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Julian Palzer,1,2 Benedikt Mues,2 Richard Goerg,1,2 Merel Aberle,3 Sander S Rensen,3 Steven WM Olde Damink,1,3 Rianne DW Vaes,3 Thorsten Cramer,1,3 Thomas Schmitz-Rode,2 Ulf P Neumann,1,3 Ioana Slabu,2 Anjali A Roeth1,3 1Department of General, Visceral and Transplant Surgery, RWTH Aachen University Hospital, Aachen, Germany; 2Institute of Applied Medical Engineering, Helmholtz Institute Aachen, RWTH Aachen University Hospital, Aachen, Germany; 3Department of Surgery, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the NetherlandsCorrespondence: Anjali A Roeth Email aroeth@ukaachen.deIntroduction: Pancreatic ductal adenocarcinoma (PDAC) is a cancer with a meager prognosis due to its chemotherapy resistance. A new treatment method may be magnetic fluid hyperthermia (MFH). Magnetoliposomes (ML), consisting of superparamagnetic iron oxide nanoparticles (SPION) stabilized with a phospholipid-bilayer, are exposed to an alternating magnetic field (AMF) to generate heat. To optimize this therapy, we investigated the effects of MFH on human PDAC cell lines and 3D organoid cultures.Material and Methods: ML cytotoxicity was tested on Mia PaCa-2 and PANC-1 cells and on PDAC 3D organoid cultures, generated from resected tissue of patients. The MFH was achieved by AMF application with an amplitude of 40– 47 kA/m and a frequency of 270 kHz. The MFH effect on the cell viability of the cell lines and the organoid cultures was investigated at two different time points. Clonogenic assays evaluated the impairment of colony formation. Altering ML set-ups addressed differences arising from intra- vs extracellular ML locations.Results: Mia PaCa-2 and PANC-1 cells showed no cytotoxic effects at ML concentrations up to 300 μg(Fe)/mL and 225 μg(Fe)/mL, respectively. ML at a concentration of 225 μg(Fe)/mL were also non-toxic for PDAC organoid cultures. MFH treatment using exclusively extracellular ML presented the highest impact on cell viability. Clonogenic assays demonstrated remarkable impairment as long-term outcome in MFH-treated PDAC cell lines. Additionally, we successfully treated PDAC organoids with extracellular ML-derived MFH, resulting in notably reduced cell viabilities 2h and 24 h post treatment. Still, PDAC organoids seem to partly recover from MFH after 24 h as opposed to conventional 2D-cultures.Conclusion: Treatment with MFH strongly diminished pancreatic cancer cell viability in vitro, making it a promising treatment strategy. As organoids resemble the more advanced in vivo conditions better than conventional 2D cell lines, our organoid model holds great potential for further investigations.Keywords: magnetic nanoparticles, SPION, pancreatic cancer, PDAC, organoids, magnetic fluid hyperthermia

Objectives: Body composition assessment using CT images at the L3-level is increasingly applied in cancer research and has been shown to be strongly associated with long-term survival. Robust high-throughput automated segmentation is key to assess large patient cohorts and to support implementation of body composition analysis into routine clinical practice. We trained and externally validated a deep learning neural network (DLNN) to automatically segment L3-CT images., Methods: Expert-drawn segmentations of visceral and subcutaneous adipose tissue (VAT/SAT) and skeletal muscle (SM) of L3-CT-images of 3187 patients undergoing abdominal surgery were used to train a DLNN. The external validation cohort was comprised of 2535 patients with abdominal cancer. DLNN performance was evaluated with (geometric) dice similarity (DS) and Lin's concordance correlation coefficient., Results: There was a strong concordance between automatic and manual segmentations with median DS for SM, VAT, and SAT of 0.97 (IQR: 0.95-0.98), 0.98 (IQR: 0.95-0.98), and 0.95 (IQR: 0.92-0.97), respectively. Concordance correlations were excellent: SM 0.964 (0.959-0.968), VAT 0.998 (0.998-0.998), and SAT 0.992 (0.991-0.993). Bland-Altman metrics indicated only small and clinically insignificant systematic offsets; SM radiodensity: 0.23 Hounsfield units (0.5%), SM: 1.26 cm2.m-2 (2.8%), VAT: -1.02 cm2.m-2 (1.7%), and SAT: 3.24 cm2.m-2 (4.6%)., Conclusion: A robustly-performing and independently externally validated DLNN for automated body composition analysis was developed., Advances in Knowledge: This DLNN was successfully trained and externally validated on several large patient cohorts. The trained algorithm could facilitate large-scale population studies and implementation of body composition analysis into clinical practice., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Institute of Radiology.)

Background: Cancer cachexia is a multifactorial metabolic syndrome characterized by systemic inflammation and ongoing skeletal muscle loss resulting in weakness, poor quality of life, and decreased survival. Whereas lipid accumulation in skeletal muscle is associated with cancer cachexia as well as the prognosis of cancer patients, surprisingly little is known about the nature of the lipids that accumulate in the muscle during cachexia, and whether this is related to inflammation. We aimed to identify the types and distributions of intramyocellular lipids in patients with and without cancer cachexia., Methods: Rectus abdominis muscle biopsies were collected during surgery of patients with pancreatic ductal adenocarcinoma (n = 10 without cachexia, n = 20 cachectic without inflammation (CRP < 10 mg/L), n = 10 cachectic with inflammation (CRP ≥ 10 mg/L). L3-CT scans were analysed to assess body composition based on validated thresholds in Hounsfield units (HU). Muscle sections were stained with Oil-Red O and H&E to assess general lipid accumulation and atrophy. Untargeted lipidomic analyses were performed on laser-microdissected myotubes using LC-MS/MS. The spatial distribution of intramyocellular lipids with differential abundance between groups was visualized by mass-spectrometry imaging. Genes coding for inflammation markers and enzymes involved in de novo ceramide synthesis were studied by qPCR., Results: Muscle radiation attenuation was lower in cachectic patients with inflammation (median 24.3 [18.6-30.8] HU) as compared with those without inflammation (34.2 [29.3-38.7] HU, P = 0.033) or no cachexia (37.4 [33.9-42.9] HU, P = 0.012). Accordingly, intramyocellular lipid content was lower in non-cachectic patients (1.9 [1.6-2.1]%) as compared with those with cachexia with inflammation (5.5 [4.5-7.3]%, P = 0.002) or without inflammation (4.8 [2.6-6.0]%, P = 0.017). Intramyocellular lipid accumulation was associated with both local IL-6 mRNA levels (r s  = 0.57, P = 0.015) and systemic CRP levels (r s  = 0.49, P = 0.024). Compared with non-cachectic subjects, cachectic patients had a higher relative abundance of intramyocellular glycerophospholipids and a lower relative abundance of glycerolipids. Furthermore, increases in several intramyocellular lipids such as SM(d36:1), PC(34:1), and TG(48:1) were found in cachectic patients with inflammation and correlated with specific cachexia features. Altered intramyocellular lipid species such as PC(34:1), LPC(18:2), and TG(48:1) showed an uneven distribution in muscle sections of cachectic and non-cachectic patients, with areas featuring abundance of these lipids next to areas almost devoid of them., Conclusions: Intramyocellular lipid accumulation in patients with cachexia is associated with both local and systemic inflammation, and characterized by changes in defined lipid species such as glycerolipids and glycerophospholipids., (© 2024 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

Background: Cachexia-associated body composition alterations and tumor metabolic activity are both associated with survival of cancer patients. Recently, subcutaneous adipose tissue properties have emerged as particularly prognostic body composition features. We hypothesized that tumors with higher metabolic activity instigate cachexia related peripheral metabolic alterations, and investigated whether tumor metabolic activity is associated with body composition and survival in patients with non-small-cell lung cancer (NSCLC), focusing on subcutaneous adipose tissue., Methods: A retrospective analysis was performed on a cohort of 173 patients with NSCLC. 18 F-fluorodeoxyglucose positron emission tomography-computed tomography (PET-CT) scans obtained before treatment were used to analyze tumor metabolic activity (standardized uptake value (SUV) and SUV normalized by lean body mass (SUL)) as well as body composition variables (subcutaneous and visceral adipose tissue radiodensity (SAT/VAT radiodensity) and area; skeletal muscle radiodensity (SM radiodensity) and area). Subjects were divided into groups with high or low SAT radiodensity based on Youden Index of Receiver Operator Characteristics (ROC). Associations between tumor metabolic activity, body composition variables, and survival were analyzed by Mann-Whitney tests, Cox regression, and Kaplan-Meier analysis., Results: The overall prevalence of high SAT radiodensity was 50.9% (88/173). Patients with high SAT radiodensity had shorter survival compared with patients with low SAT radiodensity (mean: 45.3 vs. 50.5 months, p = 0.026). High SAT radiodensity was independently associated with shorter overall survival (multivariate Cox regression HR = 1.061, 95% CI: 1.022-1.101, p = 0.002). SAT radiodensity also correlated with tumor metabolic activity (SULpeak r s  = 0.421, p = 0.029; SUVpeak r s  = 0.370, p = 0.048). In contrast, the cross-sectional areas of SM, SAT, and VAT were not associated with tumor metabolic activity or survival., Conclusion: Higher SAT radiodensity is associated with higher tumor metabolic activity and shorter survival in patients with NSCLC. This may suggest that tumors with higher metabolic activity induce subcutaneous adipose tissue alterations such as decreased lipid density, increased fibrosis, or browning., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Background: 5-Fluorouracil (5-FU) is used as an antineoplastic agent in distinct cancer types. Increasing evidence suggests that the gut microbiota might modulate 5-FU efficacy and toxicity, potentially affecting the patient's prognosis. The current experimental study investigated 5-FU-induced microbiota alterations, as well as the potential of prebiotic fibre mixtures (M1-M4) to counteract these shifts., Methods: A pooled microbial consortium was derived from ten healthy donors, inoculated in an in vitro model of the colon, and treated with 5-FU, with or without prebiotic fibre mixtures for 72 h. Four different prebiotic fibre mixtures were tested: M1 containing short-chain galacto-oligosaccharides (sc GOS), long-chain fructo-oligosaccharides (lcFOS), and low viscosity pectin (lvPect), M2 consisting of arabinoxylan, beta-glucan, pectin, and resistant starch, M3 which was a mixture of scGOS and lcFOS, and M4 containing arabinoxylan, beta-glucan, pectin, resistant starch, and inulin., Results: We identified 5-FU-induced changes in gut microbiota composition, but not in microbial diversity. Administration of prebiotic fibre mixtures during 5-FU influenced gut microbiota composition and taxa abundance. Amongst others, prebiotic fibre mixtures successfully stimulated potentially beneficial bacteria (Bifidobacterium, Lactobacillus, Anaerostipes, Weissella, Olsenella, Senegalimassilia) and suppressed the growth of potentially pathogenic bacteria (Klebsiella, Enterobacter) in the presence of 5-FU. The short-chain fatty acid (SCFA) acetate increased slightly during 5-FU, but even more during 5-FU with prebiotic fibre mixtures, while propionate was lower due to 5-FU with or without prebiotic fibre mixtures, compared to control. The SCFA butyrate and valerate did not show differences among all conditions. The branched-chain fatty acids (BCFA) iso-butyrate and iso-valerate were higher in 5-FU, but lower in 5-FU + prebiotics, compared to control., Conclusions: These data suggest that prebiotic fibre mixtures represent a promising strategy to modulate 5-FU-induced microbial dysbiosis towards a more favourable microbiota, thereby possibly improving 5-FU efficacy and reducing toxicity, which should be evaluated further in clinical studies., (© 2024. The Author(s).)

