scRNAseq Data Analysis Code Muscle Inflammation is Regulated by NF-kB From Multiple Cells to Control Distinct States of Wasting in Cancer Cachexia

Although cancer cachexia is classically characterized as a systemic inflammatory disorder, emerging evidence indicates that weight loss also associates with local tissue inflammation. We queried the regulation of this inflammation and its causality to cachexia by exploring skeletal muscle, whose atrophy strongly associates with poor outcome. Using multiple mouse models and patient samples, we show that cachectic muscle is marked by enhanced innate immunity. NF-kB activity in multiple cells, including satellite cells, myofibers, and fibro-adipogenic progenitors, promotes macrophage expansion derived equally from infiltrating monocytes and resident tissue. Moreover, NF-kB activated cells and macrophages undergo crosstalk: whereas NF-kB+ cells recruit macrophages to inhibit regeneration and promote atrophy, while interestingly also protecting myofibers, macrophages stimulate NF-kB+ cells in a feed forward loop to sustain inflammation. Together, we propose that NF-kB functions in multiple cells in the muscle microenvironment to stimulate macrophage inflammation that both promotes and protects against muscle wasting in cancer. scRNA sequencing was done for 3 datasets, one from Human Pancreatic ductal adenocarcinoma (PDAC) and other two from cachexia in mice induced by C26 colon cancer cells and cachexia in KPP mice(KrasLSL-G12D/+, Ptenf/f, Ptf1aER-Cre/+).For the human dataset Muscles from human samples were taken at time of pancreatic tumor resection, or time of other abdominal surgery for control patients. Muscle were digested in an enzymatic buffer and CD45 negative and positive cells were separated by MACs beads. Both fractions were sent for single cell RNA sequencing. Samples include CD45-/CD45+ Weight Stable PDAC(Neg_WS_PDAC,Pos_WS_PDAC),CD45-/CD45+ Control(Neg_Control,Pos_Control) and CD45-/CD45+ Cachetic PDAC(Neg_C_PDAC, Pos_C_PDAC). For cachexia in mice we induced cancer by injecting the C-26 colon cancer cells subcutaneously in CD2F1 mice. At endpoint, hindlimb muscle was extracted and enzymatically digested. MACs beads were used to sort CD45 positive and negative fractions for single cell sequencing.Samples include CD45-/+ Control and CD45-/+ C26 cachexia from two batches(S1 and S2). For cachexia in KPP mice tamoxifen was injected for 5 days to induce pancreatic tumor formation. Mice were euthanized after tumor formation and cachexia development.MACs beads were used to sort CD45 positive and negative fractions for single cell sequencing. Samples include CD45-/+ Control and CD45-/+ Kpp cachexia.