Stimulation of Glioma-Associated Microglia/Macrophages effector phagocytic synapse towards tumor clearance in glioma.

Abstract

Glioblastoma (GBM) is the most aggressive form of glioma and currently has no cure. Given that around 30% of the cells within the tumor are microglia/macrophages (glioma- associated microglia/macrophages or GAMM from now on), we decided to study them in order to shed light in possible future immunotherapies. The first discovery during this investigation was the presence of GAMM in pseudopalisades (PPs) of human GBM. These structures are thought to be very important in the contribution of the tumor invasiveness, therefore the knowledge of the role of GAMMs here might be crucial. Particularly, GAMMs were found to be traveling through the PPs towards the necrotic focus, contrasting with the tumor cells. Moreover, the myeloid cells seem to gain cellular persistence with the hypoxic gradient and travel in a haptotactic manner using the gradient of glioma cells as a cue. When they reach the necrotic focus, they shift their phenotype and phagocytose tumor material, including GFAP+ fragments and nuclei. Secondly, by means of cell cultures we achieved to translocate p65 NF-κB and promote phagocytosis of tumor glioma cells (C6) by primary microglia. Moreover, using cell lines (BV-2 and GL261) we described the putative steps of phagocytosis and the distribution of some receptors (CD11b and CD16/32) involved in the process of phagocytosis. Importantly, the distribution of Iba-1 in interacting GAMMs was also defined in the animal models. Finally, we tested two immunotherapy strategies in a immunocompetent GBM animal model (C57/BL6 intracraneally inoculated with GL261 cells), and discovered that both immunotherapies have different outcomes: while CD47 neutralizing antibody seemed to be non-effective, neutralizing SIRP1α had a beneficial outcome. This way, anti-CD47 treated animals did not have any increase on survival rate than control groups; and they showed decreased bodyweight throughout the experiment, suggesting that the therapy had some systemic side effects. On the other hand, blocking SIRP1α allowed the increase of the bodyweight of the animals throughout the experiment, and decreased the cellularity of the tumor core by increasing the phagocytic activity of GAMM at the peripheral area of tumor invasion without interfering in their infiltration capacity. In all, this thesis contributes to a better understanding of the role of GAMMs in GBM and the intrinsic phagocytic capacity they can play, possibly helping in the development of immunotherapeutic tools to fight this fatal tumor.

Date of Award	26 Jul 2019
Original language	English
Supervisor	Jose Aguilera Avila (Tutor) & Carlos Barcia Gonzalez (Director)