HCT116 colorectal liver metastases exacerbate muscle wasting in a mouse model for the study of colorectal cancer cachexia

摘要

Colorectal cancer (CRC) is often accompanied by formation of liver metastases (LM) and skeletal muscle wasting, i.e. cachexia. Despite affecting the majority of CRC patients, cachexia remains underserved, understudied and uncured. Animal models for the study of CRC-induced cachexia, in particular models containing LM, are sparse; therefore, we aimed to characterize two new models of CRC cachexia. Male NSG mice were injected subcutaneously (HCT116) or intrasplenically (mHCT116) with human HCT116 CRC tumor cells to disseminate LM, whereas experimental controls received saline ( n =5-8/group). Tumor growth was accompanied by loss of skeletal muscle mass (HCT116: ?20%; mHCT116: ?31%; quadriceps muscle) and strength (HCT116: ?20%; mHCT116: ?27%), with worsened loss of skeletal muscle mass in mHCT116 compared with HCT116 (gastrocnemius: ?19%; tibialis anterior: ?22%; quadriceps: ?21%). Molecular analyses revealed elevated protein ubiquitination in HCT116, whereas mHCT116 also displayed elevated Murf1 and atrogin-1 expression, along with reduced mitochondrial proteins PGC1α, OPA1, mitofusin 2 and cytochrome C. Further, elevated IL6 levels were found in the blood of mHCT116 hosts, which was associated with higher phosphorylation of STAT3 in skeletal muscle. To clarify whether STAT3 was a main player in muscle wasting in this model, HCT116 cells were co-cultured with C2C12 myotubes. Marked myotube atrophy (–53%) was observed, along with elevated phospho-STAT3 levels (+149%). Conversely, inhibition of STAT3 signaling by means of a JAK/STAT3 inhibitor was sufficient to rescue myotube atrophy induced by HCT116 cells (+55%). Overall, our results indicate that the formation of LM exacerbates cachectic phenotype and associated skeletal muscle molecular alterations in HCT116 tumor hosts. RESULTS HCT116 subcutaneous and metastatic tumor hosts experience weight and fat loss To assess the impact of HCT116-induced CRC growth on the development of cachexia, male NSG mice were subcutaneously injected with 3×10sup6/sup HCT116 cells (HCT116) or were intrasplenically injected with 1.25×10sup5/sup HCT116 cells (mHCT116) to disseminate LM. It is important to note that sham and mHCT116 animals were euthanized at day 24, whereas control and HCT116 animals were euthanized at day 30. By day 24 the mHCT116 hosts were displaying an average weight loss of ～2?g, which was accompanied by minimal abdominal ascites, marked decline in activity, hunched over appearance, and were therefore euthanized. There was no significant difference in initial or final body weight between experimental groups ( Fig. 1 A-C). The carcass weights demonstrated a 13% reduction ( P 0.01, Fig.?1 D) in HCT116 hosts relative to controls, whereas they were 21% decreased ( P 0.0001, Fig.?1 D) in the mHCT116 bearers compared with the sham-operated animals. Interestingly, despite being exposed to tumor conditions for a shorter time, the weight of the mHCT116 carcasses was significantly reduced compared with controls (?25%; P 0.0001) and HCT116 hosts (?15%; P 0.0001). Further, the HCT116 hosts did not display cardiac atrophy, whereas mHCT116 demonstrated significant loss in heart size relative to sham (?18%, P 0.05, Fig.?1 E). Livers of mHCT116 mice increased 98% compared with livers of HCT116 hosts ( P 0.05, Fig.?1 G), likely owing to the formation of LM (Fig. 1H). In terms of fat mass, mHCT116 hosts saw greater fat loss (?80%, P 0.001) relative to controls than did HCT116 hosts (?63%, P 0.01) ( Fig.?1 F), although the two tumor groups were not significantly different.