Tumour-associated microglia/macrophage heterogeneity in gliobastoma. (Doctoral thesis)

Glioblastoma (GBM) is the most common and aggressive primary brain tumour in adults, characterized by high degrees of both inter- and intra-tumour heterogeneity. GBM cells secrete numerous factors promoting the recruitment and infiltration of cellular players to the local tumour microenvironment. Tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment in GBM playing important roles along tumour development. Along GBM progression, these cells are supposed to be geared towards a tumour-supportive phenotype, therefore TAMs are pursued as key targets for the development of novel strategies aimed at re-educating them towards anti-tumour phenotypes. However, it is yet unclear how these immune suppressive properties are acquired and whether TAM subsets may phenotypically and functionally differently contribute to tumour development. Hence, the main goal of the present PhD project was to elucidate TAM diversity under defined temporal and spatial settings in GBM. Taking advantage of the GBM GL261 syngeneic and patient-derived orthotopic xenograft mouse models, we comprehensively studied the cellular and transcriptional heterogeneity of TAMs by combining single-cell RNA-sequencing, multicolour flow cytometry, immunohistological and functional analyses. We demonstrated that, as observed in patients, the myeloid compartment is the most affected and heterogeneous stromal compartment, with microglia and macrophage-like cells acquiring key transcriptional differences and rapidly adapting along GBM progression. Specifically, we uncovered that TAM transcriptional programmes converge over time, suggesting a context-dependent symbiosis mechanism characterized by decreased antigen-presenting cell signatures at late tumour stages. In the absence of Acod1/Irg1, a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype, we detected higher TAM diversity in the TME displaying increased immunogenicity and correlating with increased lymphocytic recruitment to the tumour site. Additionally, we uncovered that TAMs exhibit niche-specific functional adaptations in the tumour microenvironment, with microglia in the invasive landscapes displaying higher immune reactive profiles when compared to the corresponding cells in the angiogenic tumour phenotypes. Taken together, our data provide insights into the spatial and molecular heterogeneity of TAMs dynamically adapting along tumour progression or across specific tumour sites and revealing potential reactive anti-tumorigenic cell subsets that may be harnessed for therapeutic intervention in GBM.