Different Driver Mutations Influence the Composition of the Immune Tumor Microenvironment During Glioma Progression

Background: 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. Though the most number of infiltrating cells in the tumor are peripheral macrophages and microglia, recent appreciation of 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 specific oncogenic drivers, tumor was harvested at different time-points from different subtypes of GBM and also tumors arising from separate cell-of-origin, dissociated and enriched for CD45+ cells. The samples were processed for flow cytometry, qPCR and in-vitro studies. Neutrophils were isolated via gradient centrifugation and utilized for functional assays; migration, flow cytometry, neutrophil activation, co-culture etc.

Results: Flow cytometry analysis shows differences in the brain TME of both the innate and adaptive immune populations compared to healthy brain tissue, changes were also seen in spleen and bone marrow even at early stages of GBM development. There was also difference in expression levels of NFkB targets in CD45+ cells between Mesenchymal (MES) and Proneural (PN) subtypes. The neutrophil population varies not only at various time-points but also between the tumor types. In-vitro assay showed higher migration and formation of neutrophils extracellular traps (NETs) on exposure to glioma cells condition media. RT-qPCR showed differences in NFkB targets between WT and PN GBM bone-marrow derived neutrophils.

Conclusion: This novel GBM mouse model allows us to reveal the TME immune cell population from initiation to endpoint of the disease and compare between different GBM types. The dynamic changes in the TME and the differences in the expression levels of NFkB targets suggests at cross-talk. Preliminary results hints at neutrophils role as pro-tumorigenic.