Stefan Krauss

• Kømurcu, Kristina Sæterdal; Zawadzka, Malgorzata Elzbieta; Meszka, Igor; Aizenshtadt, Aleksandra; Hrušková, Helena & Aakervik, Lydia Emilie [Show all 10 contributors for this article] (2026). A Validated Mass Spectrometry Platform for Oxysterol Analysis of Single Human Gastruloids and Liver Organoids. Analytical Chemistry. ISSN 0003-2700. 98(9), p. 6792–6804. doi: 10.1021/acs.analchem.5c07140. Full text in Research Archive
• Bentzen, Heidi Beate; Gaillard, Maxence; Nachman, Iftach; Reumann, Daniel; Gadegaard, Nikolaj & David, Laurent [Show all 14 contributors for this article] (2026). A guide to using embedded ethics in human stem-cell-based embryo model research. Nature Cell Biology. ISSN 1465-7392. 28(4), p. 665–673. doi: 10.1038/s41556-026-01909-9. Full text in Research Archive
• Sowa, Sven T.; Kücükdisli, Murat; Mostinski, Yelena; Schaller, David A.; Vinagreiro, Carolina S. & Cirillo, Davide [Show all 16 contributors for this article] (2025). Deconstruction of Dual-Site Tankyrase Inhibitors Provides Insights into Binding Energetics and Suggests Critical Hotspots for Ligand Optimization. Journal of Medicinal Chemistry. ISSN 0022-2623. 68(7), p. 7263–7279. doi: 10.1021/acs.jmedchem.4c02845. Full text in Research Archive
• Martinez, Mikel Amirola; Combriat, Thomas Michel Daniel; Ferencevic, Katharina; Wilhelmsen, Ingrid; Dalmao-Fernandez, Andrea & Olsen, Petter Angell [Show all 9 contributors for this article] (2025). Aspects of zone-like identity and holotomographic tracking of human stem cell-derived liver sinusoidal endothelial cells. Frontiers in Cell and Developmental Biology. 13. doi: 10.3389/fcell.2025.1528991. Full text in Research Archive Show summary

  Introduction: Liver sinusoidal endothelial cells (LSEC) are specialized endothelial cells with unique metabolic and barrier functions adapted to the needs of the liver sinusoid. LSECs are highly sensitive to their environment, and this fragile nature causes challenges in analyzing their phenotype under in vitro conditions. Methods: In this work, we first differentiated LSEC-like cells (scLSECs) from two human pluripotent stem cell lines and characterized them by a panel of qPCR markers, immunohistochemistry, substrate oxidation for energy metabolism, scavenger function, and nitric oxide secretion. We then introduced holotomography, a technique that allows to recover quantitative and three-dimensional information about the refractive indexes of cell components, as a tool to image and track scLSEC in vitro in a minimally intrusive, label-free manner. Results and discussion: Holotomography and developed machine learning-based algorithm for image processing allowed us to describe and monitor changes in intracellular pore-like structures over time. Finally, we tested the possibility of inducing aspects of zone-specific LSEC phenotype and metabolism using culture-based treatments, which resulted in modest shifts in marker expression and metabolic activity. The presented strategy provides an advanced tool kit for investigating liver endothelial cells.

• Nøvik, Steffen; Hirayama-Shoji, Kayoko; Combriat, Thomas; Sakalys, Domantas; Aashamar, Erik & Louch, William E. [Show all 9 contributors for this article] (2025). Bioimpedance and electrophysiology measurements of engineered heart tissues outside the culture medium. Biosensors and Bioelectronics. ISSN 0956-5663. 287, p. 117634–117634. doi: 10.1016/j.bios.2025.117634. Full text in Research Archive
• Pabst, Oliver; Schülke, Christin; Balasingham, Ilangko; Krauss, Stefan Johannes Karl; Glover, Joel Clinton & Martinsen, Ørjan Grøttem (2025). Recording and modeling of pinched hysteresis loops, the fingerprint of a memristor, in neurons. Scientific Reports. 15. doi: 10.1038/s41598-025-21035-0. Full text in Research Archive Show summary

  A variant of the Hodgkin-Huxley model has been proposed in which time-varying Na+ and K+ ion conductances are replaced by memristors, based on the proposal that ion channels are memristors. This hypothesis predicts that current-voltage plots of neurons subjected to sinusoidal stimulation should exhibit a pinched hysteresis loop, the fingerprint of a memristor. We tested this using whole-cell patch clamp recordings from human neurons derived from neural progenitor cells and observed pinched hysteresis loops in 16% of all recorded neurons. Recordings with current clamp and voltage clamp with a holding potential of 0 mV yielded a higher success rate than voltage clamp recordings with a holding potential of around − 60 mV. In addition, more neurons exhibited pinched hysteresis loops when the applied voltage or current amplitude was high, and the frequency was low. The recording of pinched hysteresis loops in neurons gives evidence that these contain memristors and this is the first time to be shown experimentally. This is not just an indication that the memristor-based Hodgkin-Huxley model is valid; it also demonstrates that models of neurons based on memristors actually resemble nature. We then subjected simulated neurons expressing Hodgkin-Huxley voltage-gated Na+ and K+ channels to sinusoidal membrane potential oscillations and found that the presence of pinched hysteresis loops depended strongly on the density of voltage-gated K+ channels and on the membrane capacitance but not on the presence of voltage-gated Na+ channels. Because Na+ channels are expected to be largely inactivated in neurons at a holding potential of 0 mV, these findings together suggest that it is the presence of voltage-gated K+ channels that is critical for the observed memristive behavior.

• Ho, Richard David Jun-Geet; Mossige, Joachim; Ponomartcev, Sergei; Natalia, Smirnova,; Sunny, Dai, Keqing & Hu, Xian [Show all 9 contributors for this article] (2025). [2509.08929] Spontaneous elongation of 3D gastruloids from local cell polarity alignment. arXiv. doi: https:/arxiv.org/abs/2509.08929. Full text in Research Archive Show summary

  Gastruloids are 3D stem cell aggregate models for early embryogenesis that provide a unique platform to study how collective cell dynamics drive tissue symmetry breaking and axial elongation. Using 3D light sheet imaging, we show that a pulse of Chiron, a Wnt activator, induces coherent alignment of cell polarity during elongation. While nuclear elongation occurs with or without treatment, only Chiron-treated gastruloids exhibit quasi-long-range alignment of nuclear axes, linking cell polarity coherence to tissue-scale remodeling. A minimal physical model of polarized cells, incorporating alignment-dependent torques and polarity-mediated adhesion, reproduces symmetry breaking and elongation, demonstrating that local cell polarity alignment alone can drive tissue-scale convergence-extension flows.

