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Updated: Jul 30, 2025
Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq
Published on: August 25, 2020
Single-cell molecular profiling using ex vivo functional readouts fuels precision oncology in glioblastoma
Dena Panovska1, Pouya Nazari1, Basiel Cole1
1Department of Imaging and Pathology, KU Leuven, Herestraat 49, Box 1032, Leuven, Belgium.
Background:
Functional profiling of freshly isolated glioblastoma (GBM) 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 rapidly evaluating acute molecular drug responses at single-cell resolution.
Results:
The PROSPERO assay was developed by correlating short-term single-cell molecular signatures using mass cytometry by time-of-flight (CyTOF) to long-term cytotoxicity readouts in representative patient-derived glioblastoma cell cultures (n = 14) that were exposed to radiotherapy and the small-molecule p53/MDM2 inhibitor AMG232. The predictive model was subsequently projected to evaluate drug activity in freshly resected GBM 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 patient-derived xenograft (PDX) models. This approach also allowed the investigation of cellular plasticity, which in PDCLs highlighted therapy-induced proneural-to-mesenchymal (PMT) 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 freshly resected brain tumor samples, in addition to providing key functional insights in cellular behavior, which may ultimately complement standard, clinical biomarker evaluations.
Insights
The PROSPERO assay precisely measures molecular drug responses in brain tumors, revealing limited patient capacity to respond to therapy. This functional profiling aids precision oncology by assessing treatment efficacy at single-cell resolution.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Precision oncology for glioblastoma (GBM) requires robust methods to assess treatment efficacy.
- Current methods for evaluating treatment activity in GBM need higher resolution and specificity.
- Functional profiling of freshly isolated GBM cells is a promising next-generation approach.
Purpose of the Study:
- To introduce the PROSPERO (precision oncology by single-cell profiling using ex vivo readouts of functionality) assay.
- To evaluate the intrinsic susceptibility of high-grade brain tumor cells to therapeutic interventions.
- To rapidly assess acute molecular drug responses at single-cell resolution, moving beyond simple life/death screening.
Main Methods:
- Developed the PROSPERO assay by correlating short-term single-cell molecular signatures (mass cytometry by time-of-flight [CyTOF]) with long-term cytotoxicity in patient-derived glioblastoma cell cultures (n=14).
- Exposed cells to radiotherapy and the p53/MDM2 inhibitor AMG232.
- Validated the predictive model in freshly resected GBM samples (n=34) and patient-derived xenograft (PDX) models.
Main Results:
- PROSPERO revealed a limited capacity of glioblastoma cells to respond to therapy, indicated by a lack of key molecular marker induction and retained proliferative capacity.
- Ex vivo treatment exposure in patient samples showed limited therapeutic response.
- Investigated cellular plasticity, observing therapy-induced proneural-to-mesenchymal transitions in patient-derived cell lines (PDCLs) and heterogeneous responses in patient samples.
Conclusions:
- PROSPERO offers a precise method for evaluating therapy efficacy in brain tumors by measuring molecular drug responses via specific biomarker changes.
- The assay provides crucial functional insights into cellular behavior, complementing standard clinical biomarker evaluations.
- This approach enhances precision oncology by enabling functional profiling of freshly resected brain tumor samples.