Patients with pancreatic cancer often suffer from cachexia and experience gastrointestinal symptoms that may be related to intestinal smooth muscle cell (SMC) dysfunction. We hypothesized that pancreatic tumor organoids from cachectic patients release factors that perturb the SMC's contractile characteristics. Human visceral SMCs were exposed to conditioned medium (CM) from the pancreatic tumor organoid cultures of cachectic ( n = 2) and non-cachectic ( n = 2) patients. Contractile proteins and markers of inflammation, muscle atrophy, and proliferation were evaluated by qPCR and Western blot. SMC proliferation and migration were monitored by live cell imaging. The Ki-67-positive cell fraction was determined in the intestinal smooth musculature of pancreatic cancer patients. CM from the pancreatic tumor organoids of cachectic patients did not affect IL-1β , IL-6 , IL-8 , MCP-1 , or Atrogin-1 expression. However, CM reduced the α-SMA, γ-SMA, and SM22-α levels, which was accompanied by a reduced SMC doubling time and increased expression of S100A4 , a Ca 2 +-binding protein associated with the synthetic SMC phenotype. In line with this, Ki-67-positive nuclei were increased in the intestinal smooth musculature of patients with a low versus high L3-SMI. In conclusion, patient-derived pancreatic tumor organoids release factors that compromise the contractile SMC phenotype and increase SMC proliferation. This may contribute to the frequently observed gastrointestinal motility problems in these patients.

Background: Morbidity rates in pancreatic surgery are high, and frail patients with low aerobic capacity are especially at risk of complications and require prophylactic interventions. Previous studies of small patient cohorts receiving intra-abdominal surgery have shown that an exercise prehabilitation program increases aerobic capacity, leading to better treatment outcomes., Objective: In this study, we aim to assess the feasibility of a home-based exercise prehabilitation program in unfit patients scheduled for pancreatic surgery on a larger scale., Methods: In this multicenter study, adult patients scheduled for elective pancreatic surgery with a preoperative oxygen uptake (VO 2 ) at the ventilatory anaerobic threshold ≤13 mL/kg/min or a VO 2 at peak exercise ≤18 mL/kg/min will be recruited. A total of 30 patients will be included in the 4-week, home-based, partly supervised exercise prehabilitation program. The program comprises 25-minute high-intensity interval training on an advanced cycle ergometer 3 times a week. Training intensity will be based on steep ramp test performance (ie, a short-term maximal exercise test on a cycle ergometer), aiming to improve aerobic capacity. Twice a week, patients will perform functional task exercises to improve muscle function and functional mobility. A steep ramp test will be repeated weekly, and training intensity will be adjusted accordingly. Next to assessing the feasibility (participation rate, reasons for nonparticipation, adherence, dropout rate, reasons for dropout, adverse events, and patient and therapist appreciation) of this program, individual patients' responses to prehabilitation on aerobic capacity, functional mobility, body composition, quality of life, and immune system factors will be evaluated., Results: Recruitment for this study began in January 2022 and is expected to be completed in the summer of 2023., Conclusions: Results of this study will provide important clinical and scientific knowledge on the feasibility of a partly supervised home-based exercise prehabilitation program in a vulnerable patient population. This might ease the path to implementing prehabilitation programs in unfit patients undergoing complex abdominal surgery, such as pancreatic surgery., Trial Registration: ClinicalTrials.gov NCT05496777; https://classic.clinicaltrials.gov/ct2/show/NCT05496777., International Registered Report Identifier (irrid): DERR1-10.2196/46526., (©Nicole D Hildebrand, Allard G Wijma, Bart C Bongers, Sander S Rensen, Marcel den Dulk, Joost M Klaase, Steven W M Olde Damink. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 07.09.2023.)

Background: Computerized radiological image analysis (radiomics) enables the investigation of image-derived phenotypes by extracting large numbers of quantitative features. We hypothesized that radiomics features may contain prognostic information that enhances conventional body composition analysis. We aimed to investigate whether body composition-associated radiomics features hold additional value over conventional body composition analysis and clinical patient characteristics used to predict survival of pancreatic ductal adenocarcinoma (PDAC) patients., Methods: Computed tomography images of 304 patients undergoing elective pancreatic cancer resection were analysed. 2D radiomics features were extracted from skeletal muscle and subcutaneous and visceral adipose tissue (SAT and VAT) compartments from a single slice at the third lumbar vertebra. The study population was randomly split (80:20) into training and holdout subsets. Feature ranking with Least Absolute Shrinkage Selection Operator (LASSO) followed by multivariable stepwise Cox regression in 1000 bootstrapped re-samples of the training data was performed and tested on the holdout data. The fitted regression predictors were used as "scores" for a clinical (C-Score), body composition (B-Score), and radiomics (R-Score) model. To stratify patients into the highest 25% and lowest 25% risk of mortality compared to the middle 50%, the Harrell Concordance Index was used., Results: Based on LASSO and stepwise cox regression for overall survival, ASA ≥3 and age were the most important clinical variables and constituted the C-score, and VAT-index (VATI) was the most important body composition variable and constituted the B-score. Three radiomics features (SATI&#95;original&#95;shape2D&#95;Perimeter, VATI&#95;original&#95;glszm&#95;SmallAreaEmphasis, and VATI&#95;original&#95;firstorder&#95;Maximum) emerged as the most frequent set of features and yielded an R-Score. Of the mean concordance indices of C-, B-, and R-scores, R-score performed best (0.61, 95% CI 0.56-0.65, p<0.001), followed by the C-score (0.59, 95% CI 0.55-0.63, p<0.001) and B-score (0.55, 95% CI 0.50-0.60, p=0.03). Kaplan-Meier projection revealed that C-, B, and R-scores showed a clear split in the survival curves in the training set, although none remained significant in the holdout set., Conclusion: It is feasible to implement a data-driven radiomics approach to body composition imaging. Radiomics features provided improved predictive performance compared to conventional body composition variables for the prediction of overall survival of PDAC patients undergoing primary resection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 van der Kroft, Wee, Rensen, Brecheisen, van Dijk, Eickhoff, Roeth, Ulmer, Dekker, Neumann and Olde Damink.)

Introduction: Pancreatic cancer is associated with poor prognosis, and limited treatment options are available for the majority of patients. Natural killer (NK) cells in combination with antibodies inducing antibody-dependent cell-mediated cytotoxicity (ADCC) could be a highly effective new therapeutic option in pancreatic cancer. Accurate predictive preclinical models are needed to develop successful NK cell immunotherapy. Tumor organoids, in vitro 3D organ-like structures that retain important pathophysiological characteristics of the in vivo tumor, may provide such a model. In the current study, we assessed the cytotoxic potential of adoptive NK cells against human pancreatic cancer organoids. We hypothesized that NK cell anti-tumor responses could be enhanced by including ADCC-triggering antibodies., Methods: We performed cytotoxicity assays with healthy donor-derived IL-2-activated NK cells and pancreatic cancer organoids from four patients. A 3D cytotoxicity assay using live-cell-imaging was developed and enabled real-time assessment of the response., Results: We show that NK cells migrate to and target pancreatic cancer organoids, resulting in an increased organoid death, compared to the no NK cell controls (reaching an average fold change from baseline of 2.1±0.8 vs 1.4±0.6). After 24-hours of co-culture, organoid 2D growth increased. Organoids from 2 out of 4 patients were sensitive to NK cells, while organoids from the other two patients were relatively resistant, indicating patient-specific heterogeneity among organoid cultures. The ADCC-inducing antibodies avelumab (anti-PD-L1) and trastuzumab (anti-HER2) increased NK cell-induced organoid cell death (reaching an average fold change from baseline of 3.5±1.0 and 4.5±1.8, respectively). Moreover, combination therapy with avelumab or trastuzumab resulted in complete disintegration of organoids. Finally, inclusion of ADCC-inducing antibodies was able to overcome resistance in NK-organoid combinations with low or no kill., Discussion: These results support the use of organoids as a relevant and personalized model to study the anti-tumor response of NK cells in vitro and the potential of ADCC-inducing antibodies to enhance NK cell effector function., Competing Interests: GB is Chief Executive Officer/Chief Medical Officer/Cofounder of CiMaas, BV, Maastricht, The Netherlands. SR, LW, and SOD are shareholders of Adjutec B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Beelen, Aberle, Bruno, Olde Damink, Bos, Rensen and Wieten.)