• Delon, Ludivine; Busek, Mathias; Menezes, Pedro Duarte; Gadegaard, Nikolaj; Stokowiec, Justyna & Golovin, Alexey [Show all 10 contributors for this article] (2025). Placental Syncytium-on-Chip (PSoC)–Comparison of Forskolin or Mechanical Induced-Syncytialization. Advanced Healthcare Materials. ISSN 2192-2640. doi: 10.1002/adhm.202404462. Full text in Research Archive Show summary

  The placenta is a key embryonic structure that separates maternal and fetal blood systems. The barrier function of the human placenta is performed by villous trophoblasts, i.e. undifferentiated cytotrophoblasts and differentiated syncytiotrophoblats, whose maturation and function are influenced by wall shear stress (WSS) from the maternal blood circulation. Most in vitro placenta models rely on cyclic adenosine monophosphate inducer forskolin (FSK) to establish a placental syncytium. Here a trophoblastic BeWo cell line is used to systematically compare the effect of FSK treatment in static culture with WSS stimulation in a pumpless, recirculating organ-on-chip. It is shown that BeWo cells undergo a similar differentiation under WSS exposure to FSK treatment. A WSS of 0.1 dyn cm−2 leads to cell fusion, polarization, barrier functions, human chorionic gonadotropin (β-hCG) secretion, and increased expression of key transporters. Moreover, WSS induces favorable changes in the levels of FMS-like tyrosine kinase-1 (FLT-1) and Placental Growth Factor (PlGF) suggesting the development of a physiologically relevant placental syncytium-on-chip (PSoC) without the need for FSK. The platform is further expanded to a syncytiotrophoblast/endothelial co-culture showing physiological vascular functions under WSS. The forskolin-free PSoC presented here represents the first pumpless recirculating and scalable platform for physiological placental studies and drug testing.

• Shoji, Junya; Davis, Richard P.; Mummery, Christine L. & Krauss, Stefan Johannes Karl (2025). Global Literature Analysis of Tumor Organoid and Tumor-on-Chip Research. Cancers. 17(1), p. 1–26. doi: 10.3390/cancers17010108. Full text in Research Archive Show summary

  Background: Tumor organoid and tumor-on-chip (ToC) platforms replicate aspects of the anatomical and physiological states of tumors. They, therefore, serve as models for investigating tumor microenvironments, metastasis, and immune interactions, especially for precision drug testing. To map the changing research diversity and focus in this field, we performed a quality-controlled text analysis of categorized academic publications and clinical studies. Methods: Previously, we collected metadata of academic publications on organoids or organ-on-chip platforms from PubMed, Web of Science, Scopus, EMBASE, and bioRxiv, published between January 2011 and June 2023. Here, we selected documents from this metadata corpus that were computationally determined as relevant to tumor research and analyzed them using an in-house text analysis algorithm. Additionally, we collected and analyzed metadata from ClinicalTrials.gov of clinical studies related to tumor organoids or ToC as of March 2023. Results and Discussion: From 3551 academic publications and 139 clinical trials, we identified 55 and 24 tumor classes modeled as tumor organoids and ToC models, respectively. The research was particularly active in neural and hepatic/pancreatic tumor organoids, as well as gastrointestinal, neural, and reproductive ToC models. Comparative analysis with cancer statistics showed that lung, lymphatic, and cervical tumors were under-represented in tumor organoid research. Our findings also illustrate varied research topics, including tumor physiology, therapeutic approaches, immune cell involvement, and analytical techniques. Mapping the research geographically highlighted the focus on colorectal cancer research in the Netherlands, though overall the specific research focus of countries did not reflect regional cancer prevalence. These insights not only map the current research landscape but also indicate potential new directions in tumor model research.

• Kogler, Stian; Skottvoll, Frøydis Sved; Hruskova, Helena; Rise, Frode; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl [Show all 9 contributors for this article] (2025). Electromembrane Extraction Provides Unprecedented Selectivity for Drugs in Cell Culture Media Used in Organoid and Organ-on-Chip Systems. Analytical Chemistry. ISSN 0003-2700. 97(9), p. 4923–4931. doi: 10.1021/acs.analchem.4c04994. Full text in Research Archive Show summary

  The use of organoids and organ-on-chip technologies as nonanimal methodologies in drug discovery and personalized medicine is rapidly expanding. However, the complexity and small volumes of organoid culture samples present significant analytical challenges, e.g., in drug analysis using liquid chromatography–mass spectrometry (LC–MS). Essentially an electrophoresis across an oil membrane, electromembrane extraction (EME) offers a promising approach for measuring drugs, as it is, for example, compatible with small samples such as organoid and organ-on-chip formats. Given the potential of the technology, there is a need to assess the extraction purity of EME extracts to ensure EME’s compatibility with high-throughput, downstream analysis. This study evaluates the effectiveness of EME for sample cleanup in various common cell culture media used for organoids and organs-on-chips. The media were spiked with 90 small-molecule drugs. Using gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis), high-resolution nuclear magnetic resonance spectroscopy, and LC–MS, we demonstrate that EME provides exhaustive removal of unwanted medium components (proteins, polar molecules, and apolar/neutral molecules) while selectively extracting the spiked small-molecule drugs. The approach was demonstrated with human stem-cell-derived liver organoids, allowing simple detection and monitoring of telltale cytochrome P450 metabolism. Taken together, our observations highlight an unprecedented ability of EME to provide sample cleanup for drug analysis in matrixes compatible with organoids and organ-on-chip technology.

• Cunha, André; Schülke, Christin; Mesri, Alireza; Ruud, Simen Kristoffer; Aizenshtadt, Aleksandra & Ferrari, Giorgio [Show all 16 contributors for this article] (2024). Development of a Smart Wireless Multisensor Platform for an Optogenetic Brain Implant. Sensors. 24(2). doi: 10.3390/s24020575. Full text in Research Archive Show summary

  Implantable cell replacement therapies promise to completely restore the function of neural structures, possibly changing how we currently perceive the onset of neurodegenerative diseases. One of the major clinical hurdles for the routine implementation of stem cell therapies is poor cell retention and survival, demanding the need to better understand these mechanisms while providing precise and scalable approaches to monitor these cell-based therapies in both pre-clinical and clinical scenarios. This poses significant multidisciplinary challenges regarding planning, defining the methodology and requirements, prototyping and different stages of testing. Aiming toward an optogenetic neural stem cell implant controlled by a smart wireless electronic frontend, we show how an iterative development methodology coupled with a modular design philosophy can mitigate some of these challenges. In this study, we present a miniaturized, wireless-controlled, modular multisensor platform with fully interfaced electronics featuring three different modules: an impedance analyzer, a potentiostat and an optical stimulator. We show the application of the platform for electrical impedance spectroscopy-based cell monitoring, optical stimulation to induce dopamine release from optogenetically modified neurons and a potentiostat for cyclic voltammetry and amperometric detection of dopamine release. The multisensor platform is designed to be used as an opto-electric headstage for future in vivo animal experiments.

