Targeted nutritional intervention attenuates experimental lung cancer cachexia.

BACKGROUND: Cachexia, a syndrome with high prevalence in non-small cell lung cancer patients, impairs quality of life and reduces tolerance and responsiveness to cancer therapy resulting in decreased survival. Optimal nutritional care is pivotal in the treatment of cachexia and a recommended cornerstone of multimodal therapy. Here, we investigated the therapeutic effect of an intervention diet consisting of a specific combination of high protein, leucine, fish oil, vitamin D, galacto-oligosaccharides, and fructo-oligosaccharides on the development and progression of cachexia in an orthotopic lung cancer mouse model. METHODS: Eleven-week-old male 129S2/Sv mice were orthotopically implanted with 344P lung epithelial tumour cells or vehicle (control). Seven days post-implantation tumour-bearing (TB) mice were allocated to either intervention- or isocaloric control diet. Cachexia was defined as 5 days of consecutive body weight loss, after which mice were euthanized for tissue analyses. RESULTS: TB mice developed cachexia accompanied by significant loss of skeletal muscle mass and epididymal fat mass compared with sham operated mice. The cachectic endpoint was significantly delayed (46.0 ± 15.2 vs. 34.7 ± 11.4 days), and the amount (-1.57 ± 0.62 vs. -2.13 ± 0.57 g) and progression (-0.26 ± 0.14 vs. -0.39 ± 0.11 g/day) of body weight loss were significantly reduced by the intervention compared with control diet. Moreover, systemic inflammation (pentraxin-2 plasma levels) and alterations in molecular markers for proteolysis and protein synthesis, indicative of muscle atrophy signalling in TB-mice, were suppressed in skeletal muscle by the intervention diet. CONCLUSIONS: Together, these data demonstrate the potential of this multinutrient intervention, targeting multiple components of cachexia, as integral part of lung cancer management.

Cachexia and Sarcopenia in Oligometastatic Non-Small Cell Lung Cancer: Making a Potential Curable Disease Incurable?

Among patients with advanced NSCLC, there is a group of patients with synchronous oligometastatic disease (sOMD), defined as a limited number of metastases detected at the time of diagnosis. As cachexia and sarcopenia are linked to poor survival, incorporating this information could assist clinicians in determining whether a radical treatment should be administered. In a retrospective multicenter study, including all patients with adequately staged (FDG-PET, brain imaging) sOMD according to the EORTC definition, we aimed to assess the relationship between cachexia and/or sarcopenia and survival. Of the 439 patients that were identified between 2015 and 2021, 234 met the criteria for inclusion and were included. The median age of the cohort was 67, 52.6% were male, and the median number of metastasis was 1. Forty-six (19.7%) patients had cachexia, thirty-four (14.5%) had sarcopenia and twenty-one (9.0%) had both. With a median follow-up of 49.7 months, median PFS and OS were 8.6 and 17.3 months, respectively. Moreover, a trend toward longer PFS was found in patients without cachexia and sarcopenia compared to those with cachexia and/or sarcopenia. In multivariate analysis, cachexia and sarcopenia were not associated with an inferior survival, irrespective of receiving radical treatment. High CRP was associated with inferior survival and could be a prognostic factor, helping the decision of clinicians in selecting patients who may benefit from the addition of LRT. However, despite the homogeneous definition of oligometastatic disease and the adequate staging, our subgroups were small. Therefore, further studies are needed to better understand our hypothesis and generating findings.

Precision Medicine for More Oxygen (P4O2)-Study Design and First Results of the Long COVID-19 Extension.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic has led to the death of almost 7 million people, however, with a cumulative incidence of 0.76 billion, most people survive COVID-19. Several studies indicate that the acute phase of COVID-19 may be followed by persistent symptoms including fatigue, dyspnea, headache, musculoskeletal symptoms, and pulmonary functional-and radiological abnormalities. However, the impact of COVID-19 on long-term health outcomes remains to be elucidated. Aims: The Precision Medicine for more Oxygen (P4O2) consortium COVID-19 extension aims to identify long COVID patients that are at risk for developing chronic lung disease and furthermore, to identify treatable traits and innovative personalized therapeutic strategies for prevention and treatment. This study aims to describe the study design and first results of the P4O2 COVID-19 cohort. Methods: The P4O2 COVID-19 study is a prospective multicenter cohort study that includes nested personalized counseling intervention trial. Patients, aged 40-65 years, were recruited from outpatient post-COVID clinics from five hospitals in The Netherlands. During study visits at 3-6 and 12-18 months post-COVID-19, data from medical records, pulmonary function tests, chest computed tomography scans and biological samples were collected and questionnaires were administered. Furthermore, exposome data was collected at the patient's home and state-of-the-art imaging techniques as well as multi-omics analyses will be performed on collected data. Results: 95 long COVID patients were enrolled between May 2021 and September 2022. The current study showed persistence of clinical symptoms and signs of pulmonary function test/radiological abnormalities in post-COVID patients at 3-6 months post-COVID. The most commonly reported symptoms included respiratory symptoms (78.9%), neurological symptoms (68.4%) and fatigue (67.4%). Female sex and infection with the Delta, compared with the Beta, SARS-CoV-2 variant were significantly associated with more persisting symptom categories. Conclusions: The P4O2 COVID-19 study contributes to our understanding of the long-term health impacts of COVID-19. Furthermore, P4O2 COVID-19 can lead to the identification of different phenotypes of long COVID patients, for example those that are at risk for developing chronic lung disease. Understanding the mechanisms behind the different phenotypes and identifying these patients at an early stage can help to develop and optimize prevention and treatment strategies.

