PET imaging and single-cell RNA sequencing glioma microenvironment assessment after RT combined therapy

Introduction Glioblastoma (GBM) is the most common primary brain tumour in adults with a median survival of 11-12 months. Standard therapy consists of radiotherapy (RT) plus chemotherapy with temozolomide (TMZ), and no alternative treatment is available. Using multimodal imaging and single-cell RNA-sequencing (scRNA-seq), we investigated the role of RT associated with TMZ and metformin (MET) in tumour response in a syngenic murine GBM model. Methods GL261 GBM cell line was orthotopically injected into C57Bl/6 mice. Animals were randomly divided into four groups: vehicle, RT alone, RT with TMZ, and RT with TMZ plus MET. The therapy response was monitored using Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) with [ 18 F]FLT (cell proliferation). Immunofluorescence, scRNA- seq analysis and PCR analysis were carried out on brain samples collected after 4 weeks of treatment. Results RT alone greatly increased survival (40.5 days) compared to vehicle (26.5 days), and when combined with drugs this effect was potentiated (148 days). At 4 weeks, the addition of TMZ alone or with MET significantly decreased tumour sizes visible at MRI and [ 18 F]FLT uptake compared to RT alone-treated mice. Through IF we observed modifications of different inflammatory markers and downregulation of IBA1 in the peri-tumoural area when RT was combined with drugs. RT alone decreased GFAP levels intra-tumourally. scRNA-seq revealed different clusters of cells including tumor cells, glioma associated macrophages and microglia, dendritic cells, natural killer and B and T lymphocytes. RT drugs combination reduced cluster signals more than RT alone. On the basis of cell cycle analysis cancer cells were classified in two sub-clusters: one in phase G2/M and the other in phase S that was more susceptible to RT alone. Globally, RT alone increased the inflammatory milieu as shown by the modification in TAM cluster composition. This response was attenuated by the combination of RT with TMZ or TMZ plus MET. Discussion In conclusion, RT alone or in association with drugs leads to changes in tumour response mediated by immune cells. Imaging and molecular analysis give complementary information to understand the complexity of tumour response to therapy.