Single-cell molecular profiling using ex vivo functional readouts fuels precision oncology in glioblastoma

Abstract

Background: Functional profiling of freshly isolated tumor cells is being evaluated as a next-generation method for precision oncology. While promising, its success largely depends on the method to evaluate treatment activity which requires sufficient resolution and specificity. Methods: Here, we describe the PRecision oncology by Single-cell Profiling using Ex vivo ReadOuts of functionality (PROSPERO) assay to evaluate the intrinsic susceptibility of high-grade brain tumor cells to respond to therapy. Different from other assays, PROSPERO extends beyond life/death screening by evaluating acute molecular drug responses at single-cell resolution. Results: This was achieved by correlating long-term cytotoxicity readouts in representative patient-derived glioblastoma cell cultures (PDCLs, n=14) to radiotherapy and the small-molecule p53/MDM2 inhibitor AMG232, to short-term single-cell molecular signatures using CyTOF. The predictive model was subsequently projected to evaluate drug activity in fresh samples from patients (n=34). Here, PROSPERO revealed an overall limited capacity of tumor cells to respond to therapy, as reflected by the inability to induce key molecular markers upon ex vivo treatment exposure, while retaining proliferative capacity, insights that were validated in PDX models (n=8). This approach also allowed to investigate cellular plasticity: in PDCLs we observed therapy-induced proneural-to-mesenchymal transitions, while in patients samples this was more heterogeneous. Conclusion: PROSPERO provides a precise way to evaluate therapy efficacy by measuring molecular drug responses using specific biomarker changes, in addition to providing key functional insights in cellular behavior, which may ultimately complement standard, clinical biomarker evaluations.