A TGF-ß/KLF10 signaling axis regulates atrophy-associated genes to induce muscle wasting in pancreatic cancer.

Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to effective cancer management. Many potential therapies have been proposed and tested-including appetite stimulants, targeted cytokine blockers, and nutritional supplementation-yet highly effective therapies are lacking. Innovative approaches to treating cancer cachexia are needed. Members of the Kruppel-like factor (KLF) family play wide-ranging and important roles in the development, maintenance, and metabolism of skeletal muscle. Within the KLF family, we identified KLF10 upregulation in a multitude of wasting contexts-including in pancreatic, lung, and colon cancer mouse models as well as in human patients. We subsequently interrogated loss-of-function of KLF10 as a potential strategy to mitigate cancer associated muscle wasting. In vivo studies leveraging orthotopic implantation of pancreas cancer cells into wild-type and KLF10 KO mice revealed significant preservation of lean mass and robust suppression of pro-atrophy muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other factors implicated in atrophy, calcium signaling, and autophagy. Bioinformatics analyses identified Transforming growth factor beta (TGF-ß), a known inducer of KLF10 and cachexia promoting factor, as a key upstream regulator of KLF10. We provide direct in vivo evidence that KLF10 KO mice are resistant to the atrophic effects of TGF-ß. ChIP-based binding studies demonstrated direct binding to Trim63, a known wasting-associated atrogene. Taken together, we report a critical role for the TGF-ß/KLF10 axis in the etiology of pancreatic cancer-associated muscle wasting and highlight the utility of targeting KLF10 as a strategy to prevent muscle wasting and limit cancer-associated cachexia.

Muscle stem cells contribute to long-term tissue repletion following surgical sepsis.

BACKGROUND: Over the past decade, advances in sepsis identification and management have resulted in decreased sepsis mortality. This increase in survivorship has highlighted a new clinical obstacle: chronic critical illness (CCI), for which there are no effective treatment options. Up to half of sepsis survivors suffer from CCI, which can include multi-organ dysfunction, chronic inflammation, muscle wasting, physical and mental disabilities, and enhanced frailty. These symptoms prevent survivors from returning to regular day-to-day activities and are directly associated with poor quality of life. METHODS: Mice were subjected to cecal ligation and puncture (CLP) with daily chronic stress (DCS) as an in vivo model to study sepsis late-effects/sequelae on skeletal muscle components. Longitudinal monitoring was performed via magnetic resonance imaging, skeletal muscle and/or muscle stem cell (MuSCs) assays (e.g., post-necropsy wet muscle weights, minimum Feret diameter measurements, in vitro MuSC proliferation and differentiation, number of regenerating myofibres and numbers of Pax7-positive nuclei per myofibre), post-sepsis whole muscle metabolomics and MuSC isolation and high-content transcriptional profiling. RESULTS: We report several findings supporting the hypothesis that MuSCs/muscle regeneration are critically involved in post-sepsis muscle recovery. First, we show that genetic ablation of muscle stem cells (MuSCs) impairs post-sepsis muscle recovery (maintenance of 5-8% average lean mass loss compared with controls). Second, we observe impaired MuSCs expansion capacity and morphological defects at 26 days post-sepsis compared with control MuSCs (P < 0.001). Third, when subjected to an experimental muscle injury, sepsis-recovered mice exhibited evidence of impaired muscle regeneration compared with non-septic mice receiving the same muscle injury (CLP/DCS injured mean minimum Feret is 92.1% of control injured, P < 0.01). Fourth, we performed a longitudinal RNA sequencing study on MuSCs isolated from post-sepsis mice and found clear transcriptional differences in all post-sepsis samples compared with controls. At Day 28, CLP/DCS mice satellite cells have multiple altered metabolic pathways, such as oxidative phosphorylation, mitochondrial dysfunction, sirtuin signalling and oestrogen receptor signalling, compared with controls (P < 0.001). CONCLUSIONS: Our data show that MuSCs and muscle regeneration are required for effective post-sepsis muscle recovery and that sepsis triggers morphological, functional, and transcriptional changes in MuSCs. Moving forward, we strive to leverage a more complete understanding of post-sepsis MuSC/regenerative defects to identify and test novel therapies that promote muscle recovery and improve quality of life in sepsis survivors.

