Modelling glioblastoma migration with patient-derived cells, human iPSC-derived cortical neural spheroid, and high-content imaging

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Background: Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour in adults. Despite current advances, the existing standard of treatment is ineffective, and the survival prognosis remains just over a year following diagnosis. Migrating tumour cells have been implicated in the therapeutic resistance of GBM. They disperse by using structures such as white matter tracts and inevitably cause the recurrence of the tumour. Bulk tumour sequencing has indicated inter- and intra-tumour heterogeneity which includes the proneural, classical, and mesenchymal subtypes. These classifications with distinct molecular signature correlate with patient prognoses, and as such, this disease will benefit from personalised therapeutic approaches. Valuable cell models able to capture the invasiveness of GBM are critically needed to develop innovative therapies targeting migrating GBM cells. This project aims to develop an in vitro model mimicking the GBM microenvironment and investigate the GBM cells migration on axons.

Methods: We established an in vitro model mimicking the GBM microenvironment by co-culturing patient-derived GBM cells and human-induced pluripotent stem cell-derived cortical neural spheroids with radiating axons. Patient-derived GBM1 and GBM20, established as described in Wurdak et al. in 2010 and Polson et al. in 2018, maintained their stem cell-like characteristics as well as the molecular signature of the primary tumour of a classical/proneural subtype and a secondary mesenchymal subtype they are respectively derived from. Using HCI, we developed a robust pipeline to quantify the GBM cell infiltration of the neural spheroid in endpoint assays. Images were acquired on the Operetta CLS High-Content Imaging (HCI) system and analysed using the built-in Harmony Imaging and Analysis Software. We also performed live-imaging assays using the label-free HCI system called Phasefocus Livecyte, in which we studied the directionality, displacement, and speed of the GBM cells engaged on axons.

Results: Our data indicated that GBM cells changed morphology when cultured on axons. The live assays demonstrated that patient-derived GBM1 and GBM20 cells as well as non-cancer neural stem cell NS17 line migrated towards the neural spheroid. However, the endpoint assays showed a significant increase in infiltration of the neural spheroid with the GBM1 and GBM20 cells compared to NS17 cells. Finally, we used this model to screen for several inhibitors to pathways involved in the migration of GBM cells and found promising targets, PF 573228 (FAK inhibitor) and Motixafortide (CXCL12 inhibitor), which significantly decreased GBM20 and GBM1 infiltration of the neural spheroid respectively.

Conclusion: By modelling the GBM microenvironment with the ability of GBM cells to infiltrate the neural spheroid and to screen for compounds affecting cell migration, we have facilitated the investigation of GBM migration on axons ex vivo. The deliverable of this project is expected to bridge the gap between in vitro and in vivo drug screening, reducing the use of animal models, study time, and costs as well as potentially offer innovative precision-medicine therapies.
Date of Award1 Jul 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorDavide Danovi (Supervisor) & Ivo Lieberam (Supervisor)

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