Neutrophil- And Myeloid-derived Suppressor Cell-Conditioned Medium Causes Myotube Atrophy

Cancer can cause altered hematopoiesis resulting in excessive neutrophil production and reduced lymphoid and erythroid cell production. This can promote cancer progression by limiting production of potentially cancer-fighting cells (i.e., cytotoxic T cells) and increasing immunosuppressive cells related to neutrophils called myeloid-derived suppressor cells (MDSCs). In addition to their pro-cancer effects, we and other groups have recently found that MDSCs also contribute to the loss of skeletal muscle mass and function associated with cancer, a condition known as cancer cachexia. As of yet, it is not known if MDSCs can cause skeletal muscle atrophy directly, nor is it known if the specific immunosuppressive phenotype of MDSCs is required to produce muscle atrophy, or if neutrophils themselves can also cause muscle atrophy. The PURPOSE of this study was to test if MDSC-conditioned cell culture medium can cause myotube atrophy compared to neutrophil-conditioned medium and control mediums (fresh medium and cytokine-spiked medium). METHODS: Neutrophils were collected from a healthy male donor using magnetic activated cell sorting and were cultured in one of two conditions. 1) control neutrophils were cultured overnight in standard medium. 2) Treated neutrophils were cultured for 24 hours with the same medium with the addition of two cytokines that have been shown to induce the MDSC phenotype (10 ng/ml of IL-6 and 10 ng/ml GM-CSF). As an additional control, an equal volume of medium containing the cytokines was placed in a flask with no cells. Conditioned medium from all three conditions were then added to fully differentiated C2C12 myotubes. After 24 hours, the myotubes were imaged and myotube diameter was measured. RESULTS: Myotubes exposed to Neutrophil- (26.9 ± 9.6 um) or MDSC-conditioned medium (26.8 ± 13.6 um) were significantly smaller than myotubes grown in fresh medium (32 ± 15.4 um). Myotubes exposed to the control medium (containing cytokines, but not conditioned by cells) were not smaller than control myotubes (31.6 ± 11.8 um). CONCLUSIONS: These results show that neutrophils, whether untreated or exposed to a MDSC-inducing stimulus (IL-6 and GM-CSF), secrete muscle atrophy-producing factors. This finding may help in understanding the mechanism of muscle atrophy that occurs in cancer cachexia. UNM Research Allocations Committee