Adenosine monophosphate-activated protein kinase is elevated in human cachectic muscle and prevents cancer-induced metabolic dysfunction in mice.

BACKGROUND: Metabolic dysfunction and cachexia are associated with poor cancer prognosis. With no pharmacological treatments, it is crucial to define the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia. Adenosine monophosphate-activated protein kinase (AMPK) connects metabolic and muscle mass regulation. As AMPK could be a potential treatment target, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. We therefore established AMPK's roles in cancer-associated metabolic dysfunction, insulin resistance and cachexia. METHODS: In vastus lateralis muscle biopsies from n = 26 patients with non-small cell lung cancer (NSCLC), AMPK signalling and protein content were examined by immunoblotting. To determine the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead [KiDe]) specifically in striated muscle were inoculated with Lewis lung carcinoma (LLC) cells (wild type [WT]: n = 27, WT + LLC: n = 34, mAMPK-KiDe: n = 23, mAMPK-KiDe + LLC: n = 38). Moreover, male LLC-tumour-bearing mice were treated with (n = 10)/without (n = 9) 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13 days. Littermate mice were used as controls. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose and insulin tolerance tests, tissue-specific 2-[3H]deoxy-d-glucose (2-DG) uptake and immunoblotting. RESULTS: Patients with NSCLC presented increased muscle protein content of AMPK subunits α1, α2, ß2, γ1 and γ3 ranging from +27% to +79% compared with control subjects. In patients with NSCLC, AMPK subunit protein content correlated with weight loss (α1, α2, ß2 and γ1), fat-free mass (α1, ß2 and γ1) and fat mass (α1 and γ1). Tumour-bearing mAMPK-KiDe mice presented increased fat loss and glucose and insulin intolerance. LLC in mAMPK-KiDe mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps: -35%, soleus: -49%, extensor digitorum longus: -48%) and the heart (-29%) than that in non-tumour-bearing mice. In skeletal muscle, mAMPK-KiDe abrogated the tumour-induced increase in insulin-stimulated TBC1D4thr642 phosphorylation. The protein content of TBC1D4 (+26%), pyruvate dehydrogenase (PDH; +94%), PDH kinases (+45% to +100%) and glycogen synthase (+48%) was increased in skeletal muscle of tumour-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase II protein content and normalized phosphorylation of p70S6Kthr389 (mTORC1 substrate) and ACCser212 (AMPK substrate) and rescued cancer-induced insulin intolerance. CONCLUSIONS: Protein contents of AMPK subunits were upregulated in skeletal muscle of patients with NSCLC. AMPK activation seemed protectively inferred by AMPK-deficient mice developing metabolic dysfunction in response to cancer, including AMPK-dependent regulation of multiple proteins crucial for glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and possibly cachexia.

An optimization and refinement of the whole-gut transit assay in mice.

BACKGROUND: Gastrointestinal motility measurements in mice are currently performed under suboptimal conditions, as these nocturnal animals are measured during light conditions. In addition, other stressors, like individual housing, placement in a new cage during observation, and lack of bedding and cage enrichment cause animal discomfort and might contribute to higher variability. Here we aimed to develop a refined method of the widely-used whole-gut transit assay. METHODS: Wildtype mice (N = 24) were subjected to the standard or refined whole-gut transit assay, either with or without a standardized slowing in gastrointestinal motility induced by loperamide. The standard assay consisted of a gavage with carmine red, observation during the light period and individual housing in a new cage without cage enrichment. For the refined whole-gut transit assay, mice were gavaged with UV-fluorescent DETEX®, observed during the dark period, while pairwise housed in their home cage with cage enrichment. Time until excretion of the first colored fecal pellet was assessed, and pellets were collected to assess number, weight, and water content. KEY RESULTS: The DETEX®-containing pellets were UV-detectable, allowing to measure the mice in their active period in the dark. The refined method caused less variation (20.8% and 16.0%) compared to the standard method (29.0% and 21.7%). Fecal pellet number, weight, and water content was significantly different between the standard and refined method. CONCLUSIONS & INFERENCES: This refined whole-gut transit assay provides a reliable approach to measure whole-gut transit time in mice in a more physiological context, with reduced variability compared to the standard method.

