Abstract
Glioblastoma therapeutic challenges are in considerable part due to myriad survival mechanisms which allow malignant cells to repurpose signalling pathways within discreet microenvironments. These Darwinian adaptations facilitate invasion into brain parenchyma and perivascular space. We hypothesised that pre-clinical modelling of glioma invasion by recapitulating early events occurring immediately after surgery at the glioblastoma invasive margin, could reveal the cross-talk between malignant cells and surrounding healthy astrocytes. We first generated transgenic H1-derived neural stem cells using CRISPR/Cas9-mediated knock-in of the YFP reporter gene under the control of the GFAP promoter at the AAVS1 safe harbour locus. Reproducible ultrahigh-throughput AggreWells™ (7200 mini-wells per plate) were used to create astrocyte-glioblastoma organoids, which we term ‘Gliomasphere Matrices’. YFP-labelled astrocytes were co-cultured with 10 treatment-naïve patient-derived cell lines isolated from the 5-aminolevulinic (5ALA)-determined glioblastoma invasive margin. Co-cultures were seeded upon a sequentially constructed, time-of-flight secondary ion mass spectrometry (ToF-SIMS)-characterised decellularised human brain extract. YFP-astrocytes were purified from each of the 10 Gliomasphere Matrices using fluorescence-activated cell sorting (FACS) after 6- and 10-days co-culture. RNA-sequencing of the putatively reprogrammed YFP-astrocytes showed the characteristic expression of canonical key regulators of multiple malignant diseases including high-grade glioma such as SND1 and EFNB2 in addition to the identification of a single novel marker located at chromosome 1 (C1orf61), highly expressed in malignant glioma when compared to somatic cancers according to TCGA RNA-sequencing data. Differentiated YFP-astrocytes also overexpressed IFITM2 and IFITM10, known to be involved in priming resistance against pathogenic microorganisms. This ultimately suggests a fluctuating state between malignant transformation imposed by the highly infiltrative glioma cells and the counter-action of the normal astrocytes to these deleterious invasive cells. This multi-faceted model offers a unique opportunity to recapitulate early molecular cross-talk which facilitates glioblastoma recurrence and may be utilised for high-throughput drug screening.