Background: Pancreatic ductal adenocarcinoma (PDA) is highly malignant and often leads to a loss of skeletal muscle mass and sarcopenia, which is associated with poor outcomes. Although many clinical studies have reported IL-6 as a factor inducing sarcopenia, it remains unclear whether the IL-6, secreted from PDA, directly affects skeletal muscles.
Materials and Methods: Cell lines from KFC (LSL-Kras^G12D;p53^+/flPdx-cre) and KPC (LSL-Kras^G12D;p53^R172H/+Pdx-cre) mice and were employed. KFC cells were orthotopically injected into FVB/NJcl mice. The skeletal muscle were harvested 6 weeks after implantation. Co-culture experiments were performed using transwell inserts with PDA cells and the C2C12 myoblast cell line cultured with 10% FBS for 2 days and 2% horse serum for 5 days to induce cell differentiation. Expression of MyoD1, Myogenin, Acta1, and IL-6 were determined by qPCR analyses. C2C12 cells co-cultured with KPC cells were subjected to whole transcriptome analysis by RNA-seq. C2C12 myotubes were examined by immunofluorescence staining for myosin heavy chain (MF20 clone) and DAPI. The percentage of nuclei contained in myosin-positive myotubes was assessed as a fusion index. In the IL-6 knockdown experiment, KPC cells were infected with lentiviral constructs expressing shRNAs targeting IL-6 as well as a negative control shRNA.
Results: Orthotopic implantation of KFC PDA cells into syngeneic mice resulted in early retardation of body weight increase, independent from food intake (P<0.0001). A 19-31% loss in soleus, gastrocnemius, and quadriceps femoris muscle mass was observed (P<0.05). In vitro experiments revealed the C2C12 fusion index was significantly suppressed when co-cultured with KFC (31.9%) and KPC (48.7%) cell lines (P<0.01). Consistent with PDA cells blocking muscle differentiation, the expression of skeletal muscle differentiation markers (MyoD1, Myogenin and Acta1) was suppressed by in C2C12 co-cultured with KFC (58.2%) and KPC (86.2%) cells. Gene set enrichment analysis from transcriptome analysis of C2C12 cells co-cultured with KPC cells revealed that IL-6 signaling is strongly upregulated. Consistent with their differential effects on C2C12 differentiation, KPC cells express 1.6-fold more IL-6 than KFC cells. Importantly, C2C12 cells co-cultured with IL-6-deficient KPC cells exhibited a higher expression of skeletal differentiation markers (128.5%; P<0.05) and a larger fusion index (150.0%; P<0.01) than when co-cultured with control KPC cells.
Conclusions: The murine pancreatic cancer model demonstrated that IL-6 plays a crucial role in the development of sarcopenia. Interestingly, suppression of skeletal muscle differentiation was stronger in experiments employing the more malignant KPC cells than KFC cells. The suppression of IL-6 expression in pancreatic cancer cells alleviated the inhibition of skeletal muscle differentiation.

Yusuke Mizukami

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