P08.67 Defining brain microglia versus peripheral macrophages in a mouse model of glioblastoma using a non-myeloablative conditioning regimen

Central Nervous System (CNS) diseases and lesions are characterised by significant immune cell infiltration. In Glioblastoma Multiforme (GBM), a primary brain cancer, these Tumor Associated Macrophages and microglia (TAMM) may have pro-tumorigenic functions and play a key role in attenuating the host response to glioma. These cells can originate from the CNS itself as microglia or from the peripheral haematopoietic system as macrophages. Techniques currently used to separate the two populations in mice have used partially chimeric transplantation models or head shielded irradiation conditioning, however irradiation in particular leads to confounding pro-inflammatory effects in the peripheral environment. Furthermore, the reliability of using the cell surface markers CD45 high/low coupled with CD11b to distinguish central and peripheral populations has not been rigorously evaluated within the tumour microenvironment using non-irradiative methods. To circumvent these difficulties, we developed a Non-Myeloablative Conditioning (NMC) transplant regimen using low dose busulfan, achieving high CD45.1 donor marked peripheral blood haematopoietic chimerism (93%), without brain chimerism or blood brain barrier damage, thus maintaining CD45.2 marked homeostasis of the resident microglial population in the brain. Subsequent intracranial orthotopic implantation of a syngeneic mouse glioma line and whole brain flow cytometry, allowed us to characterise the proportion and type of peripheral immune cells infiltrating tumours. We observed that the proportion of central or peripheral TAMMs does not change significantly over time once the tumour is initially established. We also demonstrate that CD45/ CD11b, unreliably differentiates between central and peripheral TAMMs in the tumour environment, while the markers CD11b, Ly6C, MHCII, CD64 and MerTK, reliably differentiates subpopulations within peripheral derived TAMMs, (inflammatory monocytes, undifferentiated macrophages and macrophages) and central TAMMs, consisting of microglia, both in the NMC model and in un-transplanted mice with >95% purity. The combination of these techniques provides greater distinction of intra-tumoral myeloid cell subsets than previously described marker sets. The NMC transplantation model is simple and effective way of performing transplantation studies, and a powerful method of investigating immune cell interactions in CNS diseases, where a lack of perturbation of the system is important