• Kogler, Stian; Pedersen, Gustav Mathingsdal; Martínez-Ramírez, Felipe; Aizenshtadt, Aleksandra; Busek, Mathias & Krauss, Stefan Johannes Karl [Show all 8 contributors for this article] (2024). An FDA-Validated, Self-Cleaning Liquid Chromatography–Mass Spectrometry System for Determining Small-Molecule Drugs and Metabolites in Organoid/Organ-on-Chip Medium. Analytical Chemistry. ISSN 0003-2700. 96(29), p. 12129–12138. doi: 10.1021/acs.analchem.4c02246. Full text in Research Archive Show summary

  As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography–mass spectrometry (LC–MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC–MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.

• Menezes, Pedro Duarte; Hunter, Alisha; Dickson, Thomas; Hecht, Soren; Kumar, Charchit & Busek, Mathias [Show all 8 contributors for this article] (2024). Scalable, Transparent, and Micro: 3D-Printed Rapid Tooling for Injection Molded Microfluidics. Advanced Engineering Materials. ISSN 1438-1656. 26(20). doi: 10.1002/adem.202400276. Full text in Research Archive
• Hrušková, Helena; Olsen, Christine; Řemínek, Roman; Wang, Chencheng; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl [Show all 10 contributors for this article] (2024). Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin. Journal of Chromatography A. ISSN 0021-9673. 1717. doi: 10.1016/j.chroma.2024.464669. Full text in Research Archive Show summary

  Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

• Wilhelmsen, Ingrid; Combriat, Thomas Michel Daniel; Dalmao-Fernandez, Andrea; Stokowiec, Justyna; Wang, Chencheng & Olsen, Petter Angell [Show all 10 contributors for this article] (2024). The effects of TGF-β-induced activation and starvation of vitamin A and palmitic acid on human stem cell-derived hepatic stellate cells. Stem Cell Research & Therapy. 15(223). doi: 10.1186/s13287-024-03852-8. Full text in Research Archive Show summary

  Background Hepatic stellate cells (HSC) have numerous critical roles in liver function and homeostasis, while they are also known for their importance during liver injury and fibrosis. There is therefore a need for relevant in vitro human HSC models to fill current knowledge gaps. In particular, the roles of vitamin A (VA), lipid droplets (LDs), and energy metabolism in human HSC activation are poorly understood. Methods In this study, human pluripotent stem cell-derived HSCs (scHSCs), benchmarked to human primary HSC, were exposed to 48-hour starvation of retinol (ROL) and palmitic acid (PA) in the presence or absence of the potent HSC activator TGF-β. The interventions were studied by an extensive set of phenotypic and functional analyses, including transcriptomic analysis, measurement of activation-related proteins and cytokines, VA- and LD storage, and cell energy metabolism. Results The results show that though the starvation of ROL and PA alone did not induce scHSC activation, the starvation amplified the TGF-β-induced activation-related transcriptome. However, TGF-β-induced activation alone did not lead to a reduction in VA or LD stores. Additionally, reduced glycolysis and increased mitochondrial fission were observed in response to TGF-β. Conclusions scHSCs are robust models for activation studies. The loss of VA and LDs is not sufficient for scHSC activation in vitro, but may amplify the TGF-β-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.

• Combriat, Thomas Michel Daniel; Olsen, Petter Angell; Låstad, Silja Borring; Malthe-Sørenssen, Anders; Krauss, Stefan Johannes Karl & Dysthe, Dag Kristian (2024). Acoustic Wave-Induced Stroboscopic Optical Mechanotyping of Adherent Cells. Advanced Science. 11(16). doi: 10.1002/advs.202307929. Full text in Research Archive Show summary

  In this study, a novel, high content technique using a cylindrical acoustic transducer, stroboscopic fast imaging, and homodyne detection to recover the mechanical properties (dynamic shear modulus) of living adherent cells at low ultrasonic frequencies is presented. By analyzing the micro-oscillations of cells, whole populations are simultaneously mechanotyped with sub-cellular resolution. The technique can be combined with standard fluorescence imaging allowing to further cross-correlate biological and mechanical information. The potential of the technique is demonstrated by mechanotyping co-cultures of different cell types with significantly different mechanical properties.

• Aizenshtadt, Aleksandra; Wang, Chencheng; Abadpour, Shadab; Menezes, Pedro Duarte; Wilhelmsen, Ingrid & Dalmao-Fernandez, Andrea [Show all 15 contributors for this article] (2024). Pump-Less, Recirculating Organ-on-Chip (rOoC) Platform to Model the Metabolic Crosstalk between Islets and Liver. Advanced Healthcare Materials. ISSN 2192-2640. 13(13). doi: 10.1002/adhm.202303785. Full text in Research Archive Show summary

  Type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD) are epidemiologically correlated disorders with a worldwide growing prevalence. While the mechanisms leading to the onset and development of these conditions are not fully understood, predictive tissue representations for studying the coordinated interactions between central organs that regulate energy metabolism, particularly the liver and pancreatic islets, are needed. Here, a dual pump-less recirculating organ-on-chip platform that combines human pluripotent stem cell (sc)-derived sc-liver and sc-islet organoids is presented. The platform reproduces key aspects of the metabolic cross-talk between both organs, including glucose levels and selected hormones, and supports the viability and functionality of both sc-islet and sc-liver organoids while preserving a reduced release of pro-inflammatory cytokines. In a model of metabolic disruption in response to treatment with high lipids and fructose, sc-liver organoids exhibit hallmarks of steatosis and insulin resistance, while sc-islets produce pro-inflammatory cytokines on-chip. Finally, the platform reproduces known effects of anti-diabetic drugs on-chip. Taken together, the platform provides a basis for functional studies of obesity, T2DM, and MASLD on-chip, as well as for testing potential therapeutic interventions.