A novel orthotopic mouse model replicates human lung cancer cachexia.

INTRODUCTION: Cancer cachexia, highly prevalent in lung cancer, is a debilitating syndrome characterized by involuntary loss of skeletal muscle mass and is associated with poor clinical outcome, decreased survival and negative impact on tumour therapy. Various lung tumour-bearing animal models have been used to explore underlying mechanisms of cancer cachexia. However, these models do not simulate anatomical and immunological features key to lung cancer and associated muscle wasting. Overcoming these shortcomings is essential to translate experimental findings into the clinic. We therefore evaluated whether a syngeneic, orthotopic lung cancer mouse model replicates systemic and muscle-specific alterations associated with human lung cancer cachexia. METHODS: Immune competent, 11 weeks old male 129S2/Sv mice, were randomly allocated to either (1) sham control group or (2) tumour-bearing group. Syngeneic lung epithelium-derived adenocarcinoma cells (K-rasG12D ; p53R172HΔG ) were inoculated intrapulmonary into the left lung lobe of the mice. Body weight and food intake were measured daily. At baseline and weekly after surgery, grip strength was measured and tumour growth and muscle volume were assessed using micro cone beam CT imaging. After reaching predefined surrogate survival endpoint, animals were euthanized, and skeletal muscles of the lower hind limbs were collected for biochemical analysis. RESULTS: Two-third of the tumour-bearing mice developed cachexia based on predefined criteria. Final body weight (-13.7 ± 5.7%; P < 0.01), muscle mass (-13.8 ± 8.1%; P < 0.01) and muscle strength (-25.5 ± 10.5%; P < 0.001) were reduced in cachectic mice compared with sham controls and median survival time post-surgery was 33.5 days until humane endpoint. Markers for proteolysis, both ubiquitin proteasome system (Fbxo32 and Trim63) and autophagy-lysosomal pathway (Gabarapl1 and Bnip3), were significantly upregulated, whereas markers for protein synthesis (relative phosphorylation of Akt, S6 and 4E-BP1) were significantly decreased in the skeletal muscle of cachectic mice compared with control. The cachectic mice exhibited increased pentraxin-2 (P < 0.001) and CXCL1/KC (P < 0.01) expression levels in blood plasma and increased mRNA expression of IκBα (P < 0.05) in skeletal muscle, indicative for the presence of systemic inflammation. Strikingly, RNA sequencing, pathway enrichment and miRNA expression analyses of mouse skeletal muscle strongly mirrored alterations observed in muscle biopsies of patients with lung cancer cachexia. CONCLUSIONS: We developed an orthotopic model of lung cancer cachexia in immune competent mice. Because this model simulates key aspects specific to cachexia in lung cancer patients, it is highly suitable to further investigate the underlying mechanisms of lung cancer cachexia and to test the efficacy of novel intervention strategies.

The Modulatory Effects of Fatty Acids on Cancer Progression.

Cancer is the second leading cause of death worldwide and the global cancer burden rises rapidly. The risk factors for cancer development can often be attributed to lifestyle factors, of which an unhealthy diet is a major contributor. Dietary fat is an important macronutrient and therefore a crucial part of a well-balanced and healthy diet, but it is still unclear which specific fatty acids contribute to a healthy and well-balanced diet in the context of cancer risk and prognosis. In this review, we describe epidemiological evidence on the associations between the intake of different classes of fatty acids and the risk of developing cancer, and we provide preclinical evidence on how specific fatty acids can act on tumor cells, thereby modulating tumor progression and metastasis. Moreover, the pro- and anti-inflammatory effects of each of the different groups of fatty acids will be discussed specifically in the context of inflammation-induced cancer progression and we will highlight challenges as well as opportunities for successful application of fatty acid tailored nutritional interventions in the clinic.

11ß-HSD1 determines the extent of muscle atrophy in a model of acute exacerbation of COPD.