Background: Cancer cachexia is a multifactorial syndrome characterized by body weight loss and systemic inflammation. The characterization of the inflammatory response in patients with cachexia is still limited. Lipocalin-2, a protein abundant in neutrophils, has recently been implicated in appetite suppression in preclinical models of pancreatic cancer cachexia. We hypothesized that lipocalin-2 levels could be associated with neutrophil activation and nutritional status of pancreatic ductal adenocarcinoma (PDAC) patients., Methods: Plasma levels of neutrophil activation markers calprotectin, myeloperoxidase, elastase, and bactericidal/permeability-increasing protein (BPI) were compared between non-cachectic PDAC patients (n=13) and cachectic PDAC patients with high (≥26.9 ng/mL, n =34) or low (<26.9 ng/mL, n =34) circulating lipocalin-2 levels. Patients' nutritional status was assessed by the patient-generated subjective global assessment (PG-SGA) and through body composition analysis using CT-scan slices at the L3 level., Results: Circulating lipocalin-2 levels did not differ between cachectic and non-cachectic PDAC patients (median 26.7 (IQR 19.7-34.8) vs . 24.8 (16.6-29.4) ng/mL, p =0.141). Cachectic patients with high systemic lipocalin-2 levels had higher concentrations of calprotectin, myeloperoxidase, and elastase than non-cachectic patients or cachectic patients with low lipocalin-2 levels (calprotectin: 542.3 (355.8-724.9) vs . 457.5 (213.3-606.9), p =0.448 vs . 366.5 (294.5-478.5) ng/mL, p =0.009; myeloperoxidase: 30.3 (22.1-37.9) vs . 16.3 (12.0-27.5), p =0.021 vs . 20.2 (15.0-29.2) ng/mL, p =0.011; elastase: 137.1 (90.8-253.2) vs . 97.2 (28.8-215.7), p =0.410 vs . 95.0 (72.2-113.6) ng/mL, p =0.006; respectively). The CRP/albumin ratio was also higher in cachectic patients with high lipocalin-2 levels (2.3 (1.3-6.0) as compared to non-cachectic patients (1.0 (0.7-4.2), p =0.041). Lipocalin-2 concentrations correlated with those of calprotectin ( r s =0.36, p <0.001), myeloperoxidase ( r s =0.48, p <0.001), elastase ( r s =0.50, p <0.001), and BPI ( r s =0.22, p =0.048). Whereas no significant correlations with weight loss, BMI, or L3 skeletal muscle index were observed, lipocalin-2 concentrations were associated with subcutaneous adipose tissue index ( r s =-0.25, p =0.034). Moreover, lipocalin-2 tended to be elevated in severely malnourished patients compared with well-nourished patients (27.2 (20.3-37.2) vs . 19.9 (13.4-26.4) ng/mL, p =0.058)., Conclusions: These data suggest that lipocalin-2 levels are associated with neutrophil activation in patients with pancreatic cancer cachexia and that it may contribute to their poor nutritional status., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Deng, Aberle, van Bijnen, van der Kroft, Lenaerts, Neumann, Wiltberger, Schaap, Olde Damink and Rensen.)

Organoids are increasingly used to investigate patient-specific drug responsiveness, but organoid culture is complex and expensive, and carried out in rich, non-physiological media. We investigated reproducibility of drug-responsiveness of primary cell cultures in 2D versus 3D and in conventional versus physiological cell culture medium. 3D pancreatic ductal adenocarcinoma organoid cultures PANCO09b and PANCO11b were converted to primary cell cultures growing in 2D. Transformed 2D cultures were grown in physiological Plasmax medium or Advanced-DMEM/F12. Sensitivity towards gemcitabine, paclitaxel, SN-38, 5-fluorouacil, and oxaliplatin was investigated by cell viability assays. Growth rates of corresponding 2D and 3D cultures were comparable. PANCO09b had a shorter doubling time in physiological media. Chemosensitivity of PANCO09b and PANCO11b grown in 2D or 3D was similar, except for SN-38, to which PANCO11b cultured in 3D was more sensitive (2D: 8.2 ×10 -3 ± 2.3 ×10 -3 vs. 3D: 1.1 ×10 -3 ± 0.6 ×10 -3 , p = 0.027). PANCO09b and PANCO11b showed no major differences in chemosensitivity when cultured in physiological compared to conventional media, although PANCO11b was more sensitive to SN-38 in physiological media (9.8 × 10 -3 ± 0.7 × 10 -3 vs. 5.2 × 10 -3 ± 1.8 × 10 -3 , p = 0.015). Collectively, these data indicate that the chemosensitivity of organoids is not affected by culture medium composition or culture dimensions. This implies that organoid-based drug screens can be simplified to become more cost-effective.

This clinical study explored the associations between the intestinal microbiota, chemotherapy toxicity, and treatment response in postmenopausal oestrogen receptor positive breast cancer patients.Oestrogen receptor positive postmenopausal breast cancer patients were prospectively enroled in a multicentre cohort study and treated with 4 cycles of (neo)adjuvant adriamycin, cyclophosphamide (AC) followed by 4 cycles of docetaxel (D). Patients collected a faecal sample and completed a questionnaire before treatment, during AC, during D, and after completing AC-D. Chemotherapy toxicity and tumour response were determined. Intestinal microbiota was analysed by amplicon sequencing of the 16 S rRNA V4 gene-region. In total, 44 patients, including 18 neoadjuvant patients, were included, and 153 faecal samples were collected before AC-D (n = 44), during AC (n = 43), during D (n = 29), and after AC-D treatment (n = 37), 28 participants provided all four samples. In the whole group, observed species richness reduced during treatment (p = 0.042). The abundance of Proteobacteria, unclassified Enterobacterales, Lactobacillus, Ruminococcaceae NK4A214 group, Marvinbryantia, Christensenellaceae R7 group, and Ruminococcaceae UCG-005 changed significantly over time. Patients with any grade diarrhoea during docetaxel treatment had a significantly lower observed species richness compared to patients without diarrhoea. In the small group neoadjuvant treated patients, pathologic response was unrelated to baseline intestinal microbiota richness, diversity and composition. While the baseline microbiota was not predictive for pathologic response in a rather small group of neoadjuvant treated patients in our study, subsequent shifts in microbial richness, as well as the abundance of specific bacterial taxa, were observed during AC-D treatment in the whole group and the neoadjuvant group., (© 2022. The Author(s).)

Background: Previous pre-clinical research has indicated that the intestinal microbiota can potentiate anti-tumour efficacy of capecitabine and that capecitabine treatment impacts intestinal microbiota composition and diversity. Using a longitudinal design, this study explores the associations between the intestinal microbiota and treatment response in patients with metastatic colorectal cancer (mCRC) during capecitabine treatment., Patients and Methods: Patients with mCRC treated with capecitabine were prospectively enrolled in a multicentre cohort study. Patients collected a faecal sample and completed a questionnaire before, during, and after three cycles of capecitabine. Several clinical characteristics, including tumour response, toxicity and antibiotic use were recorded. Intestinal microbiota were analysed by amplicon sequencing of the 16S rRNA V4 gene-region., Results: Thirty-three patients were included. After three cycles of capecitabine, six patients (18%) achieved a partial response, 25 (76%) showed stable disease, and one (3%) experienced progressive disease. Of the 90 faecal samples were collected. Microbial diversity (α-diversity), community structure (β-diversity), and bacterial abundance on phylum and genus level were not significantly different between responders and non-responders and were not significantly affected by three cycles of capecitabine., Conclusion: This is the first clinical study with longitudinal intestinal microbiota sampling in mCRC patients that explores the role of the intestinal microbiota during treatment with capecitabine. Intestinal microbiota composition and diversity before, during, and after three cycles of capecitabine were not associated with response in this study population. Capecitabine did not induce significant changes in the microbiota composition and diversity during the treatment period. Individual effects of antibiotics during capecitabine treatment were observed., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Organoid culture systems are self-renewing, three-dimensional (3D) models derived from pluripotent stem cells, adult derived stem cells or cancer cells that recapitulate key molecular and structural characteristics of their tissue of origin. They generally form into hollow structures with apical-basolateral polarization. Mass spectrometry imaging (MSI) is a powerful analytical method for detecting a wide variety of molecules in a single experiment while retaining their spatiotemporal distribution. Here we describe a protocol for preparing organoids for MSI that (1) preserves the 3D morphological structure of hollow organoids, (2) retains the spatiotemporal distribution of a vast array of molecules (3) and enables accurate molecular identification based on tandem mass spectrometry. The protocol specifically focuses on the collection and embedding of the organoids in gelatin, and gives recommendations for MSI-specific sample preparation, data acquisition and molecular identification by tandem mass spectrometry. This method is applicable to a wide range of organoids from different origins, and takes 1 d from organoid collection to MSI data acquisition., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Introduction: Factors underlying antitumor immunity in pancreatic cancer (PC) are poorly understood. We hypothesized that not neoantigen quantity, but quality, is related to immune cell infiltration and survival., Methodology: We performed genomic and transcriptomic profiling of paired normal, tumor tissue of 13 patients with PC with distinct survival times. Additionally, neoantigens prediction and immunological profiling were performed., Results: The proportion of neoantigens with a low similarity-to-self score was higher in short-term survivors ( p < 0.0001), while mutational load and burden, similarity-to-known-pathogens, and immunogenicity of neoantigens were not associated with immune cell infiltration or survival., Discussion: No tumor mutational load or neoantigen quantity, but low similarity-to-self score, was associated with immune cell infiltration and survival., Competing Interests: SR: A shareholder of Adjutec B.V. SOD: A shareholder of Adjutec B.V. MB: Boston Scientific (Consultant, support for industry and investigator-initiated studies), Cook Medical (Consultant, support for industry and investigator-initiated studies), Pentax Medical (Consultant, support for investigator-initiated studies), Mylan (Support for investigator-initiated studies), ChiRoStim (Support for investigator-initiated studies). LB served as a paid consultant for Bristol-Myers Squibb in Pathologist Advisory Board PD-L1 CPS testing in upper GI cancer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Levink, Brosens, Rensen, Aberle, Olde Damink, Cahen, Buschow, Fuhler, Peppelenbosch and Bruno.)