• Wang, Chencheng; Abadpour, Shadab; Aizenshtadt, Aleksandra; Dalmao-Fernandez, Andrea; Høyem, Merete & Wilhelmsen, Ingrid [Show all 13 contributors for this article] (2024). Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling. Frontiers in Bioengineering and Biotechnology. 12. doi: 10.3389/fbioe.2024.1392575. Full text in Research Archive Show summary

  Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

• Wang, Chencheng; Abadpour, Shadab; Olsen, Petter Angell; Wang, Daxin; Stokowiec, Justyna & Chera, Simona [Show all 11 contributors for this article] (2024). Glucose Concentration in Regulating Induced Pluripotent Stem Cells Differentiation Toward Insulin-Producing Cells. Transplant International. ISSN 0934-0874. 37. doi: 10.3389/ti.2024.11900. Full text in Research Archive Show summary

  The generation of insulin-producing cells from human-induced pluripotent stem cells holds great potential for diabetes modeling and treatment. However, existing protocols typically involve incubating cells with un-physiologically high concentrations of glucose, which often fail to generate fully functional IPCs. Here, we investigated the influence of high (20 mM) versus low (5.5 mM) glucose concentrations on IPCs differentiation in three hiPSC lines. In two hiPSC lines that were unable to differentiate to IPCs sufficiently, we found that high glucose during differentiation leads to a shortage of NKX6.1+ cells that have co-expression with PDX1 due to insufficient NKX6.1 gene activation, thus further reducing differentiation efficiency. Furthermore, high glucose during differentiation weakened mitochondrial respiration ability. In the third iPSC line, which is IPC differentiation amenable, glucose concentrations did not affect the PDX1/NKX6.1 expression and differentiation efficiency. In addition, glucose-stimulated insulin secretion was only seen in the differentiation under a high glucose condition. These IPCs have higher KATP channel activity and were linked to sufficient ABCC8 gene expression under a high glucose condition. These data suggest high glucose concentration during IPC differentiation is necessary to generate functional IPCs. However, in cell lines that were IPC differentiation unamenable, high glucose could worsen the situation.

• Tse, Janson; O’Keefe, Ryan; Rigopolous, Angela; Carli, Annalisa L. E.; Waaler, Jo & Krauss, Stefan Johannes Karl [Show all 8 contributors for this article] (2023). A Mouse Model for the Rapid and Binomial Assessment of Putative WNT/β-Catenin Signalling Inhibitors. Biomedicines. 11(10). doi: 10.3390/biomedicines11102719. Full text in Research Archive Show summary

  Specific signalling thresholds of the WNT/β-catenin pathway affect embryogenesis and tissue homeostasis in the adult, with mutations in this pathway frequently occurring in cancer. Excessive WNT/β-catenin activity inhibits murine anterior development associated with embryonic lethality and accounts for the driver event in 80% of human colorectal cancers. Uncontrolled WNT/β-catenin signalling arises primarily from impairment mutation in the tumour suppressor gene APC that otherwise prevents prolonged stabilisation of β-catenin. Surprisingly, no inhibitor compounds for WNT/β-catenin signalling have reached clinical use in part owing to the lack of specific in vivo assays that discriminate between on-target activities and dose-limiting toxicities. Here, we present a simple in vivo assay with a binary outcome whereby the administration of candidate compounds to pregnant and phenotypically normal Apcflox/flox mice can rescue in utero death of Apcmin/flox mutant conceptus without subsequent post-mortem assessment of WNT/β-catenin signalling. Indeed, the phenotypic plasticity of born Apcmin/flox conceptus enables future refinement of our assay to potentially enable dosage finding and cross-compound comparisons. Thus, we show for the first time the suitability of endogenous WNT/β-catenin signalling during embryonic development to provide an unambiguous and sensitive mammalian in vivo model to assess the efficacy and bioavailability of potential WNT/β-catenin antagonists.

• LaLone, Vernon; Aizenshtadt, Aleksandra; Goertz, John; Skottvoll, Frøydis Sved; Mota, Marco Barbero & You, Junji [Show all 15 contributors for this article] (2023). Quantitative chemometric phenotyping of three-dimensional liver organoids by Raman spectral imaging. Cell Reports Methods. 3(4). doi: 10.1016/j.crmeth.2023.100440. Full text in Research Archive Show summary

  Confocal Raman spectral imaging (RSI) enables high-content, label-free visualization of a wide range of molecules in biological specimens without sample preparation. However, reliable quantification of the deconvoluted spectra is needed. Here we develop an integrated bioanalytical methodology, qRamanomics, to qualify RSI as a tissue phantom calibrated tool for quantitative spatial chemotyping of major classes of biomolecules. Next, we apply qRamanomics to fixed 3D liver organoids generated from stem-cell-derived or primary hepatocytes to assess specimen variation and maturity. We then demonstrate the utility of qRamanomics for identifying biomolecular response signatures from a panel of liver-altering drugs, probing drug-induced compositional changes in 3D organoids followed by in situ monitoring of drug metabolism and accumulation. Quantitative chemometric phenotyping constitutes an important step in developing quantitative label-free interrogation of 3D biological specimens.

• Olsen, Christine; Wang, Chencheng; Aizenshtadt, Aleksandra; Abadpour, Shadab; Lundanes, Elsa & Skottvoll, Frøydis Sved [Show all 11 contributors for this article] (2023). Simultaneous LC–MS determination of glucose regulatory peptides secreted by stem cell–derived islet organoids. Electrophoresis. ISSN 0173-0835. 44(21-22), p. 1682–1697. doi: 10.1002/elps.202300095. Full text in Research Archive Show summary

  Abstract For studying stem cell–derived islet organoids (SC‐islets) in an organ‐on‐chip (OoC) platform, we have developed a reversed‐phase liquid chromatography–tandem mass spectrometry (RPLC–MS/MS) method allowing for simultaneous determination of insulin, somatostatin‐14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl‐C18 separations using 2.1 mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2 µg/L for human insulin, 0.1 µg/L for somatostatin‐14, and 0.05 µg/L for glucagon. The here‐developed RPLC–MS/MS method showed that the SC‐islets have an insulin response dependent on glucose concentration, and the SC‐islets produce and release somatostatin‐14 and glucagon. The RPLC–MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC‐islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC‐islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well‐plate. Taken together, RPLC–MS/MS method is well suited for multi‐hormone measurements of SC‐islets on an OoC platform.

• Busek, Mathias; Aizenshtadt, Aleksandra; Koch, Timo; Frank, Anna; Delon, Ludivine Sylvie Claude & Martinez, Mikel Amirola [Show all 12 contributors for this article] (2023). Pump-less, recirculating organ-on-a-chip (rOoC) platform. Lab on a Chip. ISSN 1473-0197. doi: 10.1039/D2LC00919F. Full text in Research Archive
• Abadpour, Shadab; Niemi, Essi Maria; Orrhult, Linnea Strid; Hermanns, Carolin; Vries, Rick de & Nogueira, Liebert Parreiras [Show all 12 contributors for this article] (2023). Adipose-Derived Stromal Cells Preserve Pancreatic Islet Function in a Transplantable 3D Bioprinted Scaffold. Advanced Healthcare Materials. ISSN 2192-2640. 12(32). doi: 10.1002/adhm.202300640. Full text in Research Archive Show summary