ACUTE AND SUSTAINED ALTERATIONS TO THE BONE MARROW IMMUNE MICROENVIRONMENT FOLLOWING POLYMICROBIAL INFECTION.

ABSTRACT: Sepsis is a highly prevalent cause of death in intensive care units. Characterized by severe immune cell derangements, sepsis is often associated with multiorgan dysfunction. For many sepsis survivors, these deficits can persist long after clinical resolution of the underlying infection. Although many studies report on the impact of sepsis on individual immune cell subtypes, a comprehensive analysis of sepsis-induced alterations within and across the immune cell landscape is lacking. In this study, we used single-cell RNA sequencing to assess sepsis-associated transcriptional changes in immune cells isolated from bone marrow at single-cell resolution. We used a high-survival fecal-induced peritonitis sepsis model using Friend leukemia virus B mice. Single-cell RNA sequencing classified 3402 single cells from control subjects into 14 clusters representing long-term hematopoietic stem cell (HSC), short-term HSC, basophil, dendritic cell, eosinophil, erythroblast, erythrocyte, macrophage, neutrophil, natural killer cell, plasma cell, plasmacytoid dendritic cell, pre-B cell, and T memory cell lineages. One day following experimentally induced sepsis, cell type compositions shifted significantly and included notable decreases in HSC and myeloid cell abundance. In addition to proportional cell composition changes, acute sepsis induced significant transcriptional alterations in most immune cell types analyzed-changes that failed to completely resolve 1 month after sepsis. Taken together, we report widespread and persistent transcriptional changes in diverse immune cells in response to polymicrobial infection. This study will serve as a valuable resource for future work investigating acute and/or long-term sepsis-associated immune cell derangements.

Anticachectic regulator analysis reveals Perp-dependent antitumorigenic properties of 3-methyladenine in pancreatic cancer.

Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA-treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp, and (c) found that 3-MA-associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.

The wasting-associated metabolite succinate disrupts myogenesis and impairs skeletal muscle regeneration.

Background: Muscle wasting is a debilitating co-morbidity affecting most advanced cancer patients. Alongside enhanced muscle catabolism, defects in muscle repair/regeneration contribute to cancer-associated wasting. Among the factors implicated in suppression of muscle regeneration are cytokines that interfere with myogenic signal transduction pathways. Less understood is how other cancer/wasting-associated cues, such as metabolites, contribute to muscle dysfunction. This study investigates how the metabolite succinate affects myogenesis and muscle regeneration. Methods: We leveraged an established ectopic metabolite treatment (cell permeable dimethyl-succinate) strategy to evaluate the ability of intracellular succinate elevation to 1) affect myoblast homeostasis (proliferation, apoptosis), 2) disrupt protein dynamics and induce wasting-associated atrophy, and 3) modulate in vitro myogenesis. In vivo succinate supplementation experiments (2% succinate, 1% sucrose vehicle) were used to corroborate and extend in vitro observations. Metabolic profiling and functional metabolic studies were then performed to investigate the impact of succinate elevation on mitochondria function. Results: We found that in vitro succinate supplementation elevated intracellular succinate about 2-fold, and did not have an impact on proliferation or apoptosis of C2C12 myoblasts. Elevated succinate had minor effects on protein homeostasis (~25% decrease in protein synthesis assessed by OPP staining), and no significant effect on myotube atrophy. Succinate elevation interfered with in vitro myoblast differentiation, characterized by significant decreases in late markers of myogenesis and fewer nuclei per myosin heavy chain positive structure (assessed by immunofluorescence staining). While mice orally administered succinate did not exhibit changes in overall body composition or whole muscle weights, these mice displayed smaller muscle myofiber diameters (~6% decrease in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distribution), which was exacerbated when muscle regeneration was induced with barium chloride injury. Significant decreases in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distributions were observed 7 days and 28 days post injury. Elevated numbers of myogenin positive cells (3-fold increase) supportive of the differentiation defects observed in vitro were observed 28 days post injury. Metabolic profiling and functional metabolic assessment of myoblasts revealed that succinate elevation caused both widespread metabolic changes and significantly lowered maximal cellular respiration (~35% decrease). Conclusions: This study broadens the repertoire of wasting-associated factors that can directly modulate muscle progenitor cell function and strengthens the hypothesis that metabolic derangements are significant contributors to impaired muscle regeneration, an important aspect of cancer-associated muscle wasting.

