Evaluation of ONC201 Treatment Response and Tumor Immune-microenvironment using Longitudinal Biopsy Procedure in Murine Models of Diffuse Midline Glioma

Introduction: Diffuse Midline Glioma (DMG) is an inoperable pediatric pathology with 9-12 month average survival. Novel therapeutic agents appear promising in clinical trials, including ONC201, a blood brain barrier (BBB) permeable selective ClpP antagonist. Preclinical in vivo studies often depend on molecular and cellular analysis at necropsy. Longitudinal biopsy of supratentorial tumors in mice has been performed, however not applied to brainstem lesions. Real-time analysis of tumor microenvironment during therapy could offer insight to drug mechanism of action, synergism, and cellular and molecular treatment response.

Methods: A minimally-invasive biopsy system was adapted to a stereotactic frame. By testing increasing suction, a non-survival tumor-naïve mouse cohort established mechanical parameters for adequate biopsy with minimal tissue damage. A survival tumor-naïve group was separated into sham, low and high suction, with pontine biopsies at 2, 4 and 8 weeks. Sample analysis included flow cytometry and immunohistochemistry.
Ongoing Research: Subsequent survival cohort will undergo stereotactic implantation of isogenic DMG cells. Tumor growth will be tracked with bioluminescence, and mice randomized to control or ONC201. Biopsies will be performed at equivalent time-points, and weight and survival followed. Cells will be analyzed with flow-cytometry and single cell RNA sequencing for treatment response and immune cell infiltration.

Results: 10 mice were biopsied and suction level correlated with cavitation, all achieving sufficient cell count. All biopsies were intrapontine. Of the 10 tumor-naïve mice that underwent serial biopsies, two mice (high suction) were sacrificed for severe head tilt. Flow cytometry and immunohistochemistry results are pending. Longitudinal biopsy in ONC201 tumor treatment group is ongoing.

Conclusion : We have demonstrated the feasibility of longitudinal biopsy in the murine pons. An adequate amount of viable cells are attainable at multiple time points using the lowest suction level. Further real-time analysis of DMG microenvironment during ONC201 treatment is planned, and can provide valuable cellular and molecular data.