Andrew Benson Satterlee

Children with subtotally resected pediatric low-grade glioma (pLGG) often face multiple lines of treatment, which are seldom capable of eliminating the entire tumor. Genomics-based biomarkers are often used to select targeted therapies, but this paradigm only yields overall response rates of ~50%. Functional precision medicine (FPM), where patient-specific therapeutic efficacy is evaluated by directly treating individuals’ tumor outside their body, can predict individualized drug responses for some cancers, but pLGG is notoriously difficult to maintain outside the body, limiting development of FPM for pLGG. We describe the first platform that can maintain, treat, and analyze zero-passage pLGG tumor tissue ex vivo , facilitating FPM testing. Here, we tested pLGG tumors on our previously validated Screening Live Cancer Explants (SLiCE) platform, which allows engraftment and testing of diverse CNS tumor types atop organotypic brain slice cultures (OBSCs). After ensuring reproducible engraftment and maintenance of all three living pLGG tumor tissues on SLiCE, we measured MAPK pathway response to targeted therapies via immunoblotting. In the tested BRAF KIAA1549 fusion+ tumor, Western blot demonstrated maintenance of expected paradoxical MAPK upregulation in response to dabrafenib treatment. We then measured tumor response to targeted therapies on SLiCE. As expected, none of the BRAF KIAA1549 fusion+ pLGG tumors were sensitive to dabrafenib treatment. Two out of the three tumors demonstrated predicted sensitivity to trametinib, whereas one tumor did not. While no clinical correlates were measured in this proof-of-concept study, this mixed response to MEK inhibition on SLiCE is representative of heterogeneous real-world clinical responses. Together, these data demonstrate the feasibility of SLiCE to become a new functional biomarker of response in a tumor type where functional models are exceptionally rare, establishing a foundation for future individualized treatment strategies.

Functional Precision Medicine platforms face significant obstacles to clinical translation, including stringent requirements for analytical consistency. In this study, we present an automated data analysis workflow for an organotypic brain slice culture–based functional assay that addresses these concerns by combining computer vision and dose–response modeling approaches. Automation reduces analysis time by approximately 99% compared to the original manual workflow (from ~ 20 h to ~ 15 min to analyze an 11-drug assay), increasing throughput capabilities and freeing researchers from tedious analyses. Comparison of automated measurements with previously published manual results revealed that automation increased consistency both within experiments and across replicate experiments by removing human subjectivities. Our workflow demonstrates how to implement computer vision using limited computational resources and minimal programming expertise by leveraging cloud-based, user-friendly ML tools (e.g., Biodock) integrated into local scripts. It also addresses challenges in automated modeling of datasets that include diverse dose–response behaviors, such as hormesis and plateau effects. Finally, we outline a validation strategy for automated analysis in the absence of gold-standard measurements, an issue common in functional assays where manual measurements involve user subjectivity.

Functional precision medicine (FPM) offers a promising path forward in neuro-oncology, where genomic profiling alone often fails to predict therapeutic response. To bridge this gap, we developed the Screening Live Cancer Explants (SLiCE) platform, a rapid ex vivo drug screening assay that functionally tests passage-zero patient brain tumor tissues engrafted atop living organotypic brain slice cultures (OBSCs). With an assay time of just four days, SLiCE preserves key tumor characteristics not maintained in vitro , including genomic fidelity, growth, invasion, and treatment response, with higher engraftment rates and faster assay speeds than in vivo models. Our standard cryopreservation workflow enables reproducible, iterative, and on-demand testing of a single zero-passage specimen banked in multiple replicate aliquots, setting SLiCE apart from organoid and precision-cut tumor explant models. Here, we describe results from our actively accruing clinical feasibility study (NCT05978557), where we successfully engrafted and tested 35 of 36 diverse brain tumor specimens on SLiCE, achieving our study’s primary endpoint ahead of schedule. SLiCE produced multi-parametric drug sensitivity scores (DSSs), each normalized to off-target toxicity, for all samples within a clinically actionable 28-day window. Across 530 experiments, we generated 142 DSSs from unique drug-tumor combinations, forming a reference library for future benchmarking. We then further analyzed a subset of IDH-WT glioblastoma tumor specimens in which SLiCE DSSs correlated with patient response to temozolomide (AUC = 0.875, p = 0.0175) and overall survival (R² = 0.73). Additionally, this study validated surgically aspirated tumor tissue as a genomically, transcriptomically, and functionally similar tumor source compared to the standard, manually excised remnant tumor sample approved by clinical pathology. Collecting this often-discarded tumor source increased the mass of tumors accrued by nearly 5-fold and enabled collection from 11 additional patients, significantly increasing tumor tissue for downstream testing on SLiCE. These findings establish SLiCE as a scalable, clinically relevant platform for FPM in brain cancer, with potential to guide individualized treatment decisions and accelerate preclinical drug development.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults, with incidence peaking in later life. It is commonly treated with surgery followed by administration of ionizing radiation and the DNA alkylating agent temozolomide (TMZ). Even though this regimen confers some progression-free survival, there is essentially no cure with the median survival with the standard of care being about 12 months. Currently, several alternative approaches are being developed to improve upon this outcome. We have already shown EdU alone effectively treats GBM, and we now study efficacy of TMZ+EdU combination therapy. TMZ+EdU significantly improves antitumor efficacy compared to either single-agent therapy against GBM cell lines in vitro , against three different orthotopic GBM xenograft models, and against passage-zero patient GBM tumor tissues engrafted within an organotypic brain slice culture (OBSC)-based platform. Together, these data suggest that EdU could be effective alongside standard of care TMZ in patients with GBM. STATEMENT OF SIGNIFICANCE (50-WORD): By simultaneously engaging distinct DNA repair pathways, combination of TMZ and EdU produced unprecedented survival benefits and synergistic effects in preclinical GBM models, offering a promising new avenue in the treatment of GBM.

