Immune System-Related Changes in Preclinical GL261 Glioblastoma under TMZ Treatment: Explaining MRSI-Based Nosological Imaging Findings with RT-PCR Analyses

SIMPLE SUMMARY: Glioblastoma (GB) is an aggressive brain tumour with poor survival. Tumour microenvironment is a key element in GB evolution and response to therapy. We assessed presence and phenotypes of microglia/macrophages in preclinical GL261-GB microenvironment under Temozolomide (TMZ) treatment to unveil its possible relationship with MRSI-detected metabolomics changes. Microglia/macrophage polarisation towards an anti-tumour phenotype prevailed in TMZ-treated tumours. Since microglia/macrophages can represent 30–40% of the GB tumour volume, they must contribute the metabolomic pattern change. PD-L1 expression also correlated with the anti-tumour microglia/macrophage phenotype. These results highlight the potential of MRSI-detected metabolomics as non-invasive biomarker for immune system action. ABSTRACT: Glioblastomas (GB) are brain tumours with poor prognosis even after aggressive therapy. Previous work suggests that magnetic resonance spectroscopic imaging (MRSI) could act as a biomarker of efficient immune system attack onto GB, presenting oscillatory changes. Glioma-associated microglia/macrophages (GAMs) constitute the most abundant non-tumour cell type within the GB and can be polarised into anti-tumour (M1) or pro-tumour (M2) phenotypes. One of the mechanisms to mediate immunosuppression in brain tumours is the interaction between programmed cell death-1 ligand 1 (PD-L1) and programmed cell death-1 receptor (PD-1). We evaluated the subpopulations of GAMs in responding and control GB tumours to correlate PD-L1 expression to GAM polarisation in order to explain/validate MRSI-detected findings. Mice were evaluated by MRI/MRSI to assess the extent of response to treatment and with qPCR for GAMs M1 and M2 polarisation analyses. M1/M2 ratios and PD-L1 expression were higher in treated compared to control tumours. Furthermore, PD-L1 expression was positively correlated with the M1/M2 ratio. The oscillatory change in the GAMs prevailing population could be one of the key causes for the differential MRSI-detected pattern, allowing this to act as immune system activity biomarker in future work.