Fig 2: Genetic inhibition of mTORC1 signalling in TAM affects the immune profile of GL261 tumours Significantly deregulated canonical pathways in Cx3cr1-Rheb1 ?/? versus Rheb1 fl/fl GL261 tumours, as identified by the IPA software from differentially regulated genes and divided into three categories: adaptive immunity, cytokine signalling and innate immunity. Size of bubbles is indicative of ratio of differentially regulated genes. The x-axis represents -log10 (P-value) with a threshold of P = 0.05 applied.Levels of TAM-MG (P2RY12+ CD49d-) and TAM-BMDM (P2RY12- CD49d+) gated from CD45+ population in GL261 tumours from Rheb1 fl/fl (n = 4) and Cx3cr1-Rheb1 ?/? (n = 5) mice by flow cytometry (representative flow cytometry plots on top and quantification at the bottom; mean ± SEM; two-way ANOVA Tukey test).Percentage of CD8 CTL (top) and CD4 Th (CD4) cells in GL261 tumours from Rheb1 fl/fl (n = 4) and Cx3cr1-Rheb1 ?/? (n = 5) mice assessed by flow cytometry (representative flow cytometry plots, left and quantification, right; mean ± SEM; unpaired t-test).Tumour tissues from Rheb1 fl/fl (n = 4) and Cx3cr1-Rheb1 ?/? (n = 4) were stained for CD8, CD4 and FoxP3. DAB staining was quantified as % of positive cells using Definiens software. Representative images of the stained tissues are shown (mean ± SEM; unpaired parametric t-test). Scale bar is 100 µm.Data information: *P = 0.05 and **P = 0.01.

Fig 3: STAT3 and NF-?B signalling are regulated by mTOR activity in TAM-MG in human glioblastoma Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM-MG or TAM-BMDM signatures in Gravendeel-GBM transcriptomic data. Comparison carried out on all IDH-wild-type samples and in a subgroup-specific manner according to Wang's classifier. Size of circle is indicative of R-square value and bold outline represents a P = 0.05.Representative images of differentiation protocol of EPSC into EBS, YS-EBs and iMGL. Scale bar is 50 µm.Representative flow cytometry plots for changes in expression of surface markers during differentiation of EPSC to iMGL. Histogram of expression of SOX2 (stem cell marker), PU.1 (early yolk sac marker), P2RY12 (microglia-specific marker) and CD49d (macrophage specific marker) by EPSC, YS-EBs and iMGL are shown.Representative flow cytometry plots of expression of p-S6, p-STAT3 and p-NF-?B (p-P65) in iMGL following 4 h of incubation with different culture conditions.

Fig 4: Cx3cr1-Rheb1 ?/? mice display inhibition of mTORC1 signalling in GL261 TAM Representative flow cytometry plot for the expression of CD45 and CD11b in GL261 tumour, with CD45high CD11b+ TAM-BMDM (top gate) and CD45low CD11b+ TAM-MG (lower gate).Representative flow cytometry plot of YFP (left) and p-S6 (right) levels in TAM-BMDM (top) and TAM-MG (bottom) in Cx3cr1-Rheb1 ?/? (blue) versus Rheb1 fl/fl (red) GL261 tumours.Percentage of TAM-MG and TAM-BMDM expressing YFP in Cx3cr1-Rheb1 ?/? (n = 6) and Rheb1 fl/fl (n = 6) mice. MFI levels of p-S6 in P2RY12+ CD49d- TAM-MG and P2RY12- CD49d+ TAM-BMDM in Rheb1 fl/fl (n = 6) compared to Cx3cr1-Rheb1 ?/? (n = 6) GL261 tumours (mean ± SEM; two-way ANOVA Tukey test).Staining for Iba1, p-S6 and DAPI in Rheb1 fl/fl (n = 3) and Cx3cr1-Rheb1 ?/? (n = 2) tumour tissue (top) and percentage of Iba1+ cells co-expressing p-S6 in the three defined regions (bottom) (mean ± SEM; two-way ANOVA Tukey test). Scale bar is 100 µm.Isotype control stain demonstrating quenching of fluorescence from reporter gene (scale bar 50 µm).Data information: *P = 0.05, **P = 0.01, ***P = 0.001, ns non-significant.