An Automated Platform for Creating Patient-Derived Glioblastoma Organoids and High Throughput Drug Screening

Glioblastoma (GBM) is the most prevalent primary intrinsic brain tumor and amongst most lethal forms of cancer with a median survival of 14.6 months. GBM tumors exhibit vast inter- tumoral and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Studies suggest the presence of self-renewing, tumor-propagating cancer stem cells (CSCs) in GBM tumors plays a crucial role in the resistance to conventional therapies by multiple mechanisms. We present a method for creating vascularized multi-zonal patient-derived GBM organoids by precise robotic deposition. Precise deposition of CSCs and endothelial cells in a confined microenvironment creates a complex tumoral heterogeneity and vascularization. These patient-derived GBM organoids self-organize and allow three-dimensional cellular interactions, unveiling great cellular diversity and spatially defined cell phenotypes, that are not seen in typical spheroid cultures. The method also allows the incorporation of oxygen micro-sensors in the tumor organoids in high content screening 384-well setups with minimal variability. We show that sensor-embedded patient-derived organoids can be maintained and kinetically monitored for several days under controlled physiological conditions, allowing treatment-relevant high throughput screening. Network analysis of the assay’s responses reveals several novel pathways that shed light on basic mechanisms underlying the glioma stem cell responses to anti-cancer treatment and enabled us to delve into new targets that could specifically damage CSCs. Our platform offers a new approach of kinetic high content drug development for complex tumors, providing valuable information for both novel drugs and repurposing attempts.