Doxorubicin Paradoxically Ameliorates Tumor-Induced Inflammation in Young Mice

Figure 1. Model schematic diagram and body weight data. (A) Scheme of the experimental study design to investigate the effects of low dose DOX in EL4 lymphoma tumor-bearing C57BL/6N juvenile mice. One week following administration of intraperitoneal (IP) DOX (4 mg/kg/week) or equivalent volume of saline for 3 weeks, the final body weight with and without tumors was recorded, and (B) the change from baseline body weight was calculated (n = 8 per group); (C) tumors were harvested and weighed (n = 8 per group); (D) hearts were harvested from both tumor-free and EL4 lymphoma tumor-bearing mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks and hearts weights (mg) were measured and normalized to the tibial length (mm) (HW/TL) (n = 8 per group). Two-way ANOVA table demonstrates DOX, tumor, and interaction effects. Statistical significance of pairwise comparisons was determined using two-way ANOVA with Tukey’s post hoc analysis. Tumor weights were compared using unpaired Student’s two-tailed t-test. (* p < 0.05, ** p < 0.01, and **** p < 0.0001).

Figure 2. Chronic administration of DOX (4 mg/kg/week) for 3 weeks causes a similar cardiac response in tumor-free and tumor-bearing juvenile mice. Tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N male mice were administered intraperitoneal DOX (4 mg/kg/week) or saline for 3 weeks. Cardiac function was assessed by transthoracic echocardiography one week following the last injection (n = 8 per group). (A) Representative images from parasternal short axis view of the heart acquired in M-Mode. Effects of DOX and the tumor on (B) ejection fraction, (C) cardiac output, (D) fractional shortening, (E) left ventricular (LV) mass, (F) LV posterior wall during diastole, and (G) LV posterior wall during systole. Values are shown as means ± SEM. Two-way ANOVA was used to determine the main effects of DOX, tumor, and interaction. Statistical significance of pairwise comparisons was determined using two-way ANOVA with Tukey’s post hoc analysis.

Figure 3. Chronic administration of DOX (4 mg/kg/week) for 3 weeks abrogates tumor-induced upregulation of inflammatory markers in the heart. Hearts were harvested from both tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 7–8 per group). Following the extraction of total RNA, cardiac mRNA expression of (A) IL-1α, (B) IL-1β, (C) IL-6, (D) TNF-α, (E) Mcp-1, (F) Cxcl1, (G) Cxcl9, and (H) Cxcl10 were determined by real-time PCR. Values were normalized to 18S rRNA and expressed relative to saline-treated tumor-free mice. Values are shown as means ± SEM. Two-way ANOVA table demonstrates DOX, tumor, and interaction effects on the mRNA expression. Statistical significance of pairwise comparisons was determined using two-way ANOVA with Tukey’s post hoc analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 4. Chronic administration of DOX (4 mg/kg/week) for 3 weeks abrogates tumor-induced upregulation of inflammatory markers in the liver. Livers were harvested from both tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 8 per group). Following the extraction of total RNA, hepatic mRNA expression of (A) IL-1α, (B) IL-1β, (C) IL-6, (D) TNF-α, (E) Mcp-1, (F) Cxcl1, (G) Cxcl9, and (H) Cxcl10 were determined by real-time PCR. Values were normalized to 18S rRNA and expressed relative to saline-treated tumor-free mice. Values are shown as means ± SEM. Two-way ANOVA table demonstrates DOX, tumor, and interaction effects on the mRNA expression. Statistical significance of pairwise comparisons was determined using two-way ANOVA with Tukey’s post hoc analysis (* p < 0.05, ** p < 0.01).

Figure 5. Chronic administration of DOX (4 mg/kg/week) for 3 weeks abrogates tumor-induced upregulation of inflammatory markers in the kidney. Kidneys were harvested from both tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 8 per group). Following the extraction of total RNA, renal mRNA expression of (A) IL-1α, (B) IL-1β, (C) IL-6, (D) TNF-α, (E) Mcp-1, (F) Cxcl1, (G) Cxcl9, and (H) Cxcl10 were determined by real-time PCR. Values were normalized to 18S rRNA and expressed relative to saline-treated tumor-free mice. Values are shown as means ± SEM. Two-way ANOVA table demonstrates DOX, tumor, and interaction effects on the mRNA expression. Statistical significance of pairwise comparisons was determined using two-way ANOVA with Tukey’s post hoc analysis (* p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001).

Figure 6. Histopathological evaluation of tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice euthanized one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks. Representative images from hematoxylin and eosin (HE)-stained sections of (A) heart, (B) liver, and (C) kidney are shown. Scale bar = 50 µM (A and B) and 100 µM (C).

Figure 7. Divergent effects of DOX treatment on the serum level of inflammatory markers in tumor-free versus tumor-bearing mice. Serum was collected from tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 7–8 per group). Inflammatory markers (A) IL-6 and (B) TNF-alpha were measured using the Luminex platform. Data are presented as the mean ± SEM. Two-way ANOVA table demonstrates DOX, tumor, and interaction effects. Statistical significance of pairwise comparisons was determined using two-way ANOVA with Tukey’s post hoc analysis (* p < 0.05).