Figure 3.

The effects of P2X7 receptor (P2X7R) antagonism by AZ10606120 (AZ) compared to conventional temozolomide (TMZ) chemotherapy on patient-derived primary glioblastoma cultures. Glioblastoma specimens collected from patients undergoing routine tumour resection surgery were cultured and treated at 80% confluency with 15 μM AZ, 50 μM TMZ and a combination of AZ + TMZ for 72 h. The control group were untreated cells. Primary glioblastoma cultures were fixed and stained with primary rabbit anti-glial fibrillary acidic protein (GFAP) antibody overnight at 4 °C, followed by secondary goat anti-rabbit antibody conjugated to Texas Red-X for 2 h at room temperature. Cells were counterstained with DAPI for 1 h at room temperature. Images were acquired with a Nikon Ti-E inverted fluorescence motorised microscope equipped with a sCMOS Andor Zyla camera at 20 × objective. The total number of GFAP + /DAPI + cells were quantified across 16 random fields for each sample and treatment group. (A) Comparison of AZ 15 μM and AZ-TMZ co-therapy with TMZ in primary glioblastoma cultures. Results were yielded from a one-way repeated measures ANOVA with Tukey’s HSD post-hoc, F(1.81, 19.9) = 7.79, p = 0.004. N = 12 patients. Untreated versus AZ 15 μM: mean difference = 454.8 cells, 95% CI = 13.05 to 896.4, *p = 0.04; Untreated versus TMZ: mean difference = 179.3 cells, 95% CI = − 182.2 to 540.7, p = 0.47; Untreated versus AZ + TMZ: mean difference = 476.7 cells, 95% CI = 69.78 to 883.6, Δp = 0.02; AZ 15 μM versus TMZ: mean difference = − 275.5 cells, 95% CI = − 632.5 to 81.46, p = 0.15; AZ 15 μM versus AZ + TMZ: mean difference = 21.92 cells, 95% CI = − 59.3 to 103.1, p = 0.85; TMZ versus AZ + TMZ: mean difference = 297.4 cells, 95% CI = − 31.45 to 626.3, p = 0.08. Error bars represent SEM. (B) Individual patient responses to treatment with AZ 15 μM, TMZ and AZ + TMZ. (C) Image representation of primary glioblastoma cultures treated with AZ 15 μM, TMZ, AZ + TMZ and control. GFAP and DAPI channels are red and blue, respectively.