Background: Children with subtotally resected pediatric low-grade glioma (pLGG) often face multiple lines of treatment, which are seldom capable of eliminating the entire tumor. Genomics-based biomarkers are often used to select targeted therapies, but this paradigm only yields overall response rates of ∼50% optimally. Functional precision medicine (FPM), where patient-specific therapeutic efficacy is evaluated by directly treating individuals’ tumor outside their body, can predict individualized drug responses for some cancers, but pLGG is notoriously difficult to maintain outside the body, limiting development of FPM for pLGG. Methods: We describe what is, to our knowledge, the first platform that can maintain, treat, and analyze zero-passage pLGG tumor tissue ex vivo , facilitating FPM testing. We engraft pLGG tumors onto a previously validated organotypic brain slice culture (OBSC) platform. After ensuring reproducible engraftment and maintenance of living pLGG tumor tissue on OBSCs, we measured MAPK pathway response to targeted therapies via immunoblotting. We then measured tumor ex vivo response to targeted therapies. Results: Each zero-passage pLGG tumor tissue specimen exhibited reproducible growth on the OBSC platform. Western blot demonstrated each BRAF KIAA1549 fusion+ tumor exhibited expected paradoxical MAPK upregulation to dabrafenib treatment. Two of three tumors demonstrated cytotoxicity from trametinib as predicted, whereas one tumor did not. No clinical correlates were measured in this proof-of-concept study, though this mixed response to MEK inhibition may be in line with real-world clinical responses. Conclusion: The OBSC platform supports ex vivo maintenance of passage-zero pLGG tumor tissue and enables personalized drug screening to yield a new functional biomarker of pLGG drug response.

Functional Precision Medicine platforms, which investigate the dynamic behavior of a patient’s tumor ex vivo to inform personalized treatment, face unique obstacles to clinical translation. These include limited access to patient tissue and stringent demands for intra-platform accuracy and consistency. In this study, an automated data analysis pipeline addresses these concerns for an organotypic brain slice culture-based functional assay by combining computer vision and dose-response modeling approaches. A 99% reduction in analysis time increases the amount of patient tissue that can be processed on the platform. Comparing automated measurements to previously published manual results revealed that automation increased consistency both within experiments and across replicate experiments. This pipeline also explores implementing complex CV with limited resources, modeling a unique and diverse dataset, and validating automated analysis when no gold standard measurements exist, obstacles that hinder automation efforts across scientific disciplines.

Figure S1: Illustration of the proposed futile cycle induced by the incorporation of EdU by DNA polymerases and removal of it by excision repair. EdU gets incorporated into the genome of highly proliferative GBM cells in the brain. Then, EdU-containing DNA is recognized and removed by nucleotide excision repair, leaving the DNA gap to be filled in. During the resynthesis, EdU reincorporates into this repair patch and the futile cycle repeats, leading to apoptosis and thus, suppression of the growth of GBM tumor.

Figure S2: Features of glioblastoma models used to assess therapeutic potential of EdU. The features in red are less than desirable, blue are moderately desirable, and green are ideal. As such, the compounds were tested on multiple cell culture platforms to supplement the weaknesses of individual platforms.