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 (r s  = -0.60, P = 0.03 and r s  = 0.67, P = 0.01, respectively). The skeletal muscle index of the ascites donors was also correlated to both in vitro protein synthesis (r s  = 0.70, P = 0.005) and protein breakdown rates (r s  = -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., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Background: Previous preclinical and clinical research has investigated the role of intestinal microbiota in carcinogenesis. Growing evidence exists that intestinal microbiota can influence breast cancer carcinogenesis. However, the role of intestinal microbiota in breast cancer needs to be further investigated. This study aimed to identify the microbiota differences between postmenopausal breast cancer patients and controls., Patients and Methods: This prospective cohort study compared the intestinal microbiota richness, diversity, and composition in postmenopausal histologically proven ER+/HER2- breast cancer patients and postmenopausal controls. Patients scheduled for (neo)adjuvant adriamycin, cyclophosphamide (AC), and docetaxel (D), or endocrine therapy (tamoxifen) were prospectively enrolled in a multicentre cohort study in the Netherlands. Patients collected a faecal sample and completed a questionnaire before starting systemic cancer treatment. Controls, enrolled from the National Dutch Breast Cancer Screening Programme, also collected a faecal sample and completed a questionnaire. Intestinal microbiota was analysed by amplicon sequencing of the 16S rRNA V4 gene region., Results: In total, 81 postmenopausal ER+/HER2- breast cancer patients and 67 postmenopausal controls were included, resulting in 148 faecal samples. Observed species richness, Shannon index, and overall microbial community structure were not significantly different between breast cancer patients and controls. There was a significant difference in overall microbial community structure between breast cancer patients scheduled for adjuvant treatment, neoadjuvant treatment, and controls at the phylum ( p = 0.042) and genus levels ( p = 0.015). Dialister ( p = 0.001) and its corresponding family Veillonellaceae ( p = 0.001) were higher in patients scheduled for adjuvant treatment, compared to patients scheduled for neoadjuvant treatment. Additional sensitivity analysis to correct for the potential confounding effect of prophylactic antibiotic use, indicated no differences in microbial community structure between patients scheduled for neoadjuvant systemic treatment, adjuvant systemic treatment, and controls at the phylum ( p = 0.471) and genus levels ( p = 0.124)., Conclusions: Intestinal microbiota richness, diversity, and composition are not different between postmenopausal breast cancer patients and controls. The increased relative abundance of Dialister and Veillonellaceae was observed in breast cancer patients scheduled for adjuvant treatment, which might be caused by a relative decrease in other bacteria due to prophylactic antibiotic administration rather than an absolute increase.

Background: Cancer cachexia is characterized by a negative energy balance, muscle and adipose tissue wasting, insulin resistance, and systemic inflammation. Because of its strong negative impact on prognosis and its multifactorial nature that is still not fully understood, cachexia remains an important challenge in the field of cancer treatment. Recent animal studies indicate that the gut microbiota is involved in the pathogenesis and manifestation of cancer cachexia, but human data are lacking. The present study investigates gut microbiota composition, short-chain fatty acids (SCFA), and inflammatory parameters in human cancer cachexia., Methods: Faecal samples were prospectively collected in patients (N = 107) with pancreatic cancer, lung cancer, breast cancer, or ovarian cancer. Household partners (N = 76) of the patients were included as healthy controls with similar diet and environmental conditions. Patients were classified as cachectic if they lost >5% body weight in the last 6 months. Gut microbiota composition was analysed by sequencing of the 16S rRNA V4 gene region. Faecal SCFA levels were quantified by gas chromatography. Faecal calprotectin was assessed with enzyme-linked immunosorbent assay. Serum C-reactive protein and leucocyte counts were retrieved from medical records., Results: Cachexia prevalence was highest in pancreatic cancer (66.7%), followed by ovarian cancer (25%), lung cancer (20.8%), and breast cancer (17.3%). Microbial α-diversity was not significantly different between cachectic cancer patients (N = 33), non-cachectic cancer patients (N = 74), or healthy controls (N = 76) (species richness P = 0.31; Shannon effective index P = 0.46). Community structure (β-diversity) tended to differ between these groups (P = 0.053), although overall differences were subtle and no clear clustering of samples was observed. Proteobacteria (P < 0.001), an unknown genus from the Enterobacteriaceae family (P < 0.01), and Veillonella (P < 0.001) were more abundant among cachectic cancer patients. Megamonas (P < 0.05) and Peptococcus (P < 0.001) also showed differential abundance. Faecal levels of all SCFA tended to be lower in cachectic cancer patients, but only acetate concentrations were significantly reduced (P < 0.05). Faecal calprotectin levels were positively correlated with the abundance of Peptococcus, unknown Enterobacteriaceae, and Veillonella. We also identified several correlations and interactions between clinical and microbial parameters., Conclusions: This clinical study provided the first insights into the alterations of gut microbiota composition and SCFA levels that occur in cachectic cancer patients and how they are related to inflammatory parameters. These results pave the way for further research examining the role of the gut microbiota in cancer cachexia and its potential use as therapeutic target., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Systemic inflammation is thought to underlie many of the metabolic manifestations of cachexia in cancer patients. The complement system is an important component of innate immunity that has been shown to contribute to metabolic inflammation. We hypothesized that systemic inflammation in patients with cancer cachexia was associated with complement activation. Systemic C3a levels were higher in cachectic patients with inflammation ( n = 23, C-reactive protein (CRP) ≥ 10 mg/L) as compared to patients without inflammation ( n = 26, CRP < 10 mg/L) or without cachexia ( n = 13) (medians 102.4 (IQR 89.4-158.0) vs. 81.4 (IQR 47.9-124.0) vs. 61.6 (IQR 46.8-86.8) ng/mL, respectively, p = 0.0186). Accordingly, terminal complement complex (TCC) concentrations gradually increased in these patient groups (medians 2298 (IQR 2022-3058) vs. 1939 (IQR 1725-2311) vs. 1805 (IQR 1552-2569) mAU/mL, respectively, p = 0.0511). C3a and TCC concentrations were strongly correlated (r s = 0.468, p = 0.0005). Although concentrations of C1q and mannose-binding lectin did not differ between groups, C1q levels were correlated with both C3a and TCC concentrations (r s = 0.394, p = 0.0042 and r s = 0.300, p = 0.0188, respectively). In conclusion, systemic inflammation in patients with cancer cachexia is associated with the activation of key effector complement factors. The correlations between C1q and C3a/TCC suggest that the classical complement pathway could play a role in complement activation in patients with pancreatic cancer.

Introduction: Sarcopenia is associated with reduced pulmonary function in healthy adults, as well as with increased risk of pneumonia following abdominal surgery. Consequentially, postoperative pneumonia prolongs hospital admission, and increases in-hospital mortality following a range of surgical interventions. Little is known about the function of the diaphragm in the context of sarcopenia and wasting disorders or how its function is influenced by abdominal surgery. Liver surgery induces reactive pleural effusion in most patients, compromising postoperative pulmonary function. We hypothesise that both major hepatic resection and sarcopenia have a measurable impact on diaphragm function. Furthermore, we hypothesise that sarcopenia is associated with reduced preoperative diaphragm function, and that patients with reduced preoperative diaphragm function show a greater decline and reduced recovery of diaphragm function following major hepatic resection. The primary goal of this study is to evaluate whether sarcopenic patients have a reduced diaphragm function prior to major liver resection compared with non-sarcopenic patients, and to evaluate whether sarcopenic patients show a greater reduction in respiratory muscle function following major liver resection when compared with non-sarcopenic patients., Methods and Analysis: Transcostal B-mode, M-mode ultrasound and speckle tracking imaging will be used to assess diaphragm function perioperatively in 33 sarcopenic and 33 non-sarcopenic patients undergoing right-sided hemihepatectomy starting 1 day prior to surgery and up to 30 days after surgery. In addition, rectus abdominis and quadriceps femoris muscles thickness will be measured using ultrasound to measure sarcopenia, and pulmonary function will be measured using a hand-held bedside spirometer. Muscle mass will be determined preoperatively using CT-muscle volumetry of abdominal muscle and adipose tissue at the third lumbar vertebra level (L3). Muscle function will be assessed using handgrip strength and physical condition will be measured with a short physical performance battery . A rectus abdominis muscle biopsy will be taken intraoperatively to measure proteolytic and mitochondrial activity as well as inflammation and redox status. Systemic inflammation and sarcopenia biomarkers will be assessed in serum acquired perioperatively., Ethics and Dissemination: This trial is open for recruitment. The protocol was approved by the official Independent Medical Ethical Committee at Uniklinik (Rheinish Westphälische Technische Hochschule (RWTH) Aachen (reference EK309-18) in July 2019. Results will be published via international peer-reviewed journals and the findings of the study will be communicated using a comprehensive dissemination strategy aimed at healthcare professionals and patients., Trial Registration Number: ClinicalTrials. gov (EK309-18); Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pasmans, Adriaens, Olinga, Langen, Rensen, Schaap, Olde Damink, Caiment, van Loon, Blaak and Meex.)

