Fig. 7: VAMP7KO glioblastoma cells generate more necrotic tumors.

A–C Kaplan-Meier survival curve of patients bearing VAMP7 low (blue) or high (red) expression in the GBM-mark cohort⁵⁸, TCGA Stupp and TCGA MES (mesenchymal GB). p-val = 0.036; 0.044; 0.0005, respectively. D WT, VAMP7KO and ATG5KO RG2 cells were treated with BafA1 (100 nM) to inhibit degradative autophagy and serum-starved overnight. The next day, the secreted media was processed, and total secreted protein was precipitated using acetone. The total secreted fraction and cell lysates were collected and analyzed. The experiment was replicated thrice with similar results (E) Oxygen consumption rate (OCR) rate of basal respiration, maximal respiration, spare capacity and ATP production is significantly lower in VAMP7KO and ATG5KO RG2 cells compared with WT, indicating respiratory chain inefficiency in VAMP7KO and ATG5KO RG2 cells. Statistical significance was determined by ANOVA following by a Post Hoc group comparison with Bonferroni correction (ns: non-significant, *p-val < 0.05, **p-val < 0.01, ***p-val < 0.001, n = 18/group). F, G Measurement of freshly extracted tumors showed a tendency towards larger tumors in the grafting of VAMP7KO RG2 cells and significantly smaller in the grafted ATG5KO RG2 cells. Scale bar = 2 mm. Two-tailed Welch’s t-test was used to determine statistical significance. Data is derived from 4 animals/genotype from three independent cohorts presented as the mean ± SEM. *p-val < 0.05; ns: non-significant. H, I IHC for Vimentin in the fixed tumors showed significant decrease in the tumor volume of ATG5KO RG2 tumor. Scale bar = 2.5 mm. Data is derived from 6 animals/genotype from three independent cohorts presented as the mean ± SEM. **p-val < 0.01, *p-val < 0.05; ns: non-significant. Two-tailed Mann-Whitney test was used to determine statistical significance. J, K Haematoxylin-Eosin staining of the WT, VAMP7KO, and the ATG5KO RG2 tumors showed increased necrosis in the VAMP7KO RG2 tumors and a decrease in the same for the ATG5KO RG2 tumors. Scale bar = 2.5 mm. Data is derived from 6 animals/genotype from three independent cohorts presented as the mean ± SEM. **p-val < 0.01, *p-val < 0.05. Two-tailed Mann-Whitney test was used to determine statistical significance. L Tumor sections were double stained with HES and anti-Iba1 antibodies (macrophage and microglial cells) and revealed with secondary antibodies coupled to HRP and DAB. Magnification of the tumor center or periphery are shown for tumors derived from WT, VAMPKO or ATG5KO cells. Scale bar = 2.5 mm. M Quantitation of macrophage/microglia staining in the tumor center. Data is derived from 6 animals/genotype from three independent cohorts presented as the mean ± SEM.*p-val < 0.05. Two-tailed Mann-Whitney test was used to determine statistical significance. N Quantitation of macrophage/microglia staining at the tumor periphery. Data is derived from 6 animals/genotype from three independent cohorts presented as the mean ± SEM. *p-val < 0.05; ns non-significant. Two-tailed Mann-Whitney test was used to determine statistical significance.