Higher tumor mass and lower adipose mass are associated with colon‑26 adenocarcinoma‑induced cachexia in male, female and ovariectomized mice

Abstract

Cachexia is responsible for nearly 20% of all cancer‑related deaths, yet effective therapies to prevent or treat the disease are lacking. Clinical studies have shown that male patients lose weight at a faster rate than females. Additionally, an 'obesity paradox' may exist where excess adiposity may confer survival to patients with cancer cachexia. To further explore these phenomena, the aim of this study was to evaluate the role of changes of adipose tissue mass, sex status, and tumor mass on outcomes of male, female and ovariectomized (OVX) mice with C‑26 adenocarcinoma‑induced cachexia. We used EchoMRI to assess body composition and grip strength to measure muscle function. Body weights and food intake were measured daily. Mice were euthanized 19 days post‑-inoculation. Post‑necropsy, muscle fiber cross‑sectional areas were quantified and real‑time PCR was performed for genes relating to proteolysis. Survival curve, correlation and multiple linear regression analyses were performed to identify predictors of cachexia. Female and OVX tumor mice developed cachexia similarly to males, as evidenced by loss of skeletal and adipose masses, decreased grip strength, and increased proteolytic gene expression. Notably, female and OVX tumor mice had earlier onset of cachexia (≥5% weight loss) than male tumor mice. Larger tumor mass and lower adipose mass were the strongest predicting factors for increased severity of cachexia, regardless of sex or ovariectomy status. These results indicated that the impact of sex status may be subtle in comparison to the predictive effect of tumor and adipose mass in mice with C‑26‑induced cachexia.

Figure 1.

Characteristics of C-26 tumor-bearing male, female and OVX mice. (A) Daily body weights of tumor-bearing mice over the 19-day experimental period. (B) C-26 tumor cell inoculation induced body weight loss in all mice at necropsy. (C) Tumor mass expressed as a percentage of tumor-free body mass in mice. (D) Food intake of tumor mice. Food intake was calculated by dividing total food consumed each day by the number of mice in each cage (n=2 cages/group). (E and F) Lean body mass and adipose mass changes of tumor mice over the experimental period. Data are expressed as the mean ± SEM (n=9–10/group, unless otherwise indicated). Significant differences (P<0.05) are represented by different letters (a, b) using one-way ANOVA with post-hoc Tukey's test. Significant differences (P<0.016) between day 0 and day 19 within a single group are represented by * (Fig. 1F, all 3 groups). C-26, colon-26; OVX, ovariectomized.

Figure 2.

C-26 cachexia induces loss of muscle mass and function in male, female and OVX mice. (A) Muscle weights of tumor mice at necropsy. (B) Hematoxylin and eosin staining of representative gastrocnemius sections of tumor mice (magnification, ×20; scale bar, 50 µm). (C) Quantification of gastrocnemius CSA (n=3–5/group). (D) Frequency distribution of gastrocnemius CSA (n=3–5/group). (E and F) Change in forelimb and hindlimb grip strength of tumor mice from peak grip strength (day 7 post-inoculation). (G) Relative gene expression of markers related to muscle wasting; relative fold change was normalized to male control mice, which was set at 1 (data not shown). Data are expressed as mean ± SEM (n=8–10/group unless otherwise indicated). Significant differences (P<0.05) are represented by different letters (a, b) using one-way ANOVA with post-hoc Tukey's test. Significant differences (P<0.016) between Day 7 and Day 19 within a single group are represented by * as observed in E, OVX tumor. C-26, colon-26; OVX, ovariectomized.

Figure 3.

C-26 cachexia induces adipose loss, increases plasma IL-6 and decreases plasma adiponectin in male, female and OVX mice. (A) Adipose depot weights of tumor mice at necropsy. (B) Heart, liver and spleen weights of tumor mice at necropsy. (C) Plasma IL-6 levels of tumor mice at necropsy. (D) Plasma adiponectin levels of tumor mice at necropsy. Data are expressed as the mean ± SEM (n=8–10/group). Significant differences (P<0.05) are represented by different letters (a, b) using one-way ANOVA with post-hoc Tukey's test. C-26, colon-26; OVX, ovariectomized.

Figure 4.

Tumor and adipose mass are predictors of severity of C-26-induced cachexia. (A) Kaplan-Meier survival plot determining incidence of cachexia (defined as ≥5% body weight loss) of tumor mice by study day; the P-value represents a comparison of the 3 groups using a Mantel-Cox test. (B) Relationship between tumor mass (normalized to tumor-free body weight at necropsy) and percentage of peak body weight loss in tumor-bearing mice. (C) Relationship between day 8 EchoMRI adipose mass and percentage of adipose loss in tumor mice. (D) 3-D scatterplot of the relationship between the percentage of tumor mass and day 8 EchoMRI adipose mass (g) on the percentage of adipose loss in tumor mice. N=30 mice total. Correlations between continuous variables were assessed using Pearson's correlation coefficients where P<0.05 was considered statistically significant. C-26, colon-26; Dpi, days post-inoculation.