Background: Myosteatosis has been associated with shorter overall survival in cancer patients. The increase in ectopic fat might not be limited to skeletal muscle only and might also extend to other sites such as the liver, resulting in non-alcoholic fatty liver disease (NAFLD). In this study, we assessed the relationship between myosteatosis and NAFLD and their association with overall survival in patients with colorectal liver metastases undergoing partial hepatectomy., Methods: Patients were selected from a prospective cohort of 289 consecutive patients with colorectal liver metastases. All patients with a preoperative computed tomography (CT)-scan and liver biopsy obtained during surgery were included. If available a second pre-operative CT scan was used to calculate changes in body composition over time. Muscle radiation attenuation was defined as the average Hounsfield units on CT of all muscle tissue at the L3 level. Liver biopsies were graded by a liver pathologist using the steatosis, activity, and fibrosis scoring system for NAFLD., Results: Two-hundred and eighteen patients had an available liver biopsy of which 131 patients had two available pre-operative CT scans with an average time interval of 3.2 months. One-hundred and thirty-five (62%) biopsies were classified as NAFLD. In multivariable Cox-regression analysis, NAFLD [hazard ratio (HR): 1.8, 95%-confidence interval (CI) 1.0-3.0, P = 0.037], increase in myosteatosis (HR 1.8, 95%-CI 1.1-2.9, P = 0.018), and skeletal muscle loss (HR 1.7, 95%-CI 1.0-2.9, P = 0.035) were independently associated with shorter overall survival while high visceral adipose tissue fat content was associated with longer overall survival (HR: 0.7, 95%-CI 0.5-0.9, P = 0.014)., Conclusions: Ectopic fat content of liver as well as skeletal muscle tissue is independently associated with shorter overall survival in patients with colorectal liver metastases, while increased visceral adipose tissue fat content is associated with longer overall survival., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease and is characterized by different stages varying from benign fat accumulation to non-alcoholic steatohepatitis (NASH) that may progress to cirrhosis and liver cancer. In recent years, a regulatory role of long non-coding RNAs (lncRNAs) in NAFLD has emerged. Therefore, we aimed to characterize the still poorly understood lncRNA contribution to disease progression. Transcriptome analysis in 60 human liver samples with various degrees of NAFLD/NASH was combined with a functional genomics experiment in an in vitro model where we exposed HepG2 cells to free fatty acids (FFA) to induce steatosis, then stimulated them with tumor necrosis factor alpha (TNFα) to mimic inflammation. Bioinformatics analyses provided a functional prediction of novel lncRNAs. We further functionally characterized the involvement of one novel lncRNA in the nuclear-factor-kappa B (NF-κB) signaling pathway by its silencing in Hepatoma G2 (HepG2) cells. We identified 730 protein-coding genes and 18 lncRNAs that responded to FFA/TNFα and associated with human NASH phenotypes with consistent effect direction, with most being linked to inflammation. One novel intergenic lncRNA, designated lncTNF, was 20-fold up-regulated upon TNFα stimulation in HepG2 cells and positively correlated with lobular inflammation in human liver samples. Silencing lncTNF in HepG2 cells reduced NF-κB activity and suppressed expression of the NF-κB target genes A20 and NFKBIA . The lncTNF we identified in the NF-κB signaling pathway may represent a novel target for controlling liver inflammation.

Cancer cachexia is a metabolic syndrome characterized by unintended weight loss and muscle wasting. It has a strong negative impact on survival. Its underlying mechanisms involve systemic inflammation and insulin resistance, which are known to be influenced by the gut microbiota. Preclinical studies support a role for the gut microbiota in cancer cachexia by demonstrating that cachectic mice display: 1) various gut microbiota composition changes; 2) increased gut permeability and translocation of pro-inflammatory microbial compounds; 3) muscle atrophy-related processes linked to gut microbiota properties; 4) positive effects of microbiota-modulating interventions. Data on the relationships between gut microbiota, insulin resistance, and hepatic/adipose tissue metabolism in cachexia models are lacking. Nevertheless, the available data and existing evidence for the impact of gut microbiota on metabolic aberrations in human obesity urge for exploration of its role in human cancer cachexia. We provide practical recommendations and discuss the challenges for such future clinical studies., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Decrease in skeletal muscle index (SMI) during neoadjuvant chemotherapy (NACT) has been associated with worse outcome in patients with advanced ovarian cancer. To validate these findings, we tested if a decrease in SMI was a prognostic factor for a homogenous cohort of patients who received NACT in the randomized phase 3 OVHIPEC-trial., Methods: CT-scans were performed at baseline and after two cycles of neoadjuvant chemotherapy in stage III ovarian cancer patients. The SMI (skeletal muscle area in cm 2 divided by body surface area in m 2 ) was calculated using SliceOMatic software. The difference in SMI between both CT-scans (ΔSMI) was calculated. Cox-regression analyses were performed to analyze the independent effect of a difference in SMI (ΔSMI) on outcome. Log-rank tests were performed to plot recurrence-free (RFS) and overall survival (OS). The mean number of adverse events per patient were compared between groups using t-tests., Results: Paired CT-scans were available for 212 out of 245 patients (87%). Thirty-four of 74 patients (58%) in the group with a decrease in ΔSMI and 73 of 138 of the patients (53%) in the group with stable/increase in ΔSMI had died. Median RFS and OS did not differ significantly (p = 0.297 and p = 0.764) between groups. Patients with a decrease in SMI experienced more pre-operative adverse events, and more grade 3-4 adverse events., Conclusion: Decreased SMI during neoadjuvant chemotherapy was not associated with worse outcome in patients with stage III ovarian cancer included in the OVHIPEC-trial. However, a strong association between decreasing SMI and adverse events was found., Competing Interests: Declaration of Competing Interest Dr. Sonke reports institutional research support outside the submitted work, from AstraZeneca, Merck, Novartis, and Roche, during the conduct of the study. Dr. Dam reports research support outside the submitted work from, Roche, Novartis, and Amgen. Travel and speakers' fees were received from Roche, Novartis, and Teva. Dr. De Hingh reports grants from ROCHE, and grants from QP&S/RanD, outside the submitted work. Dr. Vuylsteke reports advisory board fees, travel grants, and speaker fees from MSD, BMS, Pfizer, Novartis, Lilly, and Roche, outside the submitted work. The other authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The majority of patients with pancreatic cancer develops cachexia. The mechanisms underlying cancer cachexia development and progression remain elusive, although tumour-derived factors are considered to play a major role. Pancreatic tumour organoids are in vitro three-dimensional organ-like structures that retain many pathophysiological characteristics of the in vivo tumour. We aimed to establish a pancreatic tumour organoid biobank from well-phenotyped cachectic and non-cachectic patients to enable identification of tumour-derived factors driving cancer cachexia., Methods: Organoids were generated from tumour tissue of eight pancreatic cancer patients. A comprehensive pre-operative patient assessment of cachexia-related parameters including nutritional status, physical performance, body composition, and inflammation was performed. Tumour-related and cachexia-related characteristics of the organoids were analysed using histological stainings, targeted sequencing, and real-time-quantitative PCR. Cachexia-related factors present in the circulation of the patients and in the tumour organoid secretome were analysed by enzyme-linked immunosorbent assay., Results: The established human pancreatic tumour organoids presented typical features of malignancy corresponding to the primary tumour (i.e. nuclear enlargement, multiple nucleoli, mitosis, apoptosis, and mutated KRAS and/or TP53). These tumour organoids also expressed variable levels of many known cachexia-related genes including interleukin-6 (IL-6), TNF-α, IL-8, IL-1α, IL-1β, Mcp-1, GDF15, and LIF. mRNA expression of IL-1α and IL-1β was significantly reduced in organoids from cachectic vs. non-cachectic patients (IL-1α: -3.8-fold, P = 0.009, and IL-1β: -4.7-fold, P = 0.004). LIF, IL-8, and GDF15 mRNA expression levels were significantly higher in organoids from cachectic vs. non-cachectic patients (LIF: 1.6-fold, P = 0.003; IL-8: 1.4-fold, P = 0.01; GDF15: 2.3-fold, P < 0.001). In line with the GDF15 and IL-8 mRNA expression levels, tumour organoids from cachectic patients secreted more GDF15 and IL-8 compared with organoids from non-cachectic patients (5.4 vs. 1.5 ng/mL, P = 0.01, and 7.4 vs. 1.3 ng/mL, P = 0.07, respectively)., Conclusions: This novel human pancreatic tumour organoid biobank provides a valuable tool to increase our understanding of the mechanisms driving cancer cachexia. Our preliminary characterization of the secretome of these organoids supports their application in functional studies including conditioned medium approaches and in vivo transplantation models., (© 2020 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Background: Low skeletal muscle radiation attenuation (SM-RA) is indicative of myosteatosis and diminished muscle function. It is predictive of poor outcome following oncological surgery in several cancer types. Postoperative pneumonia is a known risk factor for increased postoperative mortality. We hypothesized that low SM-RA of the respiratory muscles at the 4th thoracic-vertebra (T4) is associated with postoperative pneumonia following liver surgery., Methods: Postoperative pneumonia was identified using prospective infection control data. Computed tomography body composition analysis was performed at the L3-and T4 level to determine SM-RA. Body composition variables were corrected for confounders and related to postoperative pneumonia and admission time by multivariable logistic regression., Results: Body composition analysis of 180 patients was performed. Twenty-one patients developed postoperative pneumonia (11.6%). Multivariable analysis showed that low T4 SM-RA as well as low L3 SM-RA were significantly associated with postoperative pneumonia (OR 3.65, 95% CI 1.41-9.49, p < 0.01) and (OR 3.22, 95% CI 1.20-8.61, p = 0.02, respectively)., Conclusion: Low SM-RA at either the L3-or T4-level is associated with a higher risk of postoperative pneumonia following CLRM resection., (Copyright © 2019 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Plasma soluble E-selectin (sE-selectin) is a frequently used biomarker of systemic endothelial dysfunction. The present study explored the relationship between nonalcoholic fatty liver disease (NAFLD) and plasma sE-selectin levels., Methods: Expression of E-selectin in liver, visceral adipose tissue (VAT) and muscle was studied in relation to plasma sE-selectin in severely obese individuals (n = 74). The course of hepatic E-selectin expression in relation to hepatic steatosis and inflammation was examined in C57BL/6J LDLR -/- mice on a Western-type diet. The relationship between biomarkers of NAFLD, that is, plasma aminotransferase (ALT) and NAFLD susceptibility genes (rs738409 [PNPLA3] and rs1260326 [GCKR]), and plasma sE-selectin was studied in the combined CODAM (n = 571) and Hoorn (n = 694) studies., Results: E-selectin expression in liver, not VAT or muscle, was associated with plasma sE-selectin in severely obese individuals (β = 0.26; 95% CI: 0.05-0.47). NAFLD severity was associated with hepatic E-selectin expression (P = .02) and plasma sE-selectin (P = .003). LDLR -/- mice on a Western-type diet displayed increased hepatic E-selectin expression that followed the same course as hepatic inflammation, but not steatosis. In the CODAM study, plasma ALT was associated with plasma sE-selectin, independent of potential confounders (β = 0.25; 95% CI: 0.16-0.34). Both rs738409 and rs1260326 were associated with higher plasma sE-selectin in the combined CODAM and Hoorn studies (P = .01 and P = .004 respectively)., Conclusions: NAFLD and related markers are associated with higher expression of hepatic E-selectin and higher levels of plasma sE-selectin. Further studies are required to investigate the role of E-selectin in the pathogenesis of NAFLD and the applicability of sE-selectin as a plasma biomarker of NAFLD/NASH., (© 2020 The Authors. Liver International published by John Wiley & Sons Ltd.)