Single-cell deconstruction of post-sepsis skeletal muscle and adipose tissue microenvironments.

BACKGROUND: Persistent loss of skeletal muscle mass and function as well as altered fat metabolism are frequently observed in severe sepsis survivors. Studies examining sepsis-associated tissue dysfunction from the perspective of the tissue microenvironment are scarce. In this study, we comprehensively assessed transcriptional changes in muscle and fat at single-cell resolution following experimental sepsis induction. METHODS: Skeletal muscle and visceral white adipose tissue from control mice or mice 1 day or 1 month following faecal slurry-induced sepsis were used. Single cells were mechanically and enzymatically prepared from whole tissue, and viable cells were further isolated by fluorescence activated cell sorting. Droplet-based single-cell RNA-sequencing (scRNA-seq; 10× Genomics) was used to generate single-cell gene expression profiles of thousands of muscle and fat-resident cells. Bioinformatics analyses were performed to identify and compare individual cell populations in both tissues. RESULTS: In skeletal muscle, scRNA-seq analysis classified 1438 single cells into myocytes, endothelial cells, fibroblasts, mesenchymal stem cells, macrophages, neutrophils, T-cells, B-cells, and dendritic cells. In adipose tissue, scRNA-seq analysis classified 2281 single cells into adipose stem cells, preadipocytes, endothelial cells, fibroblasts, macrophages, dendritic cells, B-cells, T-cells, NK cells, and gamma delta T-cells. One day post-sepsis, the proportion of most non-immune cell populations was decreased, while immune cell populations, particularly neutrophils and macrophages, were highly enriched. Proportional changes of endothelial cells, neutrophils, and macrophages were validated using faecal slurry and cecal ligation and puncture models. At 1 month post-sepsis, we observed persistent enrichment/depletion of cell populations and further uncovered a cell-type and tissue-specific ability to return to a baseline transcriptomic state. Differential gene expression analyses revealed key genes and pathways altered in post-sepsis muscle and fat and highlighted the engagement of infection/inflammation and tissue damage signalling. Finally, regulator analysis identified gonadotropin-releasing hormone and Bay 11-7082 as targets/compounds that we show can reduce sepsis-associated loss of lean or fat mass. CONCLUSIONS: These data demonstrate persistent post-sepsis muscle and adipose tissue disruption at the single-cell level and highlight opportunities to combat long-term post-sepsis tissue wasting using bioinformatics-guided therapeutic interventions.

A novel transplantable model of lung cancer-associated tissue loss and disrupted muscle regeneration.

BACKGROUND: Cancer-associated muscle wasting (CAW), a symptom of cancer cachexia, is associated with approximately 20% of lung cancer deaths and remains poorly characterized on a mechanistic level. Current animal models for lung cancer-associated cachexia are limited in that they (1) primarily employ flank transplantation methods, (2) have short survival times not reflective of the patient condition, and (3) are typically performed in young mice not representative of mean patient age. This study investigates a new model for lung cancer-associated cachexia that can address these issues and also implicates muscle regeneration as a contributor to CAW. METHODS: We used tail vein injection as a method to introduce tumor cells that seed primarily in the lungs of mice. Body composition of tumor-bearing mice was longitudinally tracked using NMR-based, echo magnetic resonance imaging (echoMRI). These data were combined with histological and molecular assessments of skeletal muscle to provide a complete analysis of muscle wasting. RESULTS: In this new lung CAW model, we observed (1) progressive loss in whole body weight, (2) progressive loss of lean and fat mass, (3) a circulating cytokine/inflammatory profile similar to that seen in other models of CAW, (4) histological changes associated with muscle wasting, and (5) molecular changes in muscle that implicate suppression of muscle repair/regeneration. Finally, we show that survival can be extended without lessening CAW by titrating injected cell number. CONCLUSIONS: Overall, this study describes a new model of CAW that could be useful for further studies of lung cancer-associated wasting and accompanying changes in the regenerative capacity of muscle. Additionally, this model addresses many recent concerns with existing models such as immunocompetence, tumor location, and survival time.