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. 2022 Nov 7;10(11):e004974. doi: 10.1136/jitc-2022-004974

Figure 1.

Figure 1

Adar1 loss decreases tumor burden and prolongs survival, which is further enhanced by DNMTi treatment and correlates with increases of CD8+ T cells and NK cells in the tumor microenvironment. (A) Schematic of mouse experiment. ID8 Trp53-/- 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 4 (advanced stage of disease), and around week 6, mice began developing ascites (a buildup of fluid in the peritoneum). (B) Survival curve. shGFP/Mock n=9 (one mouse was excluded: it did not develop ascites and was identified as an outlier using the ROUT method), shGFP/DNMTi n=10 (no mice excluded/censored), shAdar1/Mock n=9 (one mouse was excluded: it did not develop ascites and was identified as an outlier using the ROUT method), shAdar1/DNMTi n=7 (three mice were censored: one mouse was sacrificed due to a severe dermatitis condition, one mouse had an accidental/fatal fall, and one mouse was still alive and well at the end of the predefined, 20-week long study). One limitation of this model is that mice do not typically develop palpable tumors, thus making it difficult to know whether each mouse was effectively inoculated prior to start of treatment. (C) Ascites fluid drained at week 7 (attempted from all mice that were still alive from each group). Ascites is a sign of advanced stage of disease and ascites volume is an indicator of tumor burden. (D) Ascites fluid collection over time. (E–H) Immunophenotyping was performed on ascites drained from mice in figure 1A via flow cytometry. For flow analysis days, all mice that were still alive had an attempted ascites drain (despite body weight and circumference), and all samples obtained were analyzed. One limitation of this model is that ascites is required for flow analysis of the TME, and the mice in different groups do not develop ascites at the same time. To minimize potential batch effect, drain and flow stain days were chosen to maximize the number of samples obtained from two or more groups at one time. Due to difference in disease progression in each group, the shAdar1 Mock group (group 3) only resulted in two data points (two mice), which is too few data points to perform statistical analysis on. Therefore, all data are shown, although statistical comparisons have been excluded for group 3. Group 1: mice injected with shGFP (control) cells and given mock treatment (n=9). Group 2: mice injected with shGFP (control) cells and given THU/DNMTi treatment (n=8). Group 3: mice injected with shAdar1 (Adar1 knockdown) cells and given mock treatment (n=2). Group 4: mice injected with shAdar1 (Adar1 knockdown) cells and given THU/DNMTi treatment (n=5). (E) Absolute lymphocyte cell count (per mL of ascites). Absolute lymphocyte cell count (per mL of ascites) plotted against survival. (F) Absolute myeloid cell count per (per mL of ascites). Absolute myeloid cell count per (per mL of ascites) plotted against survival. (G) Absolute count of CD8+ T cells (per mL of ascites). Absolute count of CD8+ T cells (per mL of ascites) plotted against survival. (H) Absolute count of NK cells (per mL of ascites). Absolute count of NK cells (per mL of ascites) plotted against survival. Spearman’s correlation was performed for statistical significance on linear regression graphs. A one-way ANOVA was performed for statistical significance on box-and-whisker graphs. Gehan-Breslow-Wilcoxon method was performed for statistical significance on Kaplan-Meier survival curve. A one-way ANOVA was performed for statistical significance on column graph. *P<0.05. ANOVA, analysis of variance; DNMTi, DNA methyltransferase inhibitor; i.p., intraperitoneally; NK, natural killer; THU, tetrahydrouridine; TME, tumor microenvironment.