Assessing the involvement of tumor-secreted factors in the inhibition of muscle differentiation

Cancer cachexia is a multifactorial syndrome characterized by involuntary and pathological weight loss, predominantly caused by muscle wasting. While tumors can elicit detrimental effects on skeletal muscle function, the contribution of specific tumor-derived mediators remains elusive. To explore this, we investigated the impact of conditioned media (CM) from four cachexia-inducing tumor cell lines (KPC, 4662, LLC, and C26) on muscle differentiation using C2C12 cells. Creatine kinase (CK) activity was measured as an indicator of muscle wasting, and global gene expression changes in C2C12 cells were analyzed via RNA sequencing. Cytokine profiling of the CM identified 111 immune factors, and mimic combinations of the most abundant cytokines from KPC CM were tested for their effects on CK activity. Additionally, the involvement of tumor-derived PGE2 was assessed via CRISPR/Cas9-mediated knockout of the Ptgs2 gene in KPC cells. CM from all tumor cell lines significantly reduced CK activity in C2C12 cells, consistent with downregulation of CKm gene expression. Global gene expression profiles revealed upregulation of immune-related pathways in C2C12 cells exposed to KPC CM. However, mixtures of the 14 most abundant cytokines in CM had minimal effects on CK activity, and tumor-derived PGE2 showed no significant effect on CK activity or muscle cell differentiation. These findings suggest that the observed muscle-wasting effects of cachexia-inducing tumor cells cannot be replicated by the most abundant cytokines present in CM alone, highlighting the need for further research to identify the key tumor-derived factors responsible for cancer-induced muscle wasting.