Interrogating drug action and immune response in organotypic glioma microenvironment

2068 Background: The glioma tumor microenvironment is characteristic of a high level of heterogeneity and a profoundly immune suppressive phenotype as driven by the presence of glioma stem cells and abundant innate immune cells of the myeloid lineages, rendering glioblastomas (GBM) recalcitrant to conventional treatments and immunotherapies. To address the full complexity of the brain tumor with its diverse components, we have developed an organotypic slice culture system to overcome several limitations of the other preclinical models and allow interrogation of drug action and immunomodulatory agents in human GBM ex vivo. Methods: We performed extensive characterization of the organotypic glioma slice system and optimization of the ex vivo tissue culture conditions, and determined the longitudinal dynamics of slice cell viability, relative tumor versus immune cellularity, and responses to a selection of therapeutic agents and cytokines by multiplexed flow cytometry and single cell transcriptome (scRNA seq) profiling. Results: Single cell transcriptomic analysis showed that both tumor and immune cell heterogeneity were preserved ex vivo for the two-week observation time, which exhibited tumor-type specific gene expression profiles suggesting cooperative interactions among cell populations. Consistent with the findings in fresh tumor specimens and an immune suppressive microenvironment, we found a scarcity of T lymphocytes in IDH mutant gliomas, whereas abundant T cells were found in recurrent GBM slices that persisted ex vivo. Additional analyses revealed that T cell infiltration in GBM was associated with the mesenchymal-like molecular phenotype, which exhibited remarkable intratumor heterogeneity highlighting the presence of stem-like cells and a unique cell population expressing aberrant cancer genes and antigen presenting molecules. We further found that T cells in organotypic GBM slices acquired a potent effector phenotype in response to cytokine-induced reprogramming of microglia and tumor-associated macrophages. In addition, scRNA seq also revealed a population of cancer-associated fibroblast-like cells that are present in all tumors sequenced, which may function to mediate the interactions between immune and tumor cells as our data suggested. Conclusions: We demonstrated that the organotypic slice culture system we developed is compatible with genomic, pharmacological, and immunotherapeutic manipulations and provided data showing that organotypic slice culture preserves glioma microenvironment ex vivo allowing acquisition of crucial information of response of heterogeneous patient tumor tissue to the agent tested. This method represents a unique mechanism by which therapy development can be facilitated in an ex vivo setting.