癌症恶病质的病因和机制尚未完全理解，目前还没有可完全逆转恶病质表型的治疗方法。白细胞介素-6（IL-6）已被广泛描述为骨骼肌生理病理途径中的关键因子，通过不同的信号通路发挥相反的作用。我们利用三维离体肌肉工程组织（X-MET）模拟癌症相关恶病质，并研究选择性抑制IL-6转信号在对抗恶病质表型中的有效性。C26腺癌细胞培养液被用作X-MET模型中导致癌症恶病质形成的可溶性因子的来源。在恶病质培养基中添加1.2 ng/mL的糖蛋白130融合嵌合体（gp130Fc），以中和IL-6转信号。C26培养液诱导X-MET中出现类似恶病质表型的现象，导致肌肉质量下降（-60%；P < 0.001），肌球蛋白表达减少（-92.4%；P < 0.005），收缩频率谱降低（-94%）。C26培养液中含有较高量的IL-6（8.61 ± 4.09 pg/mL）和IL6R（56.85 ± 10.96 pg/mL）。这些释放的因子在C26_CM X-MET系统中激活了信号传导子和转录激活因子3（STAT3）信号传导路线（磷酸化STAT3/总 +54.6%；P < 0.005），从而促进了Il-6 (+69.2%；P < 0.05)和Il6r (+43.0%；P < 0.05)基因表达的增强，表明诱导了一个前馈回路。在C26_CM X-MET中通过gp130Fc选择性中和IL-6转信号，防止了STAT3的过度活化（-55.8%； P < 0.005），抵消了横截面积的减少（+28.2%； P < 0.05），并减少了蛋白酶因子的表达，包括肌环蛋白-1（-88%； P < 0.005）和ATROGIN1（-92%； P < 0.05），从而保持了三维肌肉系统的强度并增加了收缩力（+20%）。有趣的是，选择性抑制IL-6转信号调控了参与肌肉发生和凋亡的基因调控网络，规范了促凋亡miRNA的表达，包括miR-31（-53.2%； P < 0.05）和miR-34c（-65%； P < 0.005），并导致gp130Fc处理的C26_CM X-MET中凋亡途径的减少，突显出cleaved caspase 3的敏感减少（-92.5%； P < 0.005）。IL-6转信号似乎是对抗癌症恶病质相关变化的一个有前景的靶点。X-MET模型已经被证明是一个可靠的药物筛选工具，用于寻找新的治疗方法并在临床前研究中进行测试，显著减少了动物模型的使用。© 2023 The Authors. 由Wiley Periodicals LLC出版的《癌恶病质、肌萎缩和肌肉》杂志发表。

Causes 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.We 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.C26-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.IL-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.© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.