Modelling three‐dimensional cancer‐associated cachexia and therapy: The molecular basis and therapeutic potential of interleukin‐6 transignalling blockade

Abstract

AbstractBackgroundCauses and mechanisms underlying cancer cachexia are not fully understood, and currently, no therapeutic approaches are available to completely reverse the cachectic phenotype. Interleukin‐6 (IL‐6) has been extensively described as a key factor in skeletal muscle physiopathology, exerting opposite roles through different signalling pathways.MethodsWe employed a three‐dimensional ex vivo muscle engineered tissue (X‐MET) to model cancer‐associated cachexia and to study the effectiveness of selective inhibition of IL‐6 transignalling in counteracting the cachectic phenotype. Conditioned medium (CM) derived from C26 adenocarcinoma cells was used as a source of soluble factors contributing to the establishment of cancer cachexia in the X‐MET model. A dose of 1.2 ng/mL of glycoprotein‐130 fused chimaera (gp130Fc) was added to cachectic culture medium to neutralize IL‐6 transignalling.ResultsC26‐conditioned medium induced a cachectic‐like phenotype in the X‐MET, leading to a decline of muscle mass (−60%; P < 0.001), a reduction in myosin expression (−92.4%; P < 0.005) and a reduction of the contraction frequency spectrum (−94%). C26‐conditioned medium contains elevated amounts of IL‐6 (8.61 ± 4.09 pg/mL) and IL6R (56.85 ± 10.96 pg/mL). These released factors activated the signal transducer and activator of transcription 3 (STAT3) signalling in the C26_CM X‐MET system (phosphorylated STAT3/TOTAL +54.6%; P < 0.005), which in turn promote an enhancement of Il‐6 (+69.2%; P < 0.05) and Il6r (+43%; P < 0.05) gene expression, suggesting the induction of a feed‐forward loop. The selective neutralization of IL‐6 transignalling, by gp130Fc, in C26_CM X‐MET prevented the hyperactivation of STAT3 (−55.8%; P < 0.005), countered the reduction of cross‐sectional area (+28.2%; P < 0.05) and reduced the expression of proteolytic factors including muscle ring finger‐1 (−88%; P < 0.005) and ATROGIN1 (−92%; P < 0.05), thus preserving the robustness and increasing the contractile force (+20%) of the three‐dimensional muscle system. Interestingly, the selective inhibition of IL‐6 transignalling modulated gene regulatory networks involved in myogenesis and apoptosis, normalizing the expression of pro‐apoptotic miRNAs, including miR‐31 (−53.2%; P < 0.05) and miR‐34c (−65%; P < 0.005), and resulting in the reduction of apoptotic pathways highlighted by the sensible reduction of cleaved caspase 3 (−92.5%; P < 0.005) in gp130Fc‐treated C26_CM X‐MET.ConclusionsIL‐6 transignalling appeared as a promising target to counter cancer cachexia‐related alterations. The X‐MET model has proven to be a reliable drug‐screening tool to identify novel therapeutic approaches and to test them in preclinical studies, significantly reducing the use of animal models.