Figure 5 from Trabectedin Enhances the Antitumor Effects of IL-12 in Triple-Negative Breast Cancer

NK-cell depletion reduces combination-induced efficacy and reduces intratumoral CD8a. BALB/c mice were inoculated with 4T1 cells and divided into one of two groups once tumors reached ∼50 mm³. Mice were either depleted of NK cells using anti–asialo-GM1 antibody or treated with an IgG isotype control antibody. Anti–asialo-GM1 was administered by intraperitoneal injection at 100 μg/100 μL 3 days prior to the start of IL-12 and trabectedin treatment and every 4 days thereafter. The IgG isotype control antibody was administered in nondepleted mice at the same time points and dose as the depletion antibody. NK-depleted mice were treated with PBS control (n = 6) or intraperitoneal injection of 0.5 μg IL-12 3×/week and intravenous injection of 0.15 mg/kg trabectedin 1×/week (n = 5). Nondepleted mice were similarly treated with either PBS control (n = 8) or IL-12 plus trabectedin (n = 6). A, Tumor growth curves throughout the 12-day treatment. B, Images of tumors after excision. For statistical analyses of tumor volumes, linear mixed modeling was used to model the longitudinal tumor volume for mice under each treatment. Comparisons were done at each time point and averaged across all time points using t-statistics. The Tukey–Kramer method was used for adjusting raw P values for multiple comparisons across treatment groups. Values are the mean ± SEM of tumor volumes at each time point. C, Plasma cytokine and chemokine levels in mice at day 19. Values represent individual samples and are shown as ± SEM. D, Representative images (5× magnification) of IHC staining for CD8a and CD4 performed on 4T1 tumors from mice treated with IL-12 and trabectedin ± NK-cell depletion. E, Quantification of IHC staining as reported by percent CD8a (n = 4–5) and percent CD4 (n = 3–5) positivity/slide. Values represent individual tumor slides and are shown as mean ± SEM. Statistical analyses of bar graphs were performed using two-tailed unpaired student t tests. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Trab, trabectedin.