Muscle atrophy is an extrapulmonary complication of acute exacerbations (AE) in chronic obstructive pulmonary disease (COPD). The endogenous production and therapeutic application of glucocorticoids (GCs) have been implicated as drivers of muscle loss in AE-COPD. The enzyme 11 ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) activates GCs and contributes toward GC-induced muscle wasting. To explore the potential of 11ßHSD1 inhibition to prevent muscle wasting here, the objective of this study was to ascertain the contribution of endogenous GC activation and amplification by 11ßHSD1 in skeletal muscle wasting during AE-COPD. Emphysema was induced by intratracheal (IT) instillation of elastase to model COPD in WT and 11ßHSD1/KO mice, followed by vehicle or IT-LPS administration to mimic AE. µCT scans were obtained prior and at study endpoint 48 h following IT-LPS, to assess emphysema development and muscle mass changes, respectively. Plasma cytokine and GC profiles were determined by ELISA. In vitro, myonuclear accretion and cellular response to plasma and GCs were determined in C2C12 and human primary myotubes. Muscle wasting was exacerbated in LPS-11ßHSD1/KO animals compared with WT controls. RT-qPCR and western blot analysis showed elevated catabolic and suppressed anabolic pathways in muscle of LPS-11ßHSD1/KO animals relative to WTs. Plasma corticosterone levels were higher in LPS-11ßHSD1/KO animals, whereas C2C12 myotubes treated with LPS-11ßHSD1/KO plasma or exogenous GCs displayed reduced myonuclear accretion relative to WT counterparts. This study reveals that 11ß-HSD1 inhibition aggravates muscle wasting in a model of AE-COPD, suggesting that therapeutic inhibition of 11ß-HSD1 may not be appropriate to prevent muscle wasting in this setting.

Adaptive exhaustion during prolonged intermittent hypoxia causes dysregulated skeletal muscle protein homeostasis.

Nocturnal hypoxaemia, which is common in chronic obstructive pulmonary disease (COPD) patients, is associated with skeletal muscle loss or sarcopenia, which contributes to adverse clinical outcomes. In COPD, we have defined this as prolonged intermittent hypoxia (PIH) because the duration of hypoxia in skeletal muscle occurs through the duration of sleep followed by normoxia during the day, in contrast to recurrent brief hypoxic episodes during obstructive sleep apnoea (OSA). Adaptive cellular responses to PIH are not known. Responses to PIH induced by three cycles of 8 h hypoxia followed by 16 h normoxia were compared to those during chronic hypoxia (CH) or normoxia for 72 h in murine C2C12 and human inducible pluripotent stem cell-derived differentiated myotubes. RNA sequencing followed by downstream analyses were complemented by experimental validation of responses that included both unique and shared perturbations in ribosomal and mitochondrial function during PIH and CH. A sarcopenic phenotype characterized by decreased myotube diameter and protein synthesis, and increased phosphorylation of eIF2α (Ser51) by eIF2α kinase, and of GCN-2 (general controlled non-derepressed-2), occurred during both PIH and CH. Mitochondrial oxidative dysfunction, disrupted supercomplex assembly, lower activity of Complexes I, III, IV and V, and reduced intermediary metabolite concentrations occurred during PIH and CH. Decreased mitochondrial fission occurred during CH. Physiological relevance was established in skeletal muscle of mice with COPD that had increased phosphorylation of eIF2α, lower protein synthesis and mitochondrial oxidative dysfunction. Molecular and metabolic responses with PIH suggest an adaptive exhaustion with failure to restore homeostasis during normoxia. KEY POINTS: Sarcopenia or skeletal muscle loss is one of the most frequent complications that contributes to mortality and morbidity in patients with chronic obstructive pulmonary disease (COPD). Unlike chronic hypoxia, prolonged intermittent hypoxia is a frequent, underappreciated and clinically relevant model of hypoxia in patients with COPD. We developed a novel, in vitro myotube model of prolonged intermittent hypoxia with molecular and metabolic perturbations, mitochondrial oxidative dysfunction, and consequent sarcopenic phenotype. In vivo studies in skeletal muscle from a mouse model of COPD shared responses with our myotube model, establishing the pathophysiological relevance of our studies. These data lay the foundation for translational studies in human COPD to target prolonged, nocturnal hypoxaemia to prevent sarcopenia in these patients.

Effects of a nutritional intervention on impaired behavior and cognitive function in an emphysematous murine model of COPD with endotoxin-induced lung inflammation.

One cluster of the extrapulmonary manifestations in chronic obstructive pulmonary disease (COPD) is related to the brain, which includes anxiety, depression and cognitive impairment. Brain-related comorbidities are related to worsening of symptoms and increased mortality in COPD patients. In this study, a murine model of COPD was used to examine the effects of emphysema and repetitive pulmonary inflammatory events on systemic inflammatory outcomes and brain function. In addition, the effect of a dietary intervention on brain-related parameters was assessed. Adult male C57Bl/6J mice were exposed to elastase or vehicle intratracheally (i.t.) once a week on three consecutive weeks. Two weeks after the final administration, mice were i.t. exposed to lipopolysaccharide (LPS) or vehicle for three times with a 10 day interval. A dietary intervention enriched with omega-3 PUFAs, prebiotic fibers, tryptophan and vitamin D was administered from the first LPS exposure onward. Behavior and cognitive function, the degree of emphysema and both pulmonary and systemic inflammation as well as blood-brain barrier (BBB) integrity and neuroinflammation in the brain were assessed. A lower score in the cognitive test was observed in elastase-exposed mice. Mice exposed to elastase plus LPS showed less locomotion in the behavior test. The enriched diet seemed to reduce anxiety-like behavior over time and cognitive impairments associated with the presented COPD model, without affecting locomotion. In addition, the enriched diet restored the disbalance in splenic T-helper 1 (Th1) and Th2 cells. There was a trend toward recovering elastase plus LPS-induced decreased expression of occludin in brain microvessels, a measure of BBB integrity, as well as improving expression levels of kynurenine pathway markers in the brain by the enriched diet. The findings of this study demonstrate brain-associated comorbidities - including cognitive and behavioral impairments - in this murine model for COPD. Although no changes in lung parameters were observed, exposure to the specific enriched diet in this model appeared to improve systemic immune disbalance, BBB integrity and derailed kynurenine pathway which may lead to reduction of anxiety-like behavior and improved cognition.

