbarasu; Grundy, Richard Guy; Rahman, Ruman

Abstract

Background
Glioblastoma (GBM) is a highly aggressive brain tumor with rapid subclonal diversification, harboring molecular abnormalities that vary temporo-spatially, a contributor to therapy resistance. Fluorescence guided neurosurgical resection utilizes administration of 5-aminolevulinic acid (5ALA) generating individually fluorescent tumor cells within a background population of non-neoplastic cells in the invasive tumor region. The aim of the study was to specifically isolate and interrogate the invasive GBM cell population using a novel 5ALA based method.

Methods
We have isolated the critical invasive GBM cell population by developing 5ALA-based metabolic fluorescence activated cell sorting. This allows purification and study of invasive cells from GBM without an overwhelming background “normal brain” signal to confound data. The population was studied using RNAseq, rtPCR and immunohistochemistry, with gene targets functionally interrogated on proliferation and migration assays using siRNA knockdown and known drug inhibitors.

Results
RNAseq analysis identifies specific genes such as SERPINE1 which is highly expressed in invasive GBM cells but at low levels in the surrounding normal brain parenchyma. siRNA knockdown and pharmacological inhibition with specific inhibitors of SERPINE1 reduced the capacity of GBM cells to invade in an in vitro assay. Rodent xenografts of 5ALA positive cells were established and serially transplanted, confirming tumorigenicity of the fluorescent patient derived cells but not the 5ALA negative cells.

Conclusions
Identification of unique molecular features in the invasive GBM population offer hope for developing more efficacious targeted therapies compared to targeting the tumor core and for isolating tumor sub-populations based upon intrinsic metabolic properties.

Citation

Smith, S. J., Rowlinson, J., Estevez-Cebrero, M., Onion, D., Ritchie, A., Clarke, P., Wood, K., Diksin, M., Lourdusamy, A., Grundy, R. G., & Rahman, R. (2020). Metabolism based isolation of invasive glioblastoma cells with specific gene signatures and tumorigenic potential. Neuro-Oncology Advances, 2(1), Article vdaa087. https://doi.org/10.1093/noajnl/vdaa087

• Journal Article Type: Article
• Acceptance Date: Jun 16, 2020
• Online Publication Date: Jul 13, 2020
• Publication Date: Sep 1, 2020
• Deposit Date: Jul 31, 2020
• Publicly Available Date: Aug 3, 2020
• Journal: Neuro-Oncology Advances
• Electronic ISSN: 2632-2498
• Publisher: Oxford University Press (OUP)
• Peer Reviewed: Peer Reviewed
• Volume: 2
• Issue: 1
• Article Number: vdaa087
• DOI: https://doi.org/10.1093/noajnl/vdaa087
• Keywords: Glioblastoma; 5-aminolevulinic acid; Neurosurgery; Gene expression; Heterogeneity
• Public URL: https://nottingham-repository.worktribe.com/output/4798559
• Publisher URL: https://academic.oup.com/noa/article/doi/10.1093/noajnl/vdaa087/5870782