Local inflammation precedes respiratory muscle wasting and fibrotic remodeling in a mouse model of PDAC
Cancer cachexia represents a debilitating muscle wasting condition that is highly prevalent in patients with pancreatic ductal adenocarcinoma (PDAC). Cachexia is estimated to contribute to ~30% of cancer-related deaths, with deterioration of respiratory muscles suspected to be a key contributor to cachexia-associated morbidity and mortality. In recent studies, we identified inflammation and fibrotic remodeling of respiratory accessory muscles as a key feature of human PDAC cachexia. To gain insight into mechanisms driving respiratory muscle wasting and fibrotic remodeling in response to PDAC, we conducted temporal histological and transcriptomic analyses on diaphragm muscles harvested from mice bearing orthotopic murine pancreatic (KPC) tumors at time points reflective of pre-cachexia (D8 and D10), mild-moderate cachexia (D12 and D14) and advanced cachexia (endpoint). Compared to Sham, KPC mice showed progressive diaphragm myofiber atrophy beginning on D12 (-16 to -24% , D12toendpointvs.Sham,P< 0.05), which preceded reductions in breathing frequency (-55%,P= 0.0074) and diaphragm excursion (-43%,P= 0.0006) observed at endpoint. During the pre-cachexia phase, diaphragms showed significantleukocyte infiltration (+3 fold to +13-fold; D8–endpointvs.Sham,P< 0.05) and transcriptomic enrichment of inflammatory processes associated with tissue injury that remained increased through endpoint. Diaphragm inflammation was followed by increases in PDGFR+ fibroadipogenic progenitors (+2.5 to +3.8-fold; D10–endpointvs.Sham,P< 0.05), ECM expansion (+1.5 to +1.8-fold; D14–endpointvs.Sham),P<0.05) and collagen accumulation (+3.8-fold; Endpointvs.Sham,P= 0.0013). These processes were supported by changes in the diaphragm transcriptome. Ingenuity pathway analysis predicted factors involved in inflammatory responses to tissue injury, including TGF-B1, Angiotensin, and PDGF BB, as top upstream regulators activated in diaphragms prior to and throughout cachexia progression, while PGC-1a and the insulin receptor were among the top upstream regulators predicted to be suppressed. The transcriptomic dataset further revealed progressive disturbances to networks involved in lipid, glucose and oxidative metabolism, activation of the unfolded protein response and neuromuscular junction remodeling associated with denervation. In summary, our data support leukocyte infiltration and fibroadipogenic progenitor expansion as early events that precede wasting and fibrotic remodeling of respiratory muscles in response to PDAC that may also underlie metabolic disturbances, weakness and respiratory complications. Overall design: Diaphragm muscles (three biological replicates per time point / phenotype) were harvested from  non-tumor-bearing mice (Sham/Control), and tumor-bearing mice at day 8 (KPC D8), day 10 (KPC D10), day 12 (KPC D12), day 14 (KPC D14) and humane-endpoint (KPC END) after KPC cell orthotopic injection