This study investigated whether there are differences in the composition of the cutaneous microbiome of the unaffected skin between patients with pressure ulcers compared with those without pressure ulcers. The cutaneous microbiome of the unaffected skin of 15 patients with sacral pressure ulcers compared to 15 patients without pressure ulcers was analysed. It demonstrated that the inter-individual variation in skin microbiota of patients with pressure ulcers was significantly higher (P = 0.01). The abundance of 23 species was significantly different with Staphylococcus aureus and unclassified Enterococcus the most abundant species in patients with pressure ulcers. Random Forest models showed that eight species were associated with pressure ulcers occurrence in 81% of the patients. A subset of four species gave the strongest interaction. The presence of unclassified Enterococcus had the highest association with pressure ulcer occurrence. This study is the first to demonstrate that the cutaneous microbiome is altered in patients with pressure ulcers.

Background: Severe obesity is a growing, worldwide burden and conventional therapies including radical change of diet and/or increased physical activity have limited results. Bariatric surgery has been proposed as an alternative therapy showing promising results. It leads to substantial weight loss and improvement of comorbidities such as type 2 diabetes. Increased adiposity is associated with changes in epigenetic profile, including DNA methylation. We investigated the effect of bariatric surgery on clinical profile, DNA methylation, and biological age estimated using Horvath's epigenetic clock., Results: To determine the impact of bariatric surgery and subsequent weight loss on clinical traits, a cohort of 40 severely obese individuals (BMI = 30-73 kg/m 2 ) was examined at the time of surgery and at three follow-up visits, i.e., 3, 6, and 12 months after surgery. The majority of the individuals were women (65%) and the mean age at surgery was 45.1 ± 8.1 years. We observed a significant decrease over time in BMI, fasting glucose, HbA1c, HOMA-IR, insulin, total cholesterol, triglycerides, LDL and free fatty acids levels, and a significant small increase in HDL levels (all p values < 0.05). Epigenome-wide association analysis revealed 4857 differentially methylated CpG sites 12 months after surgery (at Bonferroni-corrected p value < 1.09 × 10 -7 ). Including BMI change in the model decreased the number of significantly differentially methylated CpG sites by 51%. Gene set enrichment analysis identified overrepresentation of multiple processes including regulation of transcription, RNA metabolic, and biosynthetic processes in the cell. Bariatric surgery in severely obese patients resulted in a decrease in both biological age and epigenetic age acceleration (EAA) (mean = - 0.92, p value = 0.039)., Conclusions: Our study shows that bariatric surgery leads to substantial BMI decrease and improvement of clinical outcomes observed 12 months after surgery. These changes explained part of the association between bariatric surgery and DNA methylation. We also observed a small, but significant improvement of biological age. These epigenetic changes may be modifiable by environmental lifestyle factors and could be used as potential biomarkers for obesity and in the future for obesity related comorbidities.

Background: Intestinal microbiota have been suggested to play an important role in the pathogenesis of obesity and type 2 diabetes. Bariatric surgery improves both conditions and has been associated with changes in intestinal microbiota composition. We investigated the effect of a nonsurgical bariatric technique on intestinal microbiota composition in relation to metabolic improvement., Methods: Seventeen patients with obesity and type 2 diabetes were treated with the nonsurgical duodenal-jejunal bypass liner, which excludes the proximal 60 cm small intestine from food. Fecal samples as well as metabolic parameters reflecting obesity and type 2 diabetes were obtained from the patients at baseline, after 6 months with the device in situ, and 6 months after explantation., Results: After 6 months of treatment, both obesity and type 2 diabetes had improved with a decrease in weight from 106.1 [99.4-123.5] to 97.4 [89.4-114.0] kg and a decrease in HbA 1c from 8.5% [7.6-9.2] to 7.2% [6.3-8.1] (both p < 0.05). This was paralleled by an increased abundance of typical small intestinal bacteria such as Proteobacteria, Veillonella, and Lactobacillus spp. in feces. After removal of the duodenal-jejunal bypass liner, fecal microbiota composition was similar to that observed at baseline, despite persistent weight loss., Conclusion: Improvement of obesity and type 2 diabetes after exclusion of the proximal 60 cm small intestine by treatment with a nonsurgical duodenal-jejunal bypass liner may be promoted by changes in fecal microbiota composition.

Background: Sarcopenia is the loss of skeletal muscle mass and function that occurs with advancing age and certain diseases. It is thought to have a negative impact on survival in cancer patients. Routine computed tomography imaging is often used to quantify skeletal muscle in cancer patients. Sarcopenia is defined by a low skeletal muscle index (SMI). Skeletal muscle radiation attenuation (SMRA) is used to define muscle quality. The primary aim of this meta-analysis was to study the association between sarcopenia or SMRA and overall survival (OS) or complications in patients with ovarian cancer., Methods: Medline, Embase, CINAHL, and PEDro databases were searched from inception to 15 February 2019. Studies evaluating the prognostic effect of SMI and SMRA on ovarian cancer survival or surgical complications were included. Risk of bias and study quality were evaluated with the Quality in Prognosis Studies Instrument (QUIPS) according to the modified Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework., Results: The search strategy yielded 4262 hits in all four databases combined. Ten and eight studies were included for qualitative and quantitative analysis, respectively. Meta-analysis revealed a significant association between the SMI and OS [0.007; hazard ratio (HR): 1.11, 95% confidence interval (CI): 1.03-1.20]. SMRA was also significantly associated with OS (P < 0.001; HR: 1.14, 95% CI: 1.08-1.20). Association between the SMI and surgical complications had borderline statistical significance (0.05; HR: 1.23, 95% CI: 1.00-1.52). The risk of bias assessed with QUIPS was high in all studies. The quality of the evidence was very low., Conclusions: Whereas our meta-analysis indicated that a low SMI and low SMRA are associated with survival in ovarian cancer patients, the low quality of the source data precludes drawing definitive conclusions., (© 2019 The Authors Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

d-amino acids, the enantiomeric counterparts of l-amino acids, were long considered to be non-functional or not even present in living organisms. Nowadays, d-amino acids are acknowledged to play important roles in numerous physiological processes in the human body. The most commonly studied link between d-amino acids and human physiology concerns the contribution of d-serine and d-aspartate to neurotransmission. These d-amino acids and several others have also been implicated in regulating innate immunity and gut barrier function. Importantly, the presence of certain d-amino acids in the human body has been linked to several diseases including schizophrenia, amyotrophic lateral sclerosis, and age-related disorders such as cataract and atherosclerosis. Furthermore, increasing evidence supports a role for d-amino acids in the development, pathophysiology, and treatment of cancer. In this review, we aim to provide an overview of the various sources of d-amino acids, their metabolism, as well as their contribution to physiological processes and diseases in man, with a focus on cancer.

Clinical interest in the human intestinal microbiota has increased considerably. However, an overview of clinical studies investigating the link between the human intestinal microbiota and systemic cancer therapy is lacking. This systematic review summarizes all clinical studies describing the association between baseline intestinal microbiota and systemic cancer therapy outcome as well as therapy-related changes in intestinal microbiota composition. A systematic literature search was performed and provided 23 articles. There were strong indications for a close association between the intestinal microbiota and outcome of immunotherapy. Furthermore, the development of chemotherapy-induced infectious complications seemed to be associated with the baseline microbiota profile. Both chemotherapy and immunotherapy induced drastic changes in gut microbiota composition with possible consequences for treatment efficacy. Evidence in the field of hormonal therapy was very limited. Large heterogeneity concerning study design, study population, and methods used for analysis limited comparability and generalization of results. For the future, longitudinal studies investigating the predictive ability of baseline intestinal microbiota concerning treatment outcome and complications as well as the potential use of microbiota-modulating strategies in cancer patients are required. More knowledge in this field is likely to be of clinical benefit since modulation of the microbiota might support cancer therapy in the future.

Background: Development of obesity and obesity-related diseases, such as type 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD), is associated with altered gut microbiota composition. The aim of this study is to investigate associations among dietary compounds, intestinal cell function, and gut microbiota composition. We hypothesize that dietary lipid intake is associated with Paneth cell and goblet cell properties that affect gut microbiota composition., Objective: The primary objective of this study is to determine whether a difference in dietary intake is associated with a difference in intestinal mucin-2 expression and gut microbiota composition., Methods: This is a single-center prospective study, including 1 obese group undergoing laparoscopic Roux-en-y gastric bypass and 2 lean control groups undergoing either laparoscopic cholecystectomy or upper gastrointestinal endoscopy (n=228). During laparoscopy, biopsies will be taken of visceral fat (omentum majus), liver, muscle tissue of the abdominal wall, and subcutaneous fat. In the obese group, a small segment of the jejunum will be collected for analysis, which will be compared with an endoscopically derived jejunal biopsy from the upper gastrointestinal endoscopy control group. Stool samples for microbiota profiling will be collected at baseline and 1 year after surgery. Primary outcomes are fecal microbiota composition and mucus characteristics. Secondary outcomes include Paneth cell phenotype, body weight, diet composition, glucose tolerance, resolution of comorbidities, and weight loss 1 year after surgery., Results: This trial is currently open for recruitment. The anticipated completion date is December 2019., Conclusions: The Diet-Induced Alteration of Microbiota and Development of Obesity, NAFLD, and Diabetes study will improve insight into the pathophysiology of obesity and its associated metabolic disorders. Better understanding of weight loss failure and weight regain following bariatric surgery might also behold new therapeutic opportunities for obesity and obesity-related comorbidities., Trial Registration: Netherlands Trial Register NTR5660; https://www.trialregister.nl/trial/5540 (Archived by WebCite at http://www.webcitation.org/78l7jOZre)., International Registered Report Identifier (irrid): DERR1-10.2196/11553., (©Martine Uittenbogaart, Wouter KG Leclercq, Danielle Bonouvrie, Marleen M Romeijn, Arijan APM Luijten, Steven WM Olde Damink, Francois MH van Dielen, Sander S Rensen. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 19.06.2019.)