  Intra-portal islet transplantation is currently the only clinically approved beta cell replacement therapy, but its outcome is hindered by limited cell survival due to a multifactorial reaction against the allogeneic tissue in liver. Adipose-derived stromal cells (ASCs) can potentially improve the islet micro-environment by their immunomodulatory action. The challenge is to combine both islets and ASCs in a relatively easy and consistent long-term manner in a deliverable scaffold. W e report on manufacturing the Three-dimensional (3D) bioprinted double-layered scaffolds with primary islets and ASCs using a mix of alginate/nano-fibrillated cellulose (NFC) bioink. W e demonstrate the diffusion properties of the bioink and the supportive effect of human ASCs on islet viability, glucose sensing, insulin secretion and reducing the secretion of pro-inflammatory cytokines. Diabetic mice transplanted with islet-ASC scaffolds reached normoglycemia 7 days post transplantation with no significant difference between this group and the group received islets under the kidney capsules. In addition, animals transplanted with islet-ASC scaffolds stayed normoglycemic and showed elevated levels of C-peptide compared to mice transplanted with islet-only scaffolds. Our data presents a functional 3D bioprinted scaffold for islets and ASCs transplanted to the extrahepatic site and suggest a possible role of ASCs on improving the islet micro-environment.

• Kømurcu, Kristina Sæterdal; Wilhelmsen, Ingrid; Thorne, James L.; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Aizenshtadt, Aleksandra [Show all 7 contributors for this article] (2023). Mass spectrometry reveals that oxysterols are secreted from non-alcoholic fatty liver disease induced organoids. Journal of Steroid Biochemistry and Molecular Biology. ISSN 0960-0760. 232. doi: 10.1016/j.jsbmb.2023.106355. Full text in Research Archive Show summary

  Oxysterols are potential biomarkers for liver metabolism that are altered under disease conditions such as non-alcoholic fatty liver disease (NAFLD). We here apply sterolomics to organoids used for disease modeling of NAFLD. Using liquid chromatography-mass spectrometry with on-line sample clean-up and enrichment, we establish that liver organoids produce and secrete oxysterols. We find elevated levels of 26-hydroxycholesterol, an LXR agonist and the first oxysterol in the acidic bile acid synthesis, in medium from steatotic liver organoids compared to untreated organoids. Other upregulated sterols in medium from steatotic liver organoids are dihydroxycholesterols, such as 7α,26–dihydroxycholesterol, and 7α,25-dihydroxycholesterol. Through 26-hydroxycholesterol exposure to human stem cell-derived hepatic stellate cells, we observe a trend of expressional downregulation of the pro-inflammatory cytokine CCL2, suggesting a protective role of 26-hydroxycholesterol during early-phased NAFLD disease development. Our findings support the possibility of oxysterols serving as NAFLD indicators, demonstrating the usefulness of combining organoids and mass spectrometry for disease modeling and biomarker studies.

• Wilhelmsen, Ingrid; Martinez, Mikel Amirola; Stokowiec, Justyna; Wang, Chencheng; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl (2023). Characterization of human stem cell-derived hepatic stellate cells and liver sinusoidal endothelial cells during extended in vitro culture. Frontiers in Bioengineering and Biotechnology. 11. doi: 10.3389/fbioe.2023.1223737. Full text in Research Archive Show summary

  Background: There is a significant need for predictive and stable in vitro human liver representations for disease modeling and drug testing. Hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) are important non-parenchymal cell components of the liver and are hence of relevance in a variety of disease models, including hepatic fibrosis. Pluripotent stem cell- (PSC-) derived HSCs (scHSCs) and LSECs (scLSECs) offer an attractive alternative to primary human material; yet, the suitability of scHSCs and scLSECs for extended in vitro modeling has not been characterized. Methods: In this study, we describe the phenotypic and functional development of scHSCs and scLSECs during 14 days of 2D in vitro culture. Cell-specific phenotypes were evaluated by cell morphology, immunofluorescence, and gene- and protein expression. Functionality was assessed in scHSCs by their capacity for intracellular storage of vitamin A and response to pro-fibrotic stimuli induced by TGF-β. scLSECs were evaluated by nitric oxide- and factor VIII secretion as well as endocytic uptake of bioparticles and acetylated low-density lipoprotein. Notch pathway inhibition and co-culturing scHSCs and scLSECs were separately tested as options for enhancing long-term stability and maturation of the cells. Results and Conclusion: Both scHSCs and scLSECs exhibited a post-differentiation cell type-specific phenotype and functionality but deteriorated during extended culture with PSC line-dependent variability. Therefore, the choice of PSC line and experimental timeframe is crucial when designing in vitro platforms involving scHSCs and scLSECs. Notch inhibition modestly improved long-term monoculture in a cell line-dependent manner, while co-culturing scHSCs and scLSECs provides a strategy to enhance phenotypic and functional stability.

• Dalmao-Fernandez, Andrea; Aizenshtadt, Aleksandra; Bakke, Hege Gilbø; Krauss, Stefan Johannes Karl; Rustan, Arild Christian & Thoresen, G. Hege [Show all 7 contributors for this article] (2023). Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies. Frontiers in Bioengineering and Biotechnology. 11, p. 1–13. doi: 10.3389/fbioe.2023.1130693. Full text in Research Archive Show summary

  Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism. Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid. Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

• Kogler, Stian; Kømurcu, Kristina Sæterdal; Olsen, Christine Sandsnes; Shoji, Jun-ya; Skottvoll, Frøydis Sved & Krauss, Stefan Johannes Karl [Show all 8 contributors for this article] (2023). Organoids, organ-on-a-chip, separation science and mass spectrometry: An update. TrAC. Trends in analytical chemistry. ISSN 0165-9936. 161. doi: 10.1016/j.trac.2023.116996. Full text in Research Archive Show summary

  Organoids are 3D models of organs, grown in the laboratory from stem cells. An organ model grown/placed in microfluidic devices is commonly termed an “organ-on-a-chip”. Organoids and organ-on-a-chip devices are becoming important tools for studying physiology, disease modeling, drug discovery, personalized medicine, toxicology, and organ development/embryogenesis. We review how mass spectrometry is used for studying organoids and organ-on-a-chip-derived material. We first focus on proteomics, metabolomics/lipidomics, and hormones, typically discussing liquid chromatography-mass spectrometry (LC-MS) approaches. We then review work on mass spectrometry imaging (MSI) of organoids and discuss organ-on-a-chip coupled with mass spectrometry. The review focuses on research developments from the past four years. Mass spectrometric analysis of organoids and organ-on-a-chip has allowed novel insights on development and disease of e.g. brain, liver, and tumors, demonstrating potential for replacing or complementing animal models and other traditional model systems. Additional applications are emerging, e.g. related to sports doping and environmental toxicology.

