Doles, Jason D.

AbstractBackgroundOver 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.MethodsMice 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.ResultsWe 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).ConclusionsOur 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.

BACKGROUND A patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO’s ability to predict clinical response to gastrointestinal (GI) cancers. METHODS We generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments. RESULTS We report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures. CONCLUSION While we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers. TRIAL REGISTRATION Not applicable. FUNDING The Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).

Abstract Cachexia is a multifactorial syndrome characterized by extensive body weight, lean muscle and fat mass loss. Cachexia worsens cancer patient prognoses and contributes to poor chemotherapy tolerance and response rates. About 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 cachexia therapies. A major contributing factor could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged murine model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative transcriptomic analyses of the muscles identified 3-methyladenine (3-MA), a known autophagy inhibitor, as a candidate anti-wasting compound. In vitro analyses confirmed anti-wasting effects while in vivo analysis demonstrated that 3-MA could rescue muscle wasting and suppress tumor growth. Surprisingly, transcriptome analyses of 3MA-treated tumor cells did not reveal autophagy-related signatures but instead implicated Perp as a novel 3-MA target. Probing further, we 1) observed significantly higher expression of Perp in pancreatic cancer cell lines compared to control cells, 2) noted a survival disadvantage associated with elevated PERP in the TCGA database, 3) found that PERP knockdown in cancer cells led to reduced tumor proliferation, and 4) confirmed that 3-MA-associated Perp reduction inhibited tumor growth. Moreover, we also demonstrate that survival benefits associated with 3MA is independent of tumor progression. Finally, we queried Perp expression in human tumor samples and observed increased Perp expression in adenocarcinoma regions compared to tumor adjacent tissue. Taken together, we report a novel mechanism linking 3-MA to Perp inhibition, and further implicate Perp as a novel tumor promoting factor in pancreatic cancer. Citation Format: Aneesha Dasgupta, Paige C. Arneson-Wissink, Rebecca E. Schmitt, Dong Seong Cho, Alexandra M. Ducharme, Tara L. Hogenson, Eugene W. Krueger, William R. Bamlet, Lizhi Zhang, Gina Razidlo, Martin E. Fernandez-Zapico, Jason D. Doles. Identification of perp as a tumor promoter in pancreatic cancer via the utilization of an aged model of cancer cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3995.

Abstract Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to better cancer therapy. Many potential therapies have been suggested and tested - including appetite stimulants, targeted cytokine blockers, and nutritional supplementation, but effective therapies have still not found a place in the clinic. Thus, a new approach to treating cancer cachexia is warranted. Members of the Kruppel-like factor family play wide-ranging and important roles in the development, maintenance, and metabolism of muscle. Upon review both our own and multiple publically available datasets, we identified KLF10 upregulation in a multitude of cachectic muscles - including in pancreatic, lung, and colon cancer mouse models and in human cachexia patients. Thus, in this study, we interrogated loss-of-function of KLF10 in the context of pancreatic cancer associated muscle wasting. Ex vivo experiments on satellite cells isolated from KLF10 Knockout (KO) mice showed resistance to the effect of KPC conditioned media on atrophy. In vivo studies using a KLF10 KO mouse model showed that upon orthotopic implantation of T4-KPC cells, the mice demonstrated a significant preservation of lean mass and significant alterations in the expression of muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other genes implicated in atrophy, calcium signaling, and autophagy. Pathway analysis of RNA-seq data from these mice identified TGF-B, a known inducer of KLF10, as an important upstream regulator of the response. Further in vivo experimentation showed KLF10 KO mice to be resistant to the atrophic effects of TGF-B injections. In conclusion, we report a novel role of KLF10 in the manifestation of cachexia in pancreatic cancer. Citation Format: Daniel F. Gibbard, Aneesha Dasgupta, Rebecca E. Schmitt, Alexandra M. Ducharme, Paige C. Arneson, Elizabeth S. Bruinsma, John R. Hawse, Jason D. Doles. TGF-B/KLF10 signaling regulates muscle wasting in pancreatic cancer cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3985.

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.

AbstractBackgroundPersistent 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.MethodsSkeletal 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.ResultsIn 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.ConclusionsThese 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.

AbstractBackgroundMuscle 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.MethodsWe leveraged an established ectopic metabolite treatment (cell permeable dimethyl‐succinate) strategy to evaluate the ability of intracellular succinate elevation to (i) affect myoblast homeostasis (proliferation and apoptosis), (ii) disrupt protein dynamics and induce wasting‐associated atrophy, and (iii) modulate in vitro myogenesis. In vivo succinate supplementation experiments (2% succinate and 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.ResultsWe 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 O‐propargyl‐puromycin 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 and 28 days post injury. Elevated numbers of myogenin positive cells (three‐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).ConclusionsThis 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.

Obesity is a serious health concern and is associated with a reduced quality of life and a number of chronic diseases, including diabetes, heart disease, stroke, and cancer. With obesity rates on the rise worldwide, adipose tissue biology has become a top biomedical research priority. Despite steady growth in obesity-related research, more investigation into the basic biology of adipose tissue is needed to drive innovative solutions aiming to curtail the obesity epidemic. Adipose progenitor cells (APCs) play a central role in adipose tissue homeostasis and coordinate adipose tissue expansion and remodeling. Although APCs are well studied, defining and characterizing APC subsets remains ambiguous because of ill-defined cellular heterogeneity within this cellular compartment. In this study, we used single-cell RNA sequencing to create a cellular atlas of APC heterogeneity in mouse visceral adipose tissue. Our analysis identified two distinct populations of adipose tissue–derived stem cells (ASCs) and three distinct populations of preadipocytes (PAs). We identified novel cell surface markers that, when used in combination with traditional ASC and preadipocyte markers, could discriminate between these APC subpopulations by flow cytometry. Prospective isolation and molecular characterization of these APC subpopulations confirmed single-cell RNA sequencing gene expression signatures, and ex vivo culture revealed differential expansion/differentiation capabilities. Obese visceral adipose tissue featured relative expansion of less mature ASC and PA subpopulations, and expression analyses revealed major obesity-associated signaling alterations within each APC subpopulation. Taken together, our study highlights cellular and transcriptional heterogeneity within the APC pool, provides new tools to prospectively isolate and study these novel subpopulations, and underscores the importance of considering APC diversity when studying the etiology of obesity.

Duchenne muscular dystrophy (DMD) is a musculoskeletal disorder that causes severe morbidity and reduced lifespan. Individuals with DMD have an X-linked mutation that impairs their ability to produce functional dystrophin protein in muscle. No cure exists for this disease and the few therapies that are available do not dramatically delay disease progression. Thus, there is a need to better understand the mechanisms underlying DMD which may ultimately lead to improved treatment options. The muscular dystrophy (MDX) mouse model is frequently used to explore DMD disease traits. Though some studies of metabolism in dystrophic mice exist, few have characterized metabolic profiles of supporting cells in the diseased environment. Using nontargeted metabolomics we characterized metabolic alterations in muscle satellite cells (SCs) and serum of MDX mice. Additionally, live-cell imaging revealed MDX-derived adipose progenitor cell (APC) defects. Finally, metabolomic studies revealed a striking elevation of acylcarnitines in MDX APCs, which we show can inhibit APC proliferation. Together, these studies highlight widespread metabolic alterations in multiple progenitor cell types and serum from MDX mice and implicate dystrophy-associated metabolite imbalances in APCs as a potential contributor to adipose tissue disequilibrium in DMD.