Oncogenic Drivers Shape the Glioma Immune Landscape

Dinorah Friedmann-Morvinski^1,3

Introduction: Glioblastoma (GBM) is an aggressive, highly invasive primary brain tumor with near total fatality. GBM remains a challenge for prognosis despite intensive therapies. Transduction by oncogenic lentiviral-vectors, irrespective of the initiating cell population, astrocytes, mature neurons or neural stem cells (NSCs) share a common stem-like cancer cell population that can originate from dedifferentiation of mature transformed cells in GBM tumors. We believe that the tumor microenvironment (TME) may contribute to the process of tumor reprogramming. Although most of the infiltrating cells in the tumor are peripheral macrophages and microglia, recent appreciation of the effects of neutrophils in cancer directed our efforts in understanding their role in GBM.

Material and methods: Transgenic-CRE mice were injected with lentivirus carrying different oncogenic drivers (GBM subtypes) or initiated in different cell-of-origin. Tumors were harvested at different time-points, dissociated and enriched for CD45+ cells. The samples were processed for flow cytometry analysis, qPCR and in-vitro studies. Neutrophils (NF) were isolated via gradient centrifugation and utilized for functional assays; migration, flow cytometry, neutrophil activation, and cytotoxic effect.

Results and discussion: Flow cytometry analysis revealed differences in the brain TME in both the innate and adaptive immune subpopulations compared to healthy brain tissue. Temporal changes were also observed in spleen and bone marrow even at early stages of GBM development. The NF population varies not only at various time-points but also between the tumor subtypes. In-vitro assays showed higher migration and formation of neutrophils extracellular traps (NETs) on exposure to glioma cells condition media. In vivo depletion of NF at an early time point resulted in faster tumor development, while depletion at a later stage did not have an effect on tumor development.

Conclusion: Our lentiviral GBM mouse model allowed us to reveal the longitudinal TME immune cell populations, from initiation to endpoint of the disease, and compare between different GBM subtypes (oncogenic drivers). The dynamic changes in the TME and the differences in the expression levels of NF-kB targets suggest a cross-talk between tumor cells and the TME. Preliminary results hint at neutrophils acquiring a pro-tumorigenic phenotype as tumor progresses.