• Shoji, Jun-ya; Davis, Richard P.; Mummery, Christine L. & Krauss, Stefan Johannes Karl (2023). Global Meta-Analysis of Organoid and Organ-on-Chip Research. Advanced Healthcare Materials. ISSN 2192-2640. doi: 10.1002/adhm.202301067. Full text in Research Archive Show summary

  Abstract Organoids and cells in organ‐on‐chip platforms replicate higher‐level anatomical, physiological, or pathological states of tissues and organs. These technologies are widely regarded by academia, the pharmacological industry and regulators as key biomedical developments. To map advances in this emerging field, a meta‐analysis based on a quality‐controlled text‐mining algorithm is performed. The analysis covers titles, keywords, and abstracts of categorized academic publications in the literature and preprint databases published after 2010. The algorithm identifies and tracks 149 and 107 organs or organ substructures modeled as organoids and organ‐on‐chip, respectively, stem cell sources, as well as 130 diseases, and 16 groups of organisms other than human and mouse in which organoid/organ‐on‐chip technology is applied. The meta‐analysis illustrates changing diversity and focus in organoid/organ‐on‐chip research and captures its geographical distribution. The downloadable dataset provided is a robust framework for researchers to interrogate with their own questions.

• Faci, Pável Vázquez; Shoji, Kayoko; Nøvik, Steffen; Krauss, Stefan Johannes Karl & Rayner, Simon (2022). Globally Accessible Distributed Data Sharing (GADDS): A decentralized FAIR platform to facilitate data sharing in the life sciences. Bioinformatics. ISSN 1367-4803. 38(15), p. 3812–3817. doi: 10.1093/bioinformatics/btac362. Full text in Research Archive
• Nøvik, Steffen; Drageseth, Magnus Flø; Grøndalen, Magnus Børresen; Nilsen, Ola; Krauss, Stefan Johannes Karl & Martinsen, Ørjan Grøttem [Show all 7 contributors for this article] (2022). A CMOS Multi-Electrode Array for Four-Electrode Bioimpedance Measurements. IEEE Transactions on Biomedical Circuits and Systems. ISSN 1932-4545. 16(6), p. 1276–1286. doi: 10.1109/TBCAS.2022.3214243. Full text in Research Archive Show summary

  This work demonstrates how a multi-electrode array (MEA) dedicated to four-electrode bioimpedance measurements can be implemented on a complementary metal–oxide–semiconductor (CMOS) chip. As a proof of concept, an 8 × 8 pixel array along with dedicated amplifiers was designed and fabricated in the TSMC 180 nm process. Each pixel in the array contains a circular current carrying (CC) electrode that can act as a current source or sink. In order to measure a differential voltage between the pixels, each CC electrode is surrounded by a ring shaped pick up (PU) electrode. The differential voltages can be measured by an on-board instrumentation amplifier, while the currents can be measured with an on-bard transimpedance amplifier. Openings in the passivation layer exposed the aluminum top metal layer, and a metal stack of zinc, nickel and gold was deposited in an electroless plating process. The chips were then wire bonded to a ceramic package and prepared for wet experiments by encapsulating the bonding wires and pads in the photoresist SU-8. Measurements in liquids with different conductivities were performed to demonstrate the functionality of the chip.

• Brinch, Shoshy Alam; Amundsen-Isaksen, Enya; Espada, Sandra; Hammarström, Clara Louise; Aizenshtadt, Aleksandra & Olsen, Petter Angell [Show all 18 contributors for this article] (2022). The Tankyrase Inhibitor OM-153 Demonstrates Antitumor Efficacy and a Therapeutic Window in Mouse Models. Cancer Research Communications. 2(4), p. 233–245. doi: 10.1158/2767-9764.CRC-22-0027. Full text in Research Archive
• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean; Skottvoll, Frøydis Sved; Berg, Henriette Engen & Abadpour, Shadab [Show all 14 contributors for this article] (2022). "Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry. Analytical Chemistry. ISSN 0003-2700. 94(50), p. 17677–17684. doi: 10.1021/acs.analchem.2c04530. Full text in Research Archive Show summary

  Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the “organ-in-a-column” units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated “on column” with heroin, followed by on-line monitoring of the drug’s phase 1 metabolism. Enzymatic metabolism of heroin produced in the “organ-in-a-column” units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

• Busek, Mathias; Aizenshtadt, Aleksandra; Martinez, Mikel Amirola; Delon, Ludivine Sylvie Claude & Krauss, Stefan Johannes Karl (2022). Academic User View: Organ-on-a-Chip Technology. Biosensors. ISSN 2079-6374. 12(2). doi: 10.3390/bios12020126. Full text in Research Archive Show summary

  The pleasantness of gentle stroking (CT-targeted touch) varies highly between individuals and studies, indicating that relevant factors may not be accounted for. We propose that the affective value of a touch event is determined by how well its perceived purpose matches the goals of the touch receiver. The perceived purpose or meaning of touch is in turn informed by the sensory characteristics together with the setting, person factors, and the touchee’s expectations. Affective touch is often a sign of affection, intended to soothe or show support. In a typical lab study however, the toucher is a stranger and its purpose is research. The purpose of laboratory touch is nevertheless compatible with the goal of participants, namely to contribute to research. To fully understand how the perception of affective touch emerges, more studies should directly manipulate participants’ beliefs about the purpose of touch.

• Skottvoll, Frøydis Sved; Aizenshtadt, Aleksandra; Hansen, Frederik Andre; Martinez, Mikel Amirola; Andersen, Jannike Mørch & Bogen, Inger Lise [Show all 11 contributors for this article] (2022). Direct Electromembrane Extraction-Based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids. Analysis & Sensing. 2(2). doi: 10.1002/anse.202100051. Full text in Research Archive Show summary

  This work introduces a strategy for organoid analysis - direct Electromembrane Extraction based Mass Spectrometry (dEME-MS) – for coupling liver organoids with mass spectrometry (MS). dEME-MS comprises electrophoresis of selected small molecules from a culture chamber across an oil membrane, and to a MS compatible solution. This enables clean micro-extraction of drugs and their metabolites as produced in the liver organoids to capillary liquid chromatography-mass spectrometry. Applying dEME-MS, proof-of-concept of directly measuring methadone metabolism is demonstrated on adult liver organoids. With 50 liver organoids and 1 μM methadone, methadone metabolism was monitored from 0 to 24 hours (11 time points). All analytes had <0.4 % variance in retention times with >100 measurements. dEME-MS is capable of automated and selective monitoring of drug metabolism in liver organoids, and could serve as a valuable tool for automated drug discovery efforts.

• Busek, Mathias; Aizenshtadt, Aleksandra; Koch, Timo; Frank, Anna; Delon, Ludivine Sylvie Claude & Martinez, Mikel Amirola [Show all 12 contributors for this article] (2022). Pump-less, recirculating organ-on-a-chip (rOoC) platform. Lab on a Chip. ISSN 1473-0197. p. 345–361. doi: 10.1039/d2lc00919f. Full text in Research Archive Show summary

  We developed a novel, pump-less directional flow recirculating organ-on-a-chip (rOoC) platform that creates controlled unidirectional gravity-driven flow by a combination of a 3D-tilting system and an optimized microfluidic layout. The rOoC platform was assembled utilizing a layer-to-layer fabrication technology based on thermoplastic materials. It features two organoid compartments supported by two independent perfusion channels and separated by a hydrogel barrier. We developed a computational model to predict wall shear stress values and then measured the flow rate in the microfluidic channels with micro-Particle-Image-Velocimetry (μPIV). The suitability of the rOoC for functional culture of endothelial cells was tested using HUVECs seeded in the perfusion channels. HUVECs aligned in response to the directional flow, formed a barrier and were able to sprout into the organoid compartments. Next, we demonstrated the viability of human stem-cell derived liver organoids in the organoid compartments. Finally, we show the possibility to circulate immune cells in the microfluidic channels that retain viability without being trapped or activated. The rOoC platform allows growing and connecting of two or more tissue or organ representations on-chip with the possibility of applying gradients, endothelial barriers, microvasculature and circulating cells independent of external tubing and support systems.

• Skottvoll, Frøydis Sved; Hansen, Frederik; Harrison, Sean; Boger, Ida Caroline Sneis; Mrsa, Ago & Restan, Magnus Saed [Show all 14 contributors for this article] (2021). Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies. Analytical Chemistry. ISSN 0003-2700. 93, p. 3576–3585. doi: 10.1021/acs.analchem.0c05082. Full text in Research Archive Show summary

  Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 μL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.

• Olsen, Petter Angell & Krauss, Stefan Johannes Karl (2021). The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates. Genes. 12(12). doi: 10.3390/genes12121920. Full text in Research Archive Show summary

  HEK293 cells are one of the most widely used cell lines in research, and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA, and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here, we show that HEK293 cells display an unexpected distribution of key components of the WNT/β-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. The reduction of E1B-55k protein levels leads to the disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/β-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/β-catenin signaling components and on transcriptional regulation of WNT/β-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line.

• Zhang, Yun; Shen, Lichong; Dreißigacker, Katja; Zhu, Honglin; Trinh-Minh, Thuong & Meng, Xianyi [Show all 22 contributors for this article] (2021). Targeting of canonical WNT signaling ameliorates experimental sclerodermatous chronic graft-versus-host disease. Blood. ISSN 0006-4971. 137(17), p. 2403–2416. doi: 10.1182/blood.2020008720. Full text in Research Archive
• Busek, Mathias; Nøvik, Steffen; Aizenshtadt, Aleksandra; Martinez, Mikel Amirola; Combriat, Thomas Michel Daniel & Grünzner, Stefan [Show all 7 contributors for this article] (2021). Thermoplastic elastomer (Tpe)–poly(methyl methacrylate) (pmma) hybrid devices for active pumping pdms-free organ-on-a-chip systems. Biosensors. ISSN 2079-6374. 11(5). doi: 10.3390/bios11050162. Full text in Research Archive Show summary

  Polydimethylsiloxane (PDMS) has been used in microfluidic systems for years, as it can be easily structured and its flexibility makes it easy to integrate actuators including pneumatic pumps. In addition, the good optical properties of the material are well suited for analytical systems. In addition to its positive aspects, PDMS is well known to adsorb small molecules, which limits its usability when it comes to drug testing, e.g., in organ-on-a-chip (OoC) systems. Therefore, alternatives to PDMS are in high demand. In this study, we use thermoplastic elastomer (TPE) films thermally bonded to laser-cut poly(methyl methacrylate) (PMMA) sheets to build up multilayered microfluidic devices with integrated pneumatic micro-pumps. We present a low-cost manufacturing technology based on a conventional CO2 laser cutter for structuring, a spin-coating process for TPE film fabrication, and a thermal bonding process using a pneumatic hot-press. UV treatment with an Excimer lamp prior to bonding drastically improves the bonding process. Optimized bonding parameters were characterized by measuring the burst load upon applying pressure and via profilometer-based measurement of channel deformation. Next, flow and long-term stability of the chip layout were measured using microparticle Image Velocimetry (uPIV). Finally, human endothelial cells were seeded in the microchannels to check biocompatibility and flow-directed cell alignment. The presented device is compatible with a real-time live-cell analysis system.

• Leenders, Ruben G. G.; Brinch, Shoshy Alam; Sowa, Sven T.; Amundsen-Isaksen, Enya; Galera-Prat, Albert & Murthy, Sudarshan [Show all 15 contributors for this article] (2021). Development of a 1,2,4-Triazole-Based Lead Tankyrase Inhibitor: Part II. Journal of Medicinal Chemistry. ISSN 0022-2623. 64(24), p. 17936–17949. doi: 10.1021/acs.jmedchem.1c01264. Full text in Research Archive Show summary

  Tankyrase 1 and 2 (TNKS1/2) catalyze post-translational modification by poly-ADP-ribosylation of a plethora of target proteins. In this function, TNKS1/2 also impact the WNT/β-catenin and Hippo signaling pathways that are involved in numerous human disease conditions including cancer. Targeting TNKS1/2 with small-molecule inhibitors shows promising potential to modulate the involved pathways, thereby potentiating disease intervention. Based on our 1,2,4-triazole-based lead compound 1 (OM-1700), further structure–activity relationship analyses of East-, South- and West-single-point alterations and hybrids identified compound 24 (OM-153). Compound 24 showed picomolar IC50 inhibition in a cellular (HEK293) WNT/β-catenin signaling reporter assay, no off-target liabilities, overall favorable absorption, distribution, metabolism, and excretion (ADME) properties, and an improved pharmacokinetic profile in mice. Moreover, treatment with compound 24 induced dose-dependent biomarker engagement and reduced cell growth in the colon cancer cell line COLO 320DM.

