Journal: bioRxiv

Article Title: PI3Kγ inhibition suppresses microglia/TAM accumulation in glioblastoma microenvironment to promote exceptional temozolomide response

doi: 10.1101/2020.05.14.097121

Figure Lengend Snippet: (A) Upper: Representative images of in vitro colony formation after co-culturing of freshly isolated mMG gl or mMG nb with murine GL261 glioblastoma cells. Bottom: Fold-change in colony forming units (CFU) with or without mMG gl or mMG nb co-culturing. (B) Effect of orthotopic co-implantation of mMG gl or mMG nb and GL261 on mice survival. *, p<0.05; **, p<0.0001. n = 10). (C) Effects of co-culturing of GL261 with mMG gl or mMG nb on TMZ resistance, assessed by colony formation assays. *, p<0.05. (D) Ten most abundant proteins identified through unbiased proteomic profiling of conditioned media (CM) derived from immortalized human microglia (hMG). p-value of association between mRNA expression of genes encoding these CM proteins and survival in the TCGA are shown under the TCGA column. To avoid multiple comparisons, only IL11 was tested for survival association using the CGGA. p-value for this association is shown under the CGGA column. (E) Effect of IL11 neutralizing antibodies on hMG CM induced neurosphere formation in SF767. *, p<0.05. ns: not significant. (F) Effect of recombinant human IL11 (20 ng/ml) on neurosphere forming capacity of SF767 cells. *, p<0.05. (G) Survival of mice bearing GL261 cells ectopically expressing IL11 compared to mince baring GL261 cells. n=10. p=0.0003. (H) Effect of ectopic IL11 expression on TMZ resistance of SF767 cells. In vitro cell viability was determined. *, p<0.05. n=6. Error bars, SD. (I) GFP-labelled GL261intracranial tumors were sorted into tumor cells (GFP + CD45 − ), TAM (GFP-CD45 high CD11b + Gr1 + ), and microglia (GFP-CD45 low CD11b + Gr1 − ). IL11 levels were examined by qRT-PCR, and the results were displayed as heat map. (J) IL11 levels in non-myeloid cells (Iba1 − CD68 − CD11b − ) and tumor associated microglia/myeloid cells (Iba1 + CD68 + CD11b + ) sorted from three glioblastoma specimens resected from human subjects. (K) Effect of microglia co-implantation and ectopic IL11 expression on the expression of the 82 survival-associated genes. RNA was extracted from subcutaneous tumors formed by SF767-vector, SF767-IL11, and SF767 cells co-implanted with microglia and gene expression was analyzed by qRT-PCR. ΔCt between gene and actin in each sample was plotted as heat map. Three independent tumors were analyzed for each cohort.

Article Snippet: The human microglia (hMG) cell line was purchased from Applied Biological Materials (ABM).

Techniques: In Vitro, Isolation, Derivative Assay, Expressing, Recombinant, Quantitative RT-PCR, Plasmid Preparation

Journal: bioRxiv

Article Title: PI3Kγ inhibition suppresses microglia/TAM accumulation in glioblastoma microenvironment to promote exceptional temozolomide response

doi: 10.1101/2020.05.14.097121

Figure Lengend Snippet: (A) Effect of treatment with the PI3Kγ inhibitor, TG100-115, on microglia density of tumors formed after orthotopically implant of GL261 cells. Left: Representative flow cytometric analysis. Right: Quantification of microglia density in GL261. n=6; *, p<0.05. (B) ELISA analysis of IL11 protein level in orthotopically implanted GL261 tumors with or without TG100-115 treatment. *, p<0.05. (C) Effect of TG100-115 treatment on survival of mice orthotopically implanted with GL261. n=10, p=0.0002. (D) Survival of PI3Kγ −/− mice after orthotopic co-implant of GL261 cells with the microglia isolated from PI3Kγ +/+ or PI3Kγ −/− mice. n=10. ***, p<0.0001; ns: not significant. (E) (Right) qRT-PCR analysis of IL11, MYC, OLIG2, SOX2, and POU3F2 in tumor cells, TAM, and microglia isolated from GL261 tumors that underwent TG100-115 treatment. Orthotopic tumors were resected after treatment and sorted as shown in the left panel. Three independent tumors were analyzed for the treated and untreated cohorts. (F) ELISA analysis of IL levels in CM of TG100-115 treated immortalized human microglia (hMG). (G) Survival of mice orthotopically implanted with CT-2A tumor after treatment with TG100-115, TMZ, or combination of these two drugs (combo). n=10. *, p<0.05; **, p<0.01; ***, p<0.0001. (H) Effect of TG100-115 treatment on the expression of the 82 survival-associated genes. RNA was extracted from orthotopic GL261 tumors with or without TG100-115 treatment and analyzed by qRT-PCR. ΔCt between gene and actin in each sample was plotted as heat map. Three independent tumors were analyzed for the treated and untreated cohorts.

Article Snippet: The human microglia (hMG) cell line was purchased from Applied Biological Materials (ABM).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR, Expressing