Pre-to-post diagnosis weight trajectories in colorectal cancer patients with non-metastatic disease

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High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model

Normothermic Ex Vivo Liver Platform Using Porcine Slaughterhouse Livers for Disease Modeling

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments

One-carbon metabolites, B vitamins and associations with systemic inflammation and angiogenesis biomarkers among colorectal cancer patients:results from the ColoCare Study

B-vitamins involved in one-carbon metabolism have been implicated in the development of inflammation- A nd angiogenesis-related chronic diseases, such as colorectal cancer. Yet, the role of one-carbon metabolism in inflammation and angiogenesis among colorectal cancer patients remains unclear.The objective of this study was to investigate associations of components of one-carbon metabolism with inflammation and angiogenesis biomarkers among newly diagnosed colorectal cancer patients (n=238) in the prospective ColoCare Study, Heidelberg.We cross-sectionally analyzed associations between 12 B-vitamins and one-carbon metabolites and 10 inflammation and angiogenesis biomarkers from pre-surgery serum samples using multivariable linear regression models. We further explored associations among novel biomarkers in these pathways with Spearman partial correlation analyses. We hypothesized that pyridoxal-5'-phosphate (PLP) is inversely associated with inflammatory biomarkers.We observed that PLP was inversely associated with CRP (r=-0.33, plinearlinear=0.003), IL-6 (r=-0.39, plinear linear=0.02) and TNFα (r=-0.12, plinear=0.045). Similar findings were observed for 5-methyl-tetrahydrofolate and CRP (r=-0.14), SAA (r=-0.14) and TNFα (r=-0.15) among colorectal cancer patients. Folate catabolite apABG was positively correlated with IL-6 (r= 0.27, plinearlinear<0.0001), indicating higher folate utilization during inflammation.Our data support the hypothesis of inverse associations between PLP and inflammatory biomarkers among colorectal cancer patients. A better understanding of the role and inter-relation of PLP and other one-carbon metabolites with inflammatory processes among colorectal carcinogenesis and prognosis could identify targets for future dietary guidance for colorectal cancer patients.</p

Single Cell Analysis Facilitates Staging of Blimp1-Dependent Primordial Germ Cells Derived from Mouse Embryonic Stem Cells

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKitbright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKitbright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5–10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKitbright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development

Metabolites across the colorectal cancer continuum : A clinical and nutritional perspective

Colorectal cancer is one of the most prevalent cancers worldwide. Genetic, environmental, nutritional, and other lifestyle factors play a large role in the development of colorectal cancer, but the role in colorectal cancer recurrence and survival is largely unknown. Measuring metabolites, i.e. molecules present in biospecimens, such as blood and tissue samples, can help to unravel the underlying biology of the colorectal cancer continuum. The colorectal cancer continuum reflects the process in which normal cells progress into colorectal cancer cells. Therefore, the overall aim of this thesis is to better understand the association between circulating metabolites and the colorectal cancer continuum from a clinical and nutritional perspective in two international consortia.In Chapter 2, the association between plasma metabolites and colorectal cancer occurrence is studied using an untargeted metabolomics approach. Data of 268 stage I-IV colorectal cancer patients and 353 colorectal cancer-free individuals of an international consortium are included. Data are split into an independent discovery and replication set. After replication, 28 molecules are associated with colorectal cancer, including 15 molecules that could be identified as known metabolites. Levels of taurine, hypoxanthine, lysophosphatidylethanolamines (LysoPEs) (20:4) and (22:6) are shown to be higher among colorectal cancer patients compared to the colorectal cancer-free individuals. The opposite is observed for valine, leucine, bilirubin, 1-methylnicotinamide, and seven phosphatidylcholines (LysoPCs), namely LysoPC(15:0), LysoPC(16:0), LysoPC(16:0) isomer, LysoPC(P-16:0), LysoPC(16:1), LysoPC(17:0), and&nbsp; LysoPC(18:0), which show lower levels among colorectal cancer patients compared to colorectal cancer-free individuals.The association between plasma metabolites at diagnosis and colorectal cancer stage is investigated using a targeted metabolomics approach in Chapter 3. In total, 744 patients with stage I-IV colorectal cancer from an international consortium are included in the analysis. Sphingomyelin (SM) C26:0 show lower concentrations among patients with stage III compared to stage I colorectal cancer. SM C18:0 and phosphatidylcholine diacyl (PC aa) C32:0 concentrations are higher, whereas citrulline, histidine, PC aa C34:4, PC acyl-alkyl (ae) C40:1 and LysoPC a C16:0 and C17:0 are lower among stage IV compared to stage I colorectal cancer patients. No differences in plasma metabolite concentrations are observed between stage II and stage I colorectal cancer patients.In Chapter 4, two diet quality indices and three dietary patterns, and their associations with plasma metabolites in colorectal cancer patients are shown. Plasma metabolites of 195 stage I-IV colorectal cancer patients are quantified using a targeted metabolomics approach. Two widely acknowledged diet quality indices are used, the 2018 dietary cancer prevention recommendations of the World Cancer Research Fund (WCRF), i.e. the WCRF dietary score, and the Dutch Healthy Diet Index (DHD15-index), based on the Dutch dietary guidelines of 2015. A higher intake of a healthier diet is reflected by a higher WCRF score and DHD15-index. The three dietary patterns are identified based on the reported habitual dietary intake of the colorectal cancer patients by Food Frequency Questionnaire at cancer diagnosis, i.e. a Western, Carnivore and Prudent pattern. The Western and Carnivore pattern reflect high intake of items usually present in an unhealthier diet. The Western dietary pattern is characterized by a high intake of snacks, savory sauces and spreads, refined grains, pizza, high and medium-fat dairy, nuts and seeds, beer, and hard fats, and a low intake of whole grain products and potatoes. The Carnivore pattern is characterized by a high intake of red and processed meat, poultry, fish, eggs, and potatoes, and a low intake of soy and vegetarian products, and medium and high-fat dairy. The Prudent pattern reflects a healthy diet, with high consumptions of vegetables, fruits, fish, nuts and seeds, and low-fat dairy and low intakes of pastry and biscuits, and beer. Better adherence to healthier diets is associated with lower concentrations of plasma phosphatidylcholines (Chapter 4). More specifically, patients with better adherence to the WCRF dietary score, DHD guidelines and higher consumptions of the Prudent pattern show lower concentrations of plasma phosphatidylcholines. Contrary, patients with higher intakes of the Western and Carnivore patterns show higher concentrations of plasma phosphatidylcholines.Chapter 5 describes the associations between circulating concentrations of folate species at diagnosis and colorectal cancer recurrence and survival. The folate species folate, folic acid, and folate catabolites p-aminobenzoylglutamate (pABG) and p-acetamidobenzoylglutamate (apABG) are quantified using a targeted metabolomics approach in blood samples of 2024 stage I-III colorectal cancer patients within an international consortium. Circulating concentrations of folate, pABG and apABG are not associated with colorectal cancer recurrence and survival. Higher compared to lower concentrations of folic acid are associated with an increased risk of recurrences, but folic acid concentrations are not associated with colorectal cancer survival.The findings of this thesis suggest that amino acid and lipid metabolism may play important roles in the colorectal cancer continuum. In addition, higher circulating concentrations of folic acid may be associated with an increased risk of colorectal cancer recurrence. Based on the observations in this thesis, investigating metabolite concentrations across the colorectal cancer continuum may provide important leads to further study the underlying biology of colorectal cancer development and progression.This thesis also showed that national and international collaborations are valuable because of the availability of data from colorectal cancer patients from several countries and the availability of a wide variability in exposures and outcomes. Future national and international collaborations are, therefore, encouraged to further study the underlying biology of the complex interplay between metabolites, nutrition, and the colorectal cancer continuum from a clinical and nutritional perspective