• Mygland, Line; Mahmuda, Shoshy; Strand, Martin Frank; Olsen, Petter Angell; Aizenshtadt, Aleksandra & Lund, Kaja [Show all 21 contributors for this article] (2021). Identification of response signatures for tankyrase inhibitor treatment in tumor cell lines. iScience. 24(7). doi: 10.1016/j.isci.2021.102807. Full text in Research Archive Show summary

  Small-molecule tankyrase 1 and tankyrase 2 (TNKS1/2) inhibitors are effective antitumor agents in selected tumor cell lines and mouse models. Here, we characterized the response signatures and the in-depth mechanisms for the antiproliferative effect of tankyrase inhibition (TNKSi). The TNKS1/2-specific inhibitor G007-LK was used to screen 537 human tumor cell lines and a panel of particularly TNKSi-sensitive tumor cell lines was identified. Transcriptome, proteome, and bioinformatic analyses revealed the overall TNKSi-induced response signatures in the selected panel. TNKSi-mediated inhibition of wingless-type mammary tumor virus integration site/b-catenin, yes-associated protein 1 (YAP), and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signaling was validated and correlated with lost expression of the key oncogene MYC and impaired cell growth. Moreover, we show that TNKSi induces accumulation of TNKS1/2-containing b-catenin degradasomes functioning as core complexes interacting with YAP and angiomotin proteins during attenuation of YAP signaling. These findings provide a contextual and mechanistic framework for using TNKSi in anticancer treatment that warrants further comprehensive preclinical and clinical evaluations.

• Wang, Hong; Kuusela, Sara; Rinnankoski-Tuikka, Rita; Dumont, Vincent; Bouslama, Rim & Ramadan, Usama Abo [Show all 12 contributors for this article] (2020). Tankyrase inhibition ameliorates lipid disorder via suppression of PGC-1α PARylation in db/db mice. International Journal of Obesity. ISSN 0307-0565. 44(8), p. 1691–1702. doi: 10.1038/s41366-020-0573-z. Full text in Research Archive
• Kierulf-Vieira, Kirsten Strømme; Sandberg, Cecilie; Waaler, Jo; Lund, Kaja; Skaga, Erlend & Saberniak, Birthe Mikkelsen [Show all 11 contributors for this article] (2020). A small-molecule tankyrase inhibitor reduces glioma stem cell proliferation and sphere formation. Cancers. 12(6), p. 1–16. doi: 10.3390/cancers12061630. Full text in Research Archive Show summary

  Evidence suggests that the growth and therapeutic resistance of glioblastoma (GBM) may be enabled by a population of glioma stem cells (GSCs) that are regulated by typical stem cell pathways, including the WNT/β-catenin signaling pathway. We wanted to explore the effect of treating GSCs with a small-molecule inhibitor of tankyrase, G007-LK, which has been shown to be a potent modulator of the WNT/β-catenin and Hippo pathways in colon cancer. Four primary GSC cultures and two primary adult neural stem cell cultures were treated with G007-LK and subsequently evaluated through the measurement of growth characteristics, as well as the expression of WNT/β-catenin and Hippo signaling pathway-related proteins and genes. Treatment with G007-LK decreased in vitro proliferation and sphere formation in all four primary GSC cultures in a dose-dependent manner. G007-LK treatment altered the expression of key downstream WNT/β-catenin and Hippo signaling pathway-related proteins and genes. Finally, cotreatment with the established GBM chemotherapeutic compound temozolomide (TMZ) led to an additive reduction in sphere formation, suggesting that WNT/β-catenin signaling may contribute to TMZ resistance. These observations suggest that tankyrase inhibition may serve as a supplement to current GBM therapy, although more work is needed to determine the exact downstream mechanisms involved.

• Waaler, Jo; Mygland, Line; Tveita, Anders Aune; Strand, Martin Frank; Solberg, Nina & Olsen, Petter Angell [Show all 19 contributors for this article] (2020). Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models. Communications Biology. 3. doi: 10.1038/s42003-020-0916-2. Full text in Research Archive Show summary

  The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/β-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/β-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/β-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of β-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome β-catenin-mediated resistance to immune checkpoint blockade in melanoma.

• Perdreau-Dahl, Harmonie; Progida, Cinzia; Barfeld, Stefan Jörg; Guldsten, Hanne; Thiede, Bernd & Arntzen, Magnus Øverlie [Show all 10 contributors for this article] (2020). Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells. Communications Biology. 3(1). doi: 10.1038/s42003-020-0851-2. Full text in Research Archive
• Waaler, Jo; Leenders, Ruben G G; Sowa, Sven T.; Mahmuda, Shoshy; Lycke, Eric Maximilian & Nieczypor, Piotr [Show all 14 contributors for this article] (2020). Preclinical lead optimization of a 1,2,4-triazole based tankyrase inhibitor. Journal of Medicinal Chemistry. ISSN 0022-2623. 63(13), p. 6834–6846. doi: 10.1021/acs.jmedchem.0c00208. Full text in Research Archive Show summary

  Tankyrases 1 and 2 are central biotargets in the WNT/β-catenin signaling and Hippo signaling pathways. We have previously developed tankyrase inhibitors bearing a 1,2,4-triazole moiety and binding predominantly to the adenosine binding site of the tankyrase catalytic domain. Here we describe a systematic structure-guided lead optimization approach of these tankyrase inhibitors. The central 1,2,4-triazole template and trans-cyclobutyl linker of the lead compound 1 were left unchanged, while side-group East, West, and South moieties were altered by introducing different building blocks defined as point mutations. The systematic study provided a novel series of compounds reaching picomolar IC50 inhibition in WNT/β-catenin signaling cellular reporter assay. The novel optimized lead 13 resolves previous atropisomerism, solubility, and Caco-2 efflux liabilities. 13 shows a favorable ADME profile, including improved Caco-2 permeability and oral bioavailability in mice, and exhibits antiproliferative efficacy in the colon cancer cell line COLO 320DM in vitro.

• Kogler, Stian; Harrison, Sean; Aizenshtadt, Aleksandra; Skottvoll, Frøydis Sved; Abadpour, Shadab & Krauss, Stefan [Show all 10 contributors for this article] (2020). “Organoid-in-a-column” coupled on-line with liquid chromatography-mass spectrometry. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.09.08.282756. Full text in Research Archive
• Abadpour, Shadab; Aizenshtadt, Aleksandra; Olsen, Petter Angell; Shoji, Kayoko; Wilson, Steven Ray Haakon & Krauss, Stefan [Show all 7 contributors for this article] (2020). Pancreas-on-a-Chip Technology for Transplantation Applications. Current Diabetes Reports. ISSN 1534-4827. 20. doi: 10.1007/s11892-020-01357-1. Full text in Research Archive Show summary

  Abstract Purpose of Review Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. Recent Findings PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Summary Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions.

• Solberg, Nina; Melheim, Maria; Strand, Martin Frank; Olsen, Petter Angell & Krauss, Stefan (2019). MEK Inhibition Induces Canonical WNT Signaling through YAP in KRAS Mutated HCT-15 Cells, and a Cancer Preventive FOXO3/FOXM1 Ratio in Combination with TNKS Inhibition. Cancers. 11(2), p. 1–19. doi: 10.3390/cancers11020164. Full text in Research Archive Show summary

  The majority of colorectal cancers are induced by subsequent mutations in APC and KRAS genes leading to aberrant activation of both canonical WNT and RAS signaