Metastatic or xenograft colorectal cancer models induce divergent anabolic deficits and expression of pro-inflammatory effectors of muscle wasting in a tumor-type-dependent manner

Abstract

We investigated the impact of tumor burden on muscle wasting in metastatic (m) and xenograft (x) models of colorectal cancer (CRC). Male Nod SCID γ and CD2F1 mice were injected subcutaneously or intrasplenically with HCT116 or C26 tumor cells, respectively. CRC tumors resulted in significant muscle wasting regardless of tumor type or model, although muscle loss was exacerbated in mHCT116 hosts. The mHCT116 model decreased ribosomal (r)RNA content and rDNA transcription, whereas the mC26 model showed no loss of rRNA and the upregulation of rDNA transcription. The xHCT116 model reduced mTOR, RPS6, and 4E-BP1 phosphorylation, whereas the mHCT116 model had a similar effect on RPS6 and 4E-BP1 without altering mTOR phosphorylation. The C26 models caused a reduction in 4E-BP1 phosphorylation independent of mTOR. Muscle interleukin (IL)-6 mRNA was elevated in all models except xHCT116, and the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) mRNA was induced only in the mC26 model. IL-1β mRNA increased in all groups with greater expression in metastatic relative to the xenograft model regardless of tumor types. Our findings indicate that HCT116 tumor burden results in more drastic muscle wasting and anabolic deficits, whereas C26 tumor burden causes similar muscle wasting but exhibits a divergent proinflammatory phenotype. These results highlight potentially important divergence in the pathogenesis of muscle wasting among preclinical models of CRC and demonstrate that tumor burden plays a role in determining anabolic deficits and the expression of proinflammatory effectors of muscle wasting in a tumor-type-dependent manner.NEW & NOTEWORTHY We provide evidence demonstrating that colorectal tumor burden plays a role in determining anabolic deficits and the expression of proinflammatory effectors of muscle wasting in a tumor-type-dependent manner.

Keywords: cachexia; colon cancer; colorectal cancer; inflammation; muscle wasting; ribosomal RNA.

Graphical abstract

Figure 1.

Skeletal muscle wasting is exacerbated in mHCT116 compared with xHCT116 but is similar in the C26 model. Muscle wet weight of gastrocnemius muscle in HCT116 (A) and C26 models (B). Gastrocnemius muscle mass was normalized to the initial body weight for the HCT116 (C) and C26 models (D). Data are expressed as fold change from controls ± SD. A two-way ANOVA followed by a Tukey’s multiple comparisons test was performed for all direct comparisons between groups. Significant difference from control: *P ≤ 0.05, **P < 0.01, and ***P < 0.001; xenograft vs. metastasis: #P < 0.05, ##P < 0.01.

Figure 2.

Ribosomal capacity and rDNA transcription are reduced in mHCT116, but C26 tumors display elevated rDNA transcription and maintain rRNA levels. A: total rRNA content was quantified to compare between groups. Expression of 45S pre-rRNA 5′ external transcribed spacer (ETS; B) and internal transcribed spacers (ITS; C) were analyzed by qPCR. D–F: Pearson product-moment correlations between each factor and muscle mass, respectively. Data are expressed as fold change from controls ± SD. A two-way ANOVA followed by a Tukey’s multiple comparisons test was performed for all direct comparisons between groups. Significant difference from control: *P ≤ 0.05, **P < 0.01, and ***P < 0.001; xenograft vs. metastasis: #P < 0.05, ##P < 0.01, and ###P < 0.001.

Figure 3.

Intracellular signaling phosphorylation in colorectal cancer (CRC)-induced muscle wasting depends on the tumor type. A and C: quantification of Western blots for mTOR, RPS6, and 4E-BP1 phosphorylation in HCT116 and C26 model, respectively. B and D: representative image of Western blots. E and F: Pearson product-moment correlations between each factor and muscle mass, respectively. Data are expressed as fold change from controls ± SD. A two-way ANOVA followed by a Tukey’s multiple comparisons test was performed for all direct comparisons between groups. Significant difference from control: *P ≤ 0.05, **P < 0.01, and ***P < 0.001; xenograft vs. metastasis: #P < 0.05, ##P < 0.01, and ###P < 0.001.

Figure 4.

Local interleukin 6 (IL-6) expression is higher in both mHCT116 and mC26 but not TNF-α. Expression of IL-6 (A) and TNF-α mRNA (B) was analyzed by qPCR. C and D: Pearson product-moment correlations between each factor and muscle mass, respectively. Data are expressed as fold change from controls ± SD. A two-way ANOVA followed by a Tukey’s multiple comparisons test was performed for all direct comparisons between groups. Significant difference from control: *P ≤ 0.05, **P < 0.01; xenograft vs. metastasis: #P < 0.05.

Figure 5.

The expression of inflammasome-related factors is elevated in mC26 but not in mHCT116 tumor mice. Expression of NLRP3 (A), IL-1β (B), and IL-18 mRNA (C) was analyzed by qPCR. D–F: Pearson product-moment correlations between each factor and muscle mass, respectively. Data are expressed as fold change from controls ± SD. A two-way ANOVA followed by a Tukey’s multiple comparisons test was performed for all direct comparisons between groups. Significant difference from control: *P ≤ 0.05, **P < 0.01, and ***P < 0.001; xenograft vs. metastasis: #P < 0.05, ##P < 0.01, and ###P < 0.001.