Single‐Cell RNA‐Sequencing Reveals Cachectic Satellite Cell Population in Muscle of Male Mice With Cancer Cachexia

ABSTRACT Background Cancer cachexia leads to decreases in body mass, lean mass and fat mass, decreased therapeutic potential and ~20% of cancer‐related deaths. While several studies have demonstrated changes to components of the muscle microenvironment with cancer cachexia, none have comprehensively assessed changes to cellular dynamics across the duration of cachexia development. Methods Single‐cell RNA‐sequencing was performed on hindlimb muscles of male mice with 2‐, 2.5‐ and 3.5‐week subcutaneous Lewis‐lung carcinoma tumours. Cell population changes were confirmed with flow cytometry. Results Body mass (−0.51 g; p = 0.0014) and lean mass (−0.85 g; p = 0.0134) were decreased at 2.5 weeks and were significantly lower than sham. Increases in fat mass were attenuated starting at 2 weeks (0.70 g; p = 0.0408) compared to sham (1.55 g), and muscle cross‐sectional area decreased at 3.5 weeks (−14.81%; p = 0.0022) compared to sham. We report a novel cachexia‐associated satellite cell subcluster, comprising 71.1% of the population at 3.5 weeks, corresponding with a +20.33% increase in cell size ( p = 0.0266) and +19.73% increase in the proportion of activated PAX7 + MYOD + cells after 24 h cultured on individual myofibres ( p = 0.0226). This cachexia‐associated subcluster was also present in C26 tumour‐bearing mice and had a unique gene expression signature compared to other muscle wasting disorders. The cachexia‐associated subcluster was enriched for signalling pathways (IL‐17, TNF, p53, NF‐κB, FoxO, adipocytokines, NOD‐like receptor, MAPK and JAK–STAT) implicated in satellite cell dysfunction in cancer cachexia. Prior to the emergence of cachexia‐associated satellite cells, increases in CD11b + (+928.01%; p < 0.0001), Ly6C low (+1080.85%; p < 0.0001), Ly6C high (+920.33%; p = 0.0002), F4/80 + CD206 − (+299.22%; p = 0.0039), F4/80 + CD206 + (+1466.40%; p < 0.0001) immune cell populations were observed at 2 weeks compared to sham and returned to baseline by 2.5 weeks. There was also an increase in PDGFRα + fibro‐adipogenic progenitors at 2 weeks (+53.44%; p = 0.0398) and decreased CD31 + endothelial cells at 2 weeks (−57.37%; p = 0.0014) and 3.5 weeks (−39.78%; p = 0.0213) compared to sham, with no change in ITGA7 + satellite cells ( p = 0.4271). Cell communication analyses revealed a decline in cell communication with cancer cachexia in all cells except for monocytes/macrophages, and a decrease in cell adhesion‐related signalling in cachexia‐associated satellite cells, which is important for satellite cell differentiation, and may help to explain differentiation defects with cachexia. Conclusions We describe a novel satellite cell subcluster unique to cachexia. We also identified increased immune cell and fibroadipogenic progenitor content and decreased endothelial cell content that precede muscle wasting with cancer, suggesting a role for these cell populations in satellite cell dysfunction and muscle atrophy in this condition.