Effects of Cigarette Smoke on Adipose and Skeletal Muscle Tissue: In Vivo and In Vitro Studies.

Chronic obstructive pulmonary disease (COPD), often caused by smoking, is a chronic lung disease with systemic manifestations including metabolic comorbidities. This study investigates adaptive and pathological alterations in adipose and skeletal muscle tissue following cigarette smoke exposure using in vivo and in vitro models. Mice were exposed to cigarette smoke or air for 72 days and the pre-adipose cell line 3T3-L1 was utilized as an in vitro model. Cigarette smoke exposure decreased body weight, and the proportional loss in fat mass was more pronounced than the lean mass loss. Cigarette smoke exposure reduced adipocyte size and increased adipocyte numbers. Adipose macrophage numbers and associated cytokine levels, including interleukin-1ß, interleukine-6 and tumor necrosis factor-α were elevated in smoke-exposed mice. Muscle strength and protein synthesis signaling were decreased after smoke exposure; however, muscle mass was not changed. In vitro studies demonstrated that lipolysis and fatty acid oxidation were upregulated in cigarette smoke-exposed pre-adipocytes. In conclusion, cigarette smoke exposure induces a loss of whole-body fat mass and adipose atrophy, which is likely due to enhanced lipolysis.

Fatty Acids as a Tool to Boost Cancer Immunotherapy Efficacy.

Although immunotherapy represents one of the most potent therapeutic anti-cancer approaches, only a limited number of patients shows clinical benefit. Recent evidence suggests that patients' nutritional status plays a major role in immunotherapy outcome. Fatty acids are essential in a balanced diet and well-known to influence the immune response. Moreover, short-chain fatty acids (SCFAs) show beneficial effects in metabolic disorders as well as in cancer and polyunsaturated fatty acids (PUFAs) contribute to body weight and fat free mass preservation in cancer patients. In line with these data, several studies imply a role for SCFAs and PUFAs in boosting the outcome of immunotherapy. In this review, we specifically focus on mechanistic data showing that SCFAs modulate the immunogenicity of tumor cells and we discuss the direct effects of SCFAs and PUFAs on the immune system in the context of cancer. We provide preclinical and clinical evidence indicating that SCFAs and PUFAs may have the potential to boost immunotherapy efficacy. Finally, we describe the challenges and address opportunities for successful application of nutritional interventions focusing on SCFAs and PUFAs to increase the therapeutic potential of immunotherapeutic approaches for cancer.

Evaluation of the Sensitivity of Metabolic Profiling by Rapid Evaporative Ionization Mass Spectrometry: Toward More Radical Oral Cavity Cancer Resections.

Radical resection for patients with oral cavity cancer remains challenging. Rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical vapors has been reported for real-time classification of normal and tumor tissues for numerous surgical applications. However, the infiltrative pattern of invasion of oral squamous cell carcinomas (OSCC) challenges the ability of REIMS to detect low amounts of tumor cells. We evaluate REIMS sensitivity to determine the minimal amount of detected tumors cells during oral cavity cancer surgery. A total of 11 OSCC patients were included in this study. The tissue classification based on 185 REIMS ex vivo metabolic profiles from five patients was compared to histopathology classification using multivariate analysis and leave-one-patient-out cross-validation. Vapors were analyzed in vivo by REIMS during four glossectomies. Complementary desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was employed to map tissue heterogeneity on six oral cavity sections to support REIMS findings. REIMS sensitivity was assessed with a new cell-based assay consisting of mixtures of cell lines (tumor, myoblasts, keratinocytes). Our results depict REIMS classified tumor and soft tissues with 96.8% accuracy. In vivo REIMS generated intense mass spectrometric signals. REIMS detected 10% of tumor cells mixed with 90% myoblasts with 83% sensitivity and 82% specificity. DESI-MSI underlined distinct metabolic profiles of nerve features and a metabolic shift phosphatidylethanolamine PE(O-16:1/18:2))/cholesterol sulfate common to both mucosal maturation and OSCC differentiation. In conclusion, the assessment of tissue heterogeneity with DESI-MSI and REIMS sensitivity with cell mixtures characterized sensitive metabolic profiles toward in vivo tissue recognition during oral cavity cancer surgeries.

Human pancreatic tumour organoid-derived factors enhance myogenic differentiation.

BACKGROUND: Most patients with pancreatic cancer develop cachexia, which is characterized by progressive muscle loss. The mechanisms underlying muscle loss in cancer cachexia remain elusive. Pancreatic tumour organoids are 3D cell culture models that retain key characteristics of the parent tumour. We aimed to investigate the effect of pancreatic tumour organoid-derived factors on processes that determine skeletal muscle mass, including the regulation of muscle protein turnover and myogenesis. METHODS: Conditioned medium (CM) was collected from human pancreatic cancer cell lines (PK-45H, PANC-1, PK-1, and KLM-1), pancreatic tumour organoid cultures from a severely cachectic (PANCO-9a) and a non-cachectic patient (PANCO-12a), and a normal pancreas organoid culture. Differentiating C2C12 myoblasts and mature C2C12 myotubes were exposed to CM for 24 h or maintained in control medium. In myotubes, NF-kB activation was monitored using a NF-κB luciferase reporter construct, and mRNA expression of E3-ubiquitin ligases and REDD1 was analysed by RT-qPCR. C2C12 myoblast proliferation and differentiation were monitored by live cell imaging and myogenic markers and myosin heavy chain (MyHC) isoforms were assessed by RT-qPCR. RESULTS: Whereas CM from PK-1 and KLM-1 cells significantly induced NF-κB activation in C2C12 myotubes (PK-1: 3.1-fold, P < 0.001; KLM-1: 2.1-fold, P = 0.01), Atrogin-1/MAFbx and MuRF1 mRNA were only minimally and inconsistently upregulated by the CM of pancreatic cancer cell lines. Similarly, E3-ubiquitin ligases and REDD1 mRNA expression in myotubes were not altered by exposure to pancreatic tumour organoid CM. Compared with the control condition, CM from both PANCO-9a and PANCO-12a tumour organoids increased proliferation of myoblasts, which was accompanied by significant downregulation of the satellite cell marker paired-box 7 (PAX7) (PANCO-9a: -2.1-fold, P < 0.001; PANCO-12a: -2.0-fold, P < 0.001) and myogenic factor 5 (MYF5) (PANCO-9a: -2.1-fold, P < 0.001; PANCO-12a: -1.8-fold, P < 0.001) after 48 h of differentiation. Live cell imaging revealed accelerated alignment and fusion of myoblasts exposed to CM from PANCO-9a and PANCO-12a, which was in line with significantly increased Myomaker mRNA expression levels (PANCO-9a: 2.4-fold, P = 0.001; PANCO-12a: 2.2-fold, P = 0.004). These morphological and transcriptional alterations were accompanied by increased expression of muscle differentiation markers such as MyHC-IIB (PANCO-9a: 2.5-fold, P = 0.04; PANCO-12a: 3.1-fold, P = 0.006). Although the impact of organoid CM on myogenesis was not associated with the cachexia phenotype of the donor patients, it was specific for tumour organoids, as CM of control pancreas organoids did not modulate myogenic fusion. CONCLUSIONS: These data show that pancreatic tumour organoid-derived factors alter the kinetics of myogenesis, which may eventually contribute to impaired muscle mass maintenance in cancer cachexia.

Physical exercise at the crossroad between muscle wasting and the immune system: implications for lung cancer cachexia.

Cachexia is a syndrome characterized by involuntary weight loss and wasting of skeletal muscle mass. It is associated with worse overall survival and quality of life. The cancer-induced systemic inflammation and the consequent host derived catabolic stimuli, trigger cachexia by inhibiting muscle protein synthesis and enhancing muscle catabolism. The muscle itself may further promote chronic inflammation, introducing a vicious catabolic circle. Nutritional support alone plays a limited role in the treatment of cancer cachexia and should be combined with other interventions. Physical exercise lowers systemic inflammation and promotes muscle anabolism. It also attenuates the age-related physical decline in elderly and it might counteract the muscle wasting induced by the cancer cachexia syndrome. This review describes how cancer-induced systemic inflammation promotes muscle wasting and whether physical exercise may represent a suitable treatment for cancer-induced cachexia, particularly in patients with non-small cell lung cancer. We summarized pre-clinical and clinical studies investigating whether physical exercise would improve muscle performance and whether this improvement would translate in a clinically meaningful benefit for patients with cancer, in terms of survival and quality of life. Moreover, this review describes the results of studies investigating the interplay between physical exercise and the immune system, including the role of the intestinal microbiota.

Ovarian cancer ascites induces skeletal muscle wasting in vitro and reflects sarcopenia in patients.

BACKGROUND: Cachexia-associated skeletal muscle wasting or 'sarcopenia' is highly prevalent in ovarian cancer and contributes to poor outcome. Drivers of cachexia-associated sarcopenia in ovarian cancer remain elusive, underscoring the need for novel and better models to identify tumour factors inducing sarcopenia. We aimed to assess whether factors present in ascites of sarcopenic vs. non-sarcopenic ovarian cancer patients differentially affect protein metabolism in skeletal muscle cells and to determine if these effects are correlated to cachexia-related patient characteristics. METHODS: Fifteen patients with an ovarian mass and ascites underwent extensive physical screening focusing on cachexia-related parameters. Based on computed tomography-based body composition imaging, six cancer patients were classified as sarcopenic and six were not; three patients with a benign condition served as an additional non-sarcopenic control group. Ascites was collected, and concentrations of cachexia-associated factors were assessed by enzyme-linked immunosorbent assay. Subsequently, ascites was used for in vitro exposure of C2C12 myotubes followed by measurements of protein synthesis and breakdown by radioactive isotope tracing, qPCR-based analysis of atrophy-related gene expression, and NF-κB activity reporter assays. RESULTS: C2C12 protein synthesis was lower after exposure to ascites from sarcopenic patients (sarcopenia 3.1 ± 0.1 nmol/h/mg protein vs. non-sarcopenia 5.5 ± 0.2 nmol/h/mg protein, P < 0.01), and protein breakdown rates tended to be higher (sarcopenia 31.2 ± 5.2% vs. non-sarcopenia 20.9 ± 1.9%, P = 0.08). Ascites did not affect MuRF1, Atrogin-1, or REDD1 expression of C2C12 myotubes, but NF-κB activity was specifically increased in cells exposed to ascites from sarcopenic patients (sarcopenia 2.2 ± 0.4-fold compared with control vs. non-sarcopenia 1.2 ± 0.2-fold compared with control, P = 0.01). Protein synthesis and breakdown correlated with NF-κB activity (rs  = -0.60, P = 0.03 and rs  = 0.67, P = 0.01, respectively). The skeletal muscle index of the ascites donors was also correlated to both in vitro protein synthesis (rs  = 0.70, P = 0.005) and protein breakdown rates (rs  = -0.57, P = 0.04). CONCLUSIONS: Ascites of sarcopenic ovarian cancer patients induces pronounced skeletal muscle protein metabolism changes in C2C12 cells that correlate with clinical muscle measures of the patient and that are characteristic of cachexia. The use of ascites offers a new experimental tool to study the impact of both tumour-derived and systemic factors in various cachexia model systems, enabling identification of novel drivers of tissue wasting in ovarian cancer.

Hepatic Steatosis Contributes to the Development of Muscle Atrophy via Inter-Organ Crosstalk.

Individuals with hepatic steatosis often display several metabolic abnormalities including insulin resistance and muscle atrophy. Previously, we found that hepatic steatosis results in an altered hepatokine secretion profile, thereby inducing skeletal muscle insulin resistance via inter-organ crosstalk. In this study, we aimed to investigate whether the altered secretion profile in the state of hepatic steatosis also induces skeletal muscle atrophy via effects on muscle protein turnover. To investigate this, eight-week-old male C57BL/6J mice were fed a chow (4.5% fat) or a high-fat diet (HFD; 45% fat) for 12 weeks to induce hepatic steatosis, after which the livers were excised and cut into ~200-µm slices. Slices were cultured to collect secretion products (conditioned medium; CM). Differentiated L6-GLUT4myc myotubes were incubated with chow or HFD CM to measure glucose uptake. Differentiated C2C12 myotubes were incubated with chow or HFD CM to measure protein synthesis and breakdown, and gene expression via RNA sequencing. Furthermore, proteomics analysis was performed in chow and HFD CM. It was found that HFD CM caused insulin resistance in L6-GLUT4myc myotubes compared with chow CM, as indicated by a blunted insulin-stimulated increase in glucose uptake. Furthermore, protein breakdown was increased in C2C12 cells incubated with HFD CM, while there was no effect on protein synthesis. RNA profiling of C2C12 cells indicated that 197 genes were differentially expressed after incubation with HFD CM, compared with chow CM, and pathway analysis showed that pathways related to anatomical structure and function were enriched. Proteomics analysis of the CM showed that 32 proteins were differentially expressed in HFD CM compared with chow CM. Pathway enrichment analysis indicated that these proteins had important functions with respect to insulin-like growth factor transport and uptake, and affect post-translational processes, including protein folding, protein secretion and protein phosphorylation. In conclusion, the results of this study support the hypothesis that secretion products from the liver contribute to the development of muscle atrophy in individuals with hepatic steatosis.

Deep Learning Based Automated Orthotopic Lung Tumor Segmentation in Whole-Body Mouse CT-Scans.

Lung cancer is the leading cause of cancer related deaths worldwide. The development of orthotopic mouse models of lung cancer, which recapitulates the disease more realistically compared to the widely used subcutaneous tumor models, is expected to critically aid the development of novel therapies to battle lung cancer or related comorbidities such as cachexia. However, follow-up of tumor take, tumor growth and detection of therapeutic effects is difficult, time consuming and requires a vast number of animals in orthotopic models. Here, we describe a solution for the fully automatic segmentation and quantification of orthotopic lung tumor volume and mass in whole-body mouse computed tomography (CT) scans. The goal is to drastically enhance the efficiency of the research process by replacing time-consuming manual procedures with fast, automated ones. A deep learning algorithm was trained on 60 unique manually delineated lung tumors and evaluated by four-fold cross validation. Quantitative performance metrics demonstrated high accuracy and robustness of the deep learning algorithm for automated tumor volume analyses (mean dice similarity coefficient of 0.80), and superior processing time (69 times faster) compared to manual segmentation. Moreover, manual delineations of the tumor volume by three independent annotators was sensitive to bias in human interpretation while the algorithm was less vulnerable to bias. In addition, we showed that besides longitudinal quantification of tumor development, the deep learning algorithm can also be used in parallel with the previously published method for muscle mass quantification and to optimize the experimental design reducing the number of animals needed in preclinical studies. In conclusion, we implemented a method for fast and highly accurate tumor quantification with minimal operator involvement in data analysis. This deep learning algorithm provides a helpful tool for the noninvasive detection and analysis of tumor take, tumor growth and therapeutic effects in mouse orthotopic lung cancer models.

Global Deletion of 11ß-HSD1 Prevents Muscle Wasting Associated with Glucocorticoid Therapy in Polyarthritis.

Glucocorticoids provide indispensable anti-inflammatory therapies. However, metabolic adverse effects including muscle wasting restrict their use. The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) modulates peripheral glucocorticoid responses through pre-receptor metabolism. This study investigates how 11ß-HSD1 influences skeletal muscle responses to glucocorticoid therapy for chronic inflammation. We assessed human skeletal muscle biopsies from patients with rheumatoid arthritis and osteoarthritis for 11ß-HSD1 activity ex vivo. Using the TNF-α-transgenic mouse model (TNF-tg) of chronic inflammation, we examined the effects of corticosterone treatment and 11ß-HSD1 global knock-out (11ßKO) on skeletal muscle, measuring anti-inflammatory gene expression, muscle weights, fiber size distribution, and catabolic pathways. Muscle 11ß-HSD1 activity was elevated in patients with rheumatoid arthritis and correlated with inflammation markers. In murine skeletal muscle, glucocorticoid administration suppressed IL6 expression in TNF-tg mice but not in TNF-tg11ßKO mice. TNF-tg mice exhibited reductions in muscle weight and fiber size with glucocorticoid therapy. In contrast, TNF-tg11ßKO mice were protected against glucocorticoid-induced muscle atrophy. Glucocorticoid-mediated activation of catabolic mediators (FoxO1, Trim63) was also diminished in TNF-tg11ßKO compared to TNF-tg mice. In summary, 11ß-HSD1 knock-out prevents muscle atrophy associated with glucocorticoid therapy in a model of chronic inflammation. Targeting 11ß-HSD1 may offer a strategy to refine the safety of glucocorticoids.

Towards Personalized Management of Sarcopenia in COPD.

The awareness of the presence and consequences of sarcopenia has significantly increased over the past decade. Sarcopenia is defined as gradual loss of muscle mass and strength and ultimately loss of physical performance associated with aging and chronic disease. The prevalence of sarcopenia is higher in chronic obstructive pulmonary disease (COPD) compared to age-matched controls. Current literature suggests that next to physical inactivity, COPD-specific alterations in physiological processes contribute to accelerated development of sarcopenia. Sarcopenia in COPD can be assessed according to current guidelines, but during physical performance testing, ventilatory limitation should be considered. Treatment of muscle impairment can halt or even reverse sarcopenia, despite respiratory impairment. Exercise training and protein supplementation are currently at the basis of sarcopenia treatment. Furthermore, effective current and new interventions targeting the pulmonary system (eg, smoking cessation, bronchodilators and lung volume reduction surgery) may also facilitate muscle maintenance. Better understanding of disease-specific pathophysiological mechanisms involved in the accelerated development of sarcopenia in COPD will provide new leads to refine nutritional, exercise and physical activity interventions and develop pharmacological co-interventions.