Multi-omics of wellbeing

In light of major demographic trends, building and maintaining wellbeing (WB) is one of the most important societal challenges. People who feel well, function better, are less susceptible to mental illness, and thus are better able to retain competitive advantage and expand human capital. The role of genetics, and epigenetics in WB has been previously studied individually with promising results (Baselmans, Jansen, et al., 2019; Baselmans, van de Weijer, et al., 2019). However, much less is known about how these different layers interact – or if they interact at all – in shaping wellbeing. In addition, knowledge about the effects of other omics layers (and their interactions) is currently lacking. "Omics" refers to large-scale biological data, such as genomics (study of genes), transcriptomics (study of gene expression), proteomics (study of proteins), etc. Multi-omics refers to a comprehensive approach in biological research where scientists study and analyze various types of biological information, such as genes (genomics), proteins (proteomics), and other molecular factors, all at the same time. This holistic approach provides a more complete and integrated understanding of complex biological systems. This method has been proven useful in other phenotypes such as depression, childhood aggression, or ADHD. In the current study, we apply this multi-omics approach to wellbeing

Normothermic Ex Vivo Liver Platform Using Porcine Slaughterhouse Livers for Disease Modeling

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments

Dietary intake of magnesium or calcium and chemotherapy-induced peripheral neuropathy in colorectal cancer patients

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and severe side-effect in colorectal cancer (CRC) patients. This study assessed the association between habitual dietary intake of magnesium or calcium and prevalence and severity of chronic CIPN in CRC patients receiving adjuvant chemotherapy. For this prospective cohort study, 196 CRC patients were considered. Magnesium and calcium intake was determined using a food frequency questionnaire at diagnosis, during and after chemotherapy. Chronic CIPN was assessed 12 months after diagnosis using the quality of life questionnaire CIPN20. Prevalence ratios were calculated to assess the association between magnesium or calcium intake and the prevalence of CIPN. Multivariable linear regression analysis was used to assess the association between magnesium or calcium intake and severity of CIPN. CIPN was reported by 160 (82%) patients. Magnesium intake during chemotherapy was statistically significantly associated with lower prevalence of CIPN (prevalence ratio (PR) 0.53, 95% confidence interval (CI) 0.32, 0.92). Furthermore, higher dietary intake of magnesium during (β-1.08, 95% CI -1.95, -0.22) and after chemotherapy (β-0.93, 95% CI -1.81, -0.06) was associated with less severe CIPN. No associations were found for calcium intake and the prevalence and severity of CIPN. To conclude, we observed an association between higher dietary magnesium intake and lower prevalence and severity of CIPN in CRC patients

High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model