Figures & data
Figure 1. Loss of ATG7 blocks autophagy and sensitizes cells to nutrient deprivation.
(a) Lysates from B16F10, MC38, or CT26 control (Ctrl) or ATG7 knockout (KO) cells probed with indicated antibodies. (b-d) Sensitivity to nutrient deprivation of the indicated cell lines cultured in Hank’s Balanced Salt Solution (HBSS) followed by a brief recovery in complete medium. Cell abundance was measured by SRB stain. (e-g) In vitro proliferation of the indicated cell lines cultured in complete medium. Proliferation was measured by CellTiter-Glo at the indicated time points. **P < .01, ****P < .0001. Unpaired two-tailed t-test.
Figure 2. Murine tumors have differential reliance upon ATG7 when grown in immuno-competent hosts.
(a-c) In vivo tumor growth of B16F10, MC38 or CT26 tumor cells implanted in immune-deficient (Nude or NSG) mouse strains. (d-f) In vivo tumor growth of B16F10, MC38 or CT26 cell lines implanted in immune-competent (C57BL/6 or BALB/c) mouse strains. (g) In vivo tumor growth of CT26 tumor cells expressing vector control (+Vec) or ATG7 (+ATG7) implanted in BALB/c mice. Each data point represents the mean from 13 to 15 mice ± SEM. ns, not significant, *P < .05, **P < .01, ***P < .001, ****P < .0001. (a-f) ANOVA comparing Ctrl to ATG7KO. (g) Nonparametric Wilcoxon rank sum test comparing Ctrl+Vec to ATG7KO+Vec or ATG7KO+Vec to ATG7KO+ATG7.
Figure 3. CD8+ and CD4+ T cell contribution to cancer-cell dependence on ATG7 in vivo.
(a-b) Quantification of IHC staining of CD3+ (a) or CD8+ (b) cells from B16F10, MC38, or CT26 tumors grown in immunocompetent mice. Each point represents one tumor, data are displayed as chromogen positive cells per 1 mm2 viable tumor tissue and error bars represent SEM. (c-f) Ctrl or ATG7KO CT26 cells implanted in BALB/c mice treated with IgG, anti-CD8, or anti-CD4 antibodies. Data are displayed as tumor growth curves, where each data point represents the mean from 14 to 15 mice ± SEM (c-d) and Kaplan-Meier survival curves (e-f). (g) Median survival and complete response (CR) data of Figure 3e-f. CR indicates no measurable tumor at day 61 post-implant. ND indicates median survival could not be determined. (h) Statistical comparison of Figure 3c-f. Nonparametric Wilcoxon rank-sum test was used for comparison of Day 22 tumor volumes, and log-rank test was used for comparison of median survivals. ns not significant, *P < .05, **P < .01, ***P < .001, ****P < .0001. (a-b) Unpaired two-tailed t-test comparing IgG vs anti-CD8 or anti-CD4 treated.
Figure 4. Gene expression and pathway alterations upon Atg7 loss.
(a) Number of differentially upregulated or downregulated genes as measured by RNAseq comparing autophagy-deficient (without ATG7) CT26 tumors to autophagy-competent (with ATG7) CT26 tumors isolated from the indicated mouse strain (absolute log2-fold change ≥0.58, false discovery rate (FDR) ≤0.01). (b) Canonical pathways enriched in autophagy-deficient CT26 tumors, compared to autophagy-competent tumors, grown in BALB/c mice, as revealed by Ingenuity Pathway Analysis. Immune-related pathways are shown in red. (c) Cytolytic activity, defined as the log-average (geometric mean) of Gzma and Prf1 expression in transcripts per million (TPM), in autophagy-competent and autophagy-deficient CT26 tumors grown in BALB/c mice. ****P < .0001. (c) Unpaired two-tailed t-test.
Supplemental material