Background: Living tissues maintain a fine balance between protein synthesis and protein breakdown rates. Animal studies indicate that protein synthesis rates are higher in organs when compared with skeletal muscle tissue. As such, organ and tumour protein synthesis could have major effects on whole-body protein metabolism in wasting disorders such as cancer cachexia. We aimed to assess protein synthesis rates in pancreatic tumour tissue and healthy pancreas, liver, and skeletal muscle tissue in vivo in humans., Methods: In eight patients with pancreatic cancer undergoing pancreaticoduodenectomy, primed continuous infusions with L-[ring- 13 C 6 ]phenylalanine and L-[3,5- 2 H 2 ]tyrosine were started prior to surgery and continued throughout the surgical procedures. During surgery, plasma samples and biopsies from the pancreas, pancreatic tumour, liver, and vastus lateralis muscle were taken. Post-absorptive fractional protein synthesis rates were determined by measuring incorporation of labelled L-[ring- 13 C 6 ]phenylalanine in tissue protein using the weighed plasma L-[ring- 13 C 6 ]phenylalanine enrichments as the precursor pool., Results: Five male patients and three female patients with a mean age of 67 ± 2 years were included into this study. Plasma L-[ring- 13 C 6 ]phenylalanine enrichments (6-9 mole per cent excess) did not change during surgery (P = 0.60). Pancreatic tumour protein synthesis rates were 2.6-fold lower than surrounding pancreatic tissue protein synthesis rates (0.268 ± 0.053 vs. 0.694 ± 0.228%/h, respectively; P = 0.028) and 1.7-fold lower than liver protein synthesis rates (0.268 ± 0.053 vs. 0.448 ± 0.043%/h, respectively; P = 0.046). Among healthy organ samples, protein synthesis rates were 20-fold and 13-fold higher in pancreas and liver, respectively, compared with skeletal muscle tissue (0.694 ± 0.228 and 0.448 ± 0.043 vs. 0.035 ± 0.005%/h, respectively; P < 0.05)., Conclusions: Liver and pancreas tissue protein synthesis rates are higher when compared with pancreatic tumour and skeletal muscle tissue protein synthesis rates and can, therefore, strongly impact whole-body protein metabolism in vivo in humans., (© 2019 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Background: Most prognostic scoring systems for colorectal liver metastases (CRLMs) account for factors related to tumour biology. Little is known about the effects of the host phenotype to the tumour. Our objective was to delineate the relationship of systemic inflammation and body composition features [i.e. low skeletal muscle mass (sarcopenia) and low visceral adipose tissue (VAT)], two well-described host phenotypes in cancer., Methods: Clinical data and pre-operative blood samples were collected from 99 patients who underwent resection of CRLM. Pre-operative computed tomography scans were available for 97 patients; body composition was analysed at the L3 level, stratified for sex and age. Clinicopathological variables, serum C-reactive protein (CRP), and various body composition variables were evaluated. Overall survival was evaluated as a function of these same variables in multivariate Cox regression analysis., Results: Skeletal muscle was significantly correlated with VAT (r = 0.46, P < 0.001). Of patients with sarcopenia, 35 (65%) also had low VAT. C-reactive protein was elevated (≥5 mg/mL) in 42 patients (43.3%). Elevated CRP was more common in patients with sarcopenia (73.8% vs. 51.1%, P = 0.029). The most significant prognostic factors were the coincidence of elevated CRP and adverse body composition features (sarcopenia and/or low VAT; hazard ratio 4.3, 95% confidence interval 1.5-13.0, P = 0.008), as well as Fong clinical prognostic score (hazard ratio 2.9, 95% confidence interval 1.5-5.5, P = 0.002)., Conclusions: Body composition in patients with CRLM is not directly linked to the presence of systemic inflammation. However, when systemic inflammation coincides with sarcopenia and/or low VAT, prognosis is adversely affected, independent of the Fong clinical prognostic score., (© 2018 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Adaptation of the smooth muscle cell (SMC) phenotype is essential for homeostasis and is often involved in pathologies of visceral organs (e.g., uterus, bladder, gastrointestinal tract). In vitro studies of the behavior of visceral SMCs under (patho)-physiological conditions are hampered by a spontaneous, uncontrolled phenotypic modulation of visceral SMCs under regular tissue culture conditions. We aimed to develop a new visceral SMC culture model that allows controlled phenotypic modulation. Human uterine SMCs [ULTR and telomerase-immortalized human myometrial cells (hTERT-HM)] were grown to confluency and kept for up to 6 days on regular tissue culture surfaces or basement membrane (BM) matrix-coated surfaces in the presence of 0-10% serum. mRNA and protein expression and localization of SMC-specific phenotype markers and their transcriptional regulators were investigated by quantitative PCR, Western blotting, and immunofluorescence. Maintaining visceral SMCs confluent for 6 days increased α-smooth muscle actin (1.9-fold) and smooth muscle protein 22-α (3.1-fold), whereas smooth muscle myosin heavy chain was only slightly upregulated (1.3-fold). Culturing on a BM matrix-coated surface further increased these proteins and also markedly promoted mRNA expression of γ-smooth muscle actin (15.0-fold), smoothelin (3.5-fold), h-caldesmon (5.2-fold), serum response factor (7.6-fold), and myocardin (8.1-fold). Whereas additional serum deprivation only minimally affected contractile markers, platelet-derived growth factor-BB and transforming growth factor β1 consistently reduced versus increased their expression. In conclusion, we present a simple and reproducible visceral SMC culture system that allows controlled phenotypic modulation toward both the synthetic and the contractile phenotype. This may greatly facilitate the identification of factors that drive visceral SMC phenotypic changes in health and disease.

Background and Aims: We recently showed that plasma cholesteryl ester transfer protein (CETP) is mainly derived from VSIG4-positive Kupffer cells. Activation of these cells by the bacterial endotoxin lipopolysaccharide (LPS) strongly decreases CETP expression. As Kupffer cell activation plays a detrimental role in the progression of non-alcoholic fatty liver disease (NAFLD), we aimed to study if metabolic liver inflammation is also associated with a decrease in hepatic and circulating CETP., Methods: We collected plasma and liver biopsy samples at various stages of NAFLD from 93 obese individuals who underwent bariatric surgery. Liver lobular inflammation was histologically determined, and liver CETP expression, CETP positive cells, circulating CETP concentrations, and liver VSIG4 expression were quantified., Results: Mean (SD) plasma CETP concentration was 2.68 (0.89) μg/mL. In the presence of liver inflammation, compared to the absence of pathology, the difference in hepatic CETP expression was -0.03 arbitrary units (95% CI -0.26, 0.20), the difference in number of hepatic CETP positive cells (range 11-140 per mm 2 ) was -20.0 per mm 2 (95% CI -41.6, 1.9), and the difference in plasma CETP was -0.35 μg/mL (95% CI -0.80, 0.10). Hepatic VSIG4 expression was not associated with liver inflammation (0.00; 95% CI -0.15, 0.15)., Conclusions: We found no strong evidence for a strong negative association between metabolic liver inflammation and CETP-related outcomes in obese individuals, although we observed consistent trends. These data indicate that metabolic liver inflammation does not mimic the strong effects of LPS on the hepatic expression and production of CETP by Kupffer cells., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Myosteatosis, characterized by inter- and intramyocellular fat deposition, is strongly related to poor overall survival after surgery for periampullary cancer. It is commonly assessed by calculating the muscle radiation attenuation on computed tomography (CT) scans. However, since magnetic resonance imaging (MRI) is replacing CT in routine diagnostic work-up, developing methods based on MRI is important. We developed a new method using MRI-muscle signal intensity to assess myosteatosis and compared it with CT-muscle radiation attenuation., Methods: Patients were selected from a prospective cohort of 236 surgical patients with periampullary cancer. The MRI-muscle signal intensity and CT-muscle radiation attenuation were assessed at the level of the third lumbar vertebra and related to survival., Results: Forty-seven patients were included in the study. Inter-observer variability for MRI assessment was low (R 2  = 0.94). MRI-muscle signal intensity was associated with short survival: median survival 9.8 (95%-CI: 1.5-18.1) vs. 18.2 (95%-CI: 10.7-25.8) months for high vs. low intensity, respectively (p = 0.038). Similar results were found for CT-muscle radiation attenuation (low vs. high radiation attenuation: 10.8 (95%-CI: 8.5-13.1) vs. 15.9 (95%-CI: 10.2-21.7) months, respectively; p = 0.046). MRI-signal intensity correlated negatively with CT-radiation attenuation (r=-0.614, p < 0.001)., Conclusions: Myosteatosis may be adequately assessed using either MRI-muscle signal intensity or CT-muscle radiation attenuation., (Copyright © 2018 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: Obesity is a risk factor for non-alcoholic steatohepatitis (NASH). This risk has been attributed to visceral adipose tissue (vAT) expansion associated with increased proinflammatory mediators. Accumulation of CD11c + proinflammatory adipose tissue macrophages (ATM) is an important driver of vAT inflammation. We investigated the role of ATMs in hepatic inflammation during NASH development., Design: vAT isolated from lean, obese or ATM-depleted (using clodronate liposomes) obese mice was transplanted to lean ldlr -/- acceptor mice. Systemic and hepatic inflammation was assessed either after 2 weeks on standard chow or after 8 weeks on high cholesterol diet (HCD) to induce NASH., Results: Transplanting donor vAT from obese mice increased HCD-induced hepatic macrophage content compared with lean-transplanted mice, worsening liver damage. ATM depletion prior to vAT transplantation reduced this increased hepatic macrophage accumulation. On chow, vAT transplantation induced a more pronounced increase in circulating and hepatic neutrophil numbers in obese-transplanted than lean-transplanted mice, while ATM depletion prior to vAT transplantation reversed this effect. Microarray analysis of fluorescence-activated cell sorting of CD11c + and CD11c - macrophages isolated from donor adipose tissue showed that obesity resulted in enhanced expression of neutrophil chemotaxis genes specifically in CD11c + ATMs. Involvement of the neutrophil chemotaxis proteins, CXCL14 and CXCL16, was confirmed by culturing vAT. In humans, CD11c expression in vAT of obese individuals correlated with vAT expression of neutrophil chemotactic genes and with hepatic expression of neutrophil and macrophage marker genes., Conclusion: ATMs from obese vAT induce hepatic macrophage accumulation during NASH development, possibly by enhancing neutrophil recruitment., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Hepatocellular lipid accumulation characterizes nonalcoholic fatty liver disease (NAFLD). However, the types of lipids associated with disease progression are debated, as is the impact of their localization. Traditional lipidomics analysis using liver homogenates or plasma dilutes and averages lipid concentrations, and does not provide spatial information about lipid distribution. We aimed to characterize the distribution of specific lipid species related to NAFLD severity by performing label-free molecular analysis by mass spectrometry imaging (MSI). Fresh frozen liver biopsies from obese subjects undergoing bariatric surgery ( n = 23) with various degrees of NAFLD were cryosectioned and analyzed by matrix-assisted laser desorption/ionization (MALDI)-MSI. Molecular identification was verified by tandem MS. Tissue sections were histopathologically stained, annotated according to the Kleiner classification, and coregistered with the MSI data set. Lipid pathway analysis was performed and linked to local proteome networks. Spatially resolved lipid profiles showed pronounced differences between nonsteatotic and steatotic tissues. Lipid identification and network analyses revealed phosphatidylinositols and arachidonic acid metabolism in nonsteatotic regions, whereas low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) metabolism was associated with steatotic tissue. Supervised and unsupervised discriminant analysis using lipid based classifiers outperformed simulated analysis of liver tissue homogenates in predicting steatosis severity. We conclude that lipid composition of steatotic and nonsteatotic tissue is highly distinct, implying that spatial context is important for understanding the mechanisms of lipid accumulation in NAFLD. MSI combined with principal component-linear discriminant analysis linking lipid and protein pathways represents a novel tool enabling detailed, comprehensive studies of the heterogeneity of NAFLD.

Background: Lipopolysaccharide (LPS) decreases hepatic CETP (cholesteryl ester transfer protein) expression albeit that the underlying mechanism is disputed. We recently showed that plasma CETP is mainly derived from Kupffer cells (KCs). In this study, we investigated the role of KC subsets in the mechanism by which LPS reduces CETP expression., Methods and Results: In CETP-transgenic mice, LPS markedly decreased hepatic CETP expression and plasma CETP concentration without affecting hepatic macrophage number. This was paralleled by decreased expression of the resting KC markers C-type lectin domain family 4, member f ( Clec4f ) and V-set and immunoglobulin domain containing 4 ( Vsig4 ), while expression of the infiltrating monocyte marker lymphocyte antigen 6 complex locus C ( Ly6C ) was increased. Simultaneously, the ratio of plasma high-density lipoprotein-cholesterol over non-high-density lipoprotein-cholesterol transiently increased. After ablation hepatic macrophages via injection with liposomal clodronate, the reappearance of hepatic gene and protein expression of CETP coincided with Clec4f and Vsig4, but not Ly6C. Double-immunofluorescence staining showed that CETP co-localized with Clec4f + KCs and not Ly6C + monocytes. In humans, microarray gene-expression analysis of liver biopsies revealed that hepatic expression and plasma level of CETP both correlated with hepatic VSIG4 expression. LPS administration decreased the plasma CETP concentration in humans. In vitro experiments showed that LPS reduced liver X receptor-mediated CETP expression., Conclusions: Hepatic expression of CETP is exclusively confined to the resting KC subset (ie, F4/80 + Clec4f + Vsig4 + Ly6C - ). LPS activated resting KCs, leading to reduction of Clec4f and Vsig4 expression and reduction of hepatic CETP expression, consequently decreasing plasma CETP and raising high-density lipoprotein (HDL)-cholesterol. This sequence of events is consistent with the anti-inflammatory role of HDL in the response to LPS and may be relevant as a defense mechanism against bacterial infections., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Background/objectives: Mutations in the Tubby gene (TUB) cause late-onset obesity and insulin resistance in mice and syndromic obesity in humans. Although TUB gene function has not yet been fully elucidated, studies in rodents indicate that TUB is involved in the hypothalamic pathways regulating food intake and adiposity. Aside from the function in central nervous system, TUB has also been implicated in energy metabolism in adipose tissue in rodents. We aimed to determine the expression and distribution patterns of TUB in man as well as its potential association with obesity., Subjects/methods: In situ hybridization was used to localize the hypothalamic regions and cells expressing TUB mRNA. Using RT-PCR, we determined the mRNA expression level of the two TUB gene alternative splicing isoforms, the short and the long transcript variants, in the hypothalami of 12 obese and 12 normal-weight subjects, and in biopsies from visceral (VAT) and subcutaneous (SAT) adipose tissues from 53 severely obese and 24 non-obese control subjects, and correlated TUB expression with parameters of obesity and metabolic health., Results: Expression of both TUB transcripts was detected in the hypothalamus, whereas only the short TUB isoform was found in both VAT and SAT. TUB mRNA was detected in several hypothalamic regions involved in body weight regulation, including the nucleus basalis of Meynert and the paraventricular, supraoptic and tuberomammillary nuclei. We found no difference in the hypothalamic TUB expression between obese and control groups, whereas the level of TUB mRNA was significantly lower in adipose tissue of obese subjects as compared to controls. Also, TUB expression was negatively correlated with indices of body weight and obesity in a fat-depot-specific manner., Conclusions: Our results indicate high expression of TUB in the hypothalamus, especially in areas involved in body weight regulation, and the correlation between TUB expression in adipose tissue and obesity. These findings suggest a role for TUB in human obesity.

Background: An inverse relation between chemotherapy-associated liver injury (CALI) and tumour response to chemotherapy has been reported. The aim was to validate these findings, and further investigate the impact of CALI on survival in patients who underwent partial hepatectomy for colorectal liver metastases (CRLM)., Methods: Patients who received neoadjuvant chemotherapy and underwent partial hepatectomy for CRLM between 2008 and 2014 were included. Liver and tumour specimens were histologically examined for CALI (steatosis, steatohepatitis, sinusoidal dilatation [SD], nodular regeneration) and tumour regression grade (TRG). TRG 1-2 was defined as complete tumour response., Results: 166 consecutive patients were included with a median survival of 30 and 44 months for recurrence-free and overall survival, respectively. Grade 2-3 SD was found in 44 (27%) and TRG 1-2 was observed in 33 (20%) patients. Of studied CALI, only grade 2-3 SD was associated with increased TRG 3-5 (odds ratio 3.99, 95% CI 1.17-13.65, p = 0.027). CALI was not significantly related to survival. TRG 1-2 was associated with prolonged recurrence-free (hazard ratio 0.47, 95% CI 0.25-0.89, p = 0.020) and overall survival (hazard ratio 0.35, 95% CI 0.18-0.68, p = 0.002)., Conclusion: CALI was not directly related to survival. CALI was, however, associated with diminished complete tumour response, and diminished complete tumour response, in turn, was associated with decreased survival., (Copyright © 2017 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Hepatocyte apoptosis in nonalcoholic steatohepatitis (NASH) can lead to fibrosis and cirrhosis, which permanently damage the liver. Understanding the regulation of hepatocyte apoptosis is therefore important to identify therapeutic targets that may prevent the progression of NASH to fibrosis. Recently, increasing evidence has shown that long noncoding (lnc) RNAs are involved in various biological processes and that their dysregulation underlies a number of complex human diseases. By performing gene expression profiling of 4,383 lncRNAs in 82 liver samples from individuals with NASH (n = 48), simple steatosis but no NASH (n = 11), and healthy controls (n = 23), we discovered a liver-specific lncRNA (RP11-484N16.1) on chromosome 18 that showed significantly elevated expression in the liver tissue of NASH patients. This lncRNA, which we named lnc18q22.2 based on its chromosomal location, correlated with NASH grade (r = 0.51, P = 8.11 × 10 -7 ), lobular inflammation (r = 0.49, P = 2.35 × 10 -6 ), and nonalcoholic fatty liver disease activity score (r = 0.48, P = 4.69 × 10 -6 ). The association of lnc18q22.2 to liver steatosis and steatohepatitis was replicated in 44 independent liver biopsies (r = 0.47, P = 0.0013). We provided a genetic structure of lnc18q22.2 showing an extended exon 2 in liver. Knockdown of lnc18q22.2 in four different hepatocyte cell lines resulted in severe phenotypes ranging from reduced cell growth to lethality. This observation was consistent with pathway analyses of genes coexpressed with lnc18q22.2 in human liver or affected by lnc18q22.2 knockdown., Conclusion: We identified an lncRNA that can play an important regulatory role in liver function and provide new insights into the regulation of hepatocyte viability in NASH. (Hepatology 2017;66:794-808)., (© 2017 by the American Association for the Study of Liver Diseases.)