Tumour‐derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour‐bearing mice

Abstract

BackgroundCancer cachexia is a metabolic wasting syndrome that is strongly associated with a poor prognosis. The initiating factors causing fat and muscle loss are largely unknown. Previously, we found that leukaemia inhibitory factor (LIF) secreted by C26 colon carcinoma cells was responsible for atrophy in treated myotubes. In the present study, we tested whether C26 tumour‐derived LIF is required for cancer cachexia in mice by knockout of Lif in C26 cells.MethodsA C26 Lif null tumour cell line was made using CRISPR‐Cas9. Measurements of cachexia were compared in mice inoculated with C26 vs. C26Lif−/− tumour cells, and atrophy was compared in myotubes treated with medium from C26 vs. C26Lif−/− tumour cells. Levels of 25 cytokines/chemokines were compared in serum of mice bearing C26 vs. C26Lif−/− tumours and in the medium from these tumour cell lines.ResultsAt study endpoint, C26 mice showed outward signs of sickness while mice with C26Lif−/− tumours appeared healthy. Mice with C26Lif−/− tumours showed a 55–75% amelioration of body weight loss, muscle loss, fat loss, and splenomegaly compared with mice with C26 tumours (P < 0.05). The heart was not affected by LIF levels because the loss of cardiac mass was the same in C26 and C26Lif−/− tumour‐bearing mice. LIF levels in mouse serum was entirely dependent on secretion from the tumour cells. Serum levels of interleukin‐6 and G‐CSF were increased by 79‐fold and 68‐fold, respectively, in C26 mice but only by five‐fold and two‐fold, respectively, in C26Lif−/− mice, suggesting that interleukin‐6 and G‐CSF increases are dependent on tumour‐derived LIF.ConclusionsThis study shows the first use of CRISPR‐Cas9 knockout of a candidate cachexia factor in tumour cells. The results provide direct evidence for LIF as a major cachexia initiating factor for the C26 tumour in vivo. Tumour‐derived LIF was also a regulator of multiple cytokines in C26 tumour cells and in C26 tumour‐bearing mice. The identification of tumour‐derived factors such as LIF that initiate the cachectic process is immediately applicable to the development of therapeutics to treat cachexia. This is a proof of principle for studies that when carried out in human cells, will make possible an understanding of the factors causing cachexia in a patient‐specific manner.

Janus kinase inhibitors suppress cancer cachexia‐associated anorexia and adipose wasting in mice

“…34 We are encouraged that a simplified rLIF-injected mouse model is appropriate to study cachexia since a recent publication showed that genetic silencing of LIF from the C26 parental tumour line led to an anticipated decrease in systemic levels of IL-6 with suppression of the cachexia phenotype. 35 Therefore, our studies support the premise that cachexia patients have multiple circulating cachexia-inducing factors at any given time explaining why therapeutic interventions targeting a single molecule have been ineffective.…”

Pharmacological targeting of mitochondrial function and reactive oxygen species production prevents colon 26 cancer-induced cardiorespiratory muscle weakness

“…It is well-established that C26 cachexia alters the plasma levels of several soluble factors including TNFα, IL-1β and IL-6 which can accelerate the activity of key proteolytic pathways involved in the development of muscle weakness [23,24]. In this study, we confirmed that plasma levels of TNFα, IL-1β and IL-6 are increased in TB-SALINE mice compared to CON-SALINE and CON-SS-31 ( Figure 5).…”

IL-8 Released from Human Pancreatic Cancer and Tumor-Associated Stromal Cells Signals through a CXCR2-ERK1/2 Axis to Induce Muscle Atrophy

“…Therefore, the most common in vitro muscle model of cancer cachexia is the treatment of cultured skeletal muscle myotubes with media collected from cultured cancer cells (conditioned media (CM)). The most widely used cachexia-inducing cell lines are the mouse colon adenocarcinoma 26 (C26) and Lewis lung carcinoma (LLC) cells [4], and treatment of C2C12 mouse skeletal muscle myotubes with C26 or LLC CM induces significant atrophy [6][7][8]. Specific proteins identified in these CM that mediate cachexia include interleukin-6 (IL-6), leukemia inhibitory factor (LIF), tumor necrosis factor alpha (TNF-α), and heat shock protein 70 (Hsp70) [6,[9][10][11][12].…”

“…The most widely used cachexia-inducing cell lines are the mouse colon adenocarcinoma 26 (C26) and Lewis lung carcinoma (LLC) cells [4], and treatment of C2C12 mouse skeletal muscle myotubes with C26 or LLC CM induces significant atrophy [6][7][8]. Specific proteins identified in these CM that mediate cachexia include interleukin-6 (IL-6), leukemia inhibitory factor (LIF), tumor necrosis factor alpha (TNF-α), and heat shock protein 70 (Hsp70) [6,[9][10][11][12]. Further, multiple signaling pathways have been implicated in these models of muscle wasting, including STAT3, ERK1/2, NFkB, FOXO pathways, and, more recently, a TLR/MyD88/XBP1 signaling axis [6][7][8][9][10][11][12].…”

“…Specific proteins identified in these CM that mediate cachexia include interleukin-6 (IL-6), leukemia inhibitory factor (LIF), tumor necrosis factor alpha (TNF-α), and heat shock protein 70 (Hsp70) [6,[9][10][11][12]. Further, multiple signaling pathways have been implicated in these models of muscle wasting, including STAT3, ERK1/2, NFkB, FOXO pathways, and, more recently, a TLR/MyD88/XBP1 signaling axis [6][7][8][9][10][11][12]. However, as the field continues to expand into the study of additional cachexia-inducing cancer cell lines, we are likely to learn much more about additional factors and pathways driving cachexia.…”