Characterising glioma associated macrophages and microglia in glioblastoma

Abstract

Glioblastoma (GBM) is the most common and most aggressive primary brain cancer. Multimodality treatment in the form of safe maximal surgical resection together with radiotherapy and chemotherapy aim to treat GBM. Inevitably treatment has been shown to fail with reported median survival rates of 14.6 months and limited prognostic improvements. Emerging targets for therapy are focused on modulating immune cells. In particular, glioma associated macrophages and microglia (GAMMs) pose an attractive target due to the high proportional density within the tumour mass. GAMMs consist of two myeloid populations; central brain microglia and peripheral bone marrow derived macrophages. Recent evidence has explained the ontogenetic differences between these two immune cells, suggesting functional differences in GBM. However, their individual roles remain unknown due to limitations in chimeric models (irradiation) and cell surface markers (CD11b/CD45) used to track and identify these two populations. We developed our own chimeric model to address the issue of identifying microglia and macrophages using non-myeloablative busulfan pre-conditioning. Our model achieved high chimerism (>90% bone marrow), maintained homeostasis and retained the integrity of the blood-brain barrier. In doing so we highlighted in a syngeneic intracranial GBM mouse model (GL-261) a four-marker set (Ly6C/MHCII/MerTK/CD64) that reliably analysed GAMM subpopulations. The second part of this project used the ‘gold standard’ head-shielded irradiation chimeric tool typically used to investigate GAMMs as a comparison against the non-myeloablative transplant used throughout this project. As expected, mice pre-conditioned with head-shielded irradiation showed higher expression of pro-inflammatory cytokines in the peripheral blood, however in the tumour bearing hemisphere a higher propensity of macrophage recruitment was evident, which indicated that irradiation subjects mice to a non-homeostatic peripheral immune responses leading to biased conclusions and unrepresentative immune cells within the brain. After validating our chimeric model to investigate GAMMs we investigated the GAMM-glioma immune microenvironment using a timecourse study, functional transgenic knockouts and transcriptional evaluation of GAMM subpopulations. Results highlighted an array of immune cells that were deemed significant in their proportional contribution, which included: eosinophils, T and B-cells and GAMMs. We used transgenic knockout mice of each immune cell and demonstrated a worse prognosis when CCL2/CCR2 was deficient. In contrast to previous studies, mouse chimeras with CCR2-/- bone marrow (reduced macrophage trafficking), but normal brain resulted in a poor outcome in mice and indicated an important role of peripheral macrophages to treat GBM. RNA-sequencing (RNA-seq) was performed to determine the transcriptional profile of peripheral tumour macrophages and tumour microglia. The expression profile of both these populations differed, with peripheral macrophages expressing both primitive and mixed anti and pro-inflammatory genes, while tumour microglia expressed only an interferon response relative to normal microglia. The primitive expression profile of peripheral tumour macrophages and the limited pro-inflammatory response of tumour microglia indicated that peripheral tumour macrophages display potential features for immunomodulation to target GBM. However, further investigation is warranted to clarify their roles using transgenic knockouts and human tissue. Overall, this thesis provides an initial insight into the GAMM-glioma microenvironment and the necessary stepping-stone to develop future therapies that would aim to target GBM cells and improve survival for patients suffering with this devastating disease.

Date of Award	31 Dec 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Kamaly-Asl (Supervisor) & Brian Bigger (Supervisor)