Fig 1: Adar1 loss increases sensitivity to type I IFN in murine OC cell lines. (A) Schematic for growth inhibition assay. ID8 Trp53-/- Adar1 knockdown (shAdar1) and control cells (shGFP) were plated on day 0 and then treated with DNMTi for three consecutive days without media change. Cell culture supernatant was collected on day 4 for chemokine/cytokine analysis, and cell pellets were harvested on day 4 for western blot analysis. Cells were re-plated on day 4 and harvested on day 7 for western blot analysis. (B) Cell culture supernatant from day 4 was analyzed using BioLegend LEGENDplex Mouse Anti-Virus Response Panel. IFN-ß cytokine levels normalized by number of live cells. (C) Representative western blot analysis of Adar1 protein expression at day 7. (D) Growth inhibition assays showing IFN-ß or DNMTi (Dac and 5-Aza) compared with Mock (each condition performed in triplicate). (E) Schematic for colony formation assay. Cells were plated on day 0, and then anti-IFNAR1 or isotype control (10 µg/mL) was given on days 1, 3, 6, and 9. Cells were treated on days 1, 2, and 3 with either DMSO, Dac (100 nM), or 5-Aza (1 µM). IFN-ß condition was treated on day 3. Cells were incubated until day 10 when they were subsequently stained with crystal violet. (F) Colony formation assays showing Mock, 5-Aza, and IFN-ß (each condition performed in triplicate). (G) Colony formation assays using 100 µg/mL of anti-IFNAR1 or isotype control and showing Mock, 5-Aza, and IFN-ß (each condition performed in triplicate). A one-way analysis of variance was performed for statistical significance. *P<0.05; **p<0.01; ***p<0.001. 5-AZA, 5-zaacytidine; Dac, 2’-deoxy-5-azacytidine; DMSO, dimethyl sulfoxide; DNMTi, DNA methyltransferase inhibitor; IFN, interferon; IFNAR1, interferon alpha and beta receptor 1; OC, ovarian cancer.
Fig 2: Prolonged survival in mice with combined Adar1 loss and DNMT inhibition is dependent on type I IFN signaling. (A) ID8 Trp53-/- shAdar1 knockdown tumor cells were grown in tissue culture dishes and then 5e6 cells were i.p. injected into C57Bl6 wild-type mice 1 day after the first injections of antibodies. THU/DNMTi treatment began around week 2. Survival curve for the following groups: shGFP Mock/isotype control group (n=5), shGFP Mock/anti-IFNAR1 group (n=5), shAdar1 THU/DNMTi/isotype control (n=7), shAdar1 THU/DNMTi/anti-IFNAR1 (n=5). Two mice from the shGFP Mock isotype control group were censored from the study because they had not developed tumors by the study end point (week 20). (B) Ascites (a buildup of fluid in the peritoneum) collected over time for mice in figure 6A. Ascites is a sign of advanced stage of disease and ascites volume is an indicator of tumor burden in this model. (C) Survival curve (n=5 per group). ID8 Trp5-/- shGFP (control) or shAdar1 knockdown tumor cells were grown in tissue culture dishes and then 5e6 cells were i.p. injected into mice. Mock or THU/DNMTi treatment began around week 2, and around week 6, mice began developing ascites (a buildup of fluid in the peritoneum). (D) Ascites (a buildup of fluid in the peritoneum) collected over time for mice in figure 6C. Ascites is a sign of advanced stage of disease and ascites volume is an indicator of tumor burden. (E) Ascites (a buildup of fluid in the peritoneum) collected over time for C57Bl6 wild-type mice that received shAdar1 tumors with THU/DNMTi treatment and either: (1) isotype control (n=10), (2) anti-IFNAR1 (n=10), (3) anti-CD8a (n=10), or (4) anti-NK1.1 (n=10) antibodies. ID8 Trp53-/- shAdar1 knockdown tumor cells were grown in tissue culture dishes and then 5e6 cells were i.p. injected into mice 1 day after the first injections of antibodies. THU/DNMTi treatment began around week 2. (F) Average volume of ascites (a buildup of fluid in the peritoneum) for C57Bl6 wild-type mice that received shAdar1 tumors with THU/DNMTi treatment and either: (1) isotype control (n=10), (2) anti-IFNAR1 (n=10), (3) anti-CD8a (n=10), or (4) anti-NK1.1 (n=10) antibodies. ID8 Trp53-/- shAdar1 knockdown tumor cells were grown in tissue culture dishes and then 5e6 cells were i.p. injected into mice 1 day after the first injections of antibodies. THU/DNMTi treatment began around week 2. *P<0.05; **p<0.01. DNMTi, DNA methyltransferase inhibitor; IFN, interferon; IFNAR1, interferon alpha and beta receptor 1; i.p., intraperitoneally; ns, not significant; THU, tetrahydrouridine.
Fig 3: Saracatinib reduced IL-1β, CCL2, and CXCL1 release induced by CGRP in the mouse TG. (A–D) Effects of Kreb’s, 3 µM CGRP, or 1.5 μM saracatinib in the presence of 3 μM CGRP (n = 8 per group) at 20 min post treatment on IL-1β, CCL2, CXCL1, and IL-10 release (pg/mL). Abbreviations: saracatinib (SRCT). Two-tailed unpaired t-test was used for the comparison in IL-1β, CCL2, CXCL1, and IL-10 release between the CGRP group and either the Kreb’s group or the saracatinib in the presence of CGRP group. Significant differences were labeled as * p < 0.05 or ** p < 0.01.
Fig 4: mOVA2 activates DCs and mediates SIINFEKL epitope presentation to CD8 T cells.a, b Immunoblot and flow cytometry analysis of GMCSF- (a) and FLT3L- (b) BMDCs showing type I IFN-dominant activation and upregulation of costimulatory molecules upon infection with mOVA2. For co-stimulatory molecule expression, asterisks denote statistically significant differences from mock (0 h)-treated samples as determined by Two-way RM-ANOVA protected Dunnett’s post hoc test; (a) n = 4; (b) n = 3. Repeat immunoblot assays and gating strategy are shown in Supplementary Fig. 6a–c, respectively. c, d Multiplex ELISA showing cytokine profiles of infected GMCSF- (c) and FLT3L- (d) BMDCs. Asterisks denote statistically significant differences from mock (0 h)-treated samples determined by One-way ANOVA protected Dunnett’s post hoc test; n = 3. e GMCSF-BMDC/OT-I co-culture assays. Asterisks denote statistically significant differences determined by Two-way ANOVA protected Sidak’s multiple comparison test, n = 3. The assay was performed three times; representative results are shown. a–e Error bars denote SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = number of independent replicates.
Fig 5: DNMTi treatment enhances secretion of pro-inflammatory chemokines from murine OC cells and increases migration of T cells. (A) ID8 Trp53-/- Adar1 knockdown (shAdar1) and control cells (shGFP) were plated on day 0 and treated with DNMTi for three consecutive days without media change. Cell culture supernatant was collected on day 4 for chemokine/cytokine analysis with the BioLegend LEGENDplex Mouse Anti-Virus Response Panel. Chemokine/Cytokine levels were normalized by number of live cells. (C) Graphs show ratio of migrated CD4+ or CD8+ T cells to tumor cells. (D) Graphs show migration index, which is the fold change of DNMTi-treated migrated T cells to mock-treated migrated T cells (red and purple bars), or alternatively, the fold change of migrated T cells in the +CCL5 condition to the -CCL5 condition (gray bars). A one-way analysis of variance was performed for statistical significance. *P<0.05; **p<0.01; ***p<0.001. 5-AZA, 5-zaacytidine; DMSO, dimethyl sulfoxide; DNMTi, DNA methyltransferase inhibitor; IFN, interferon; OC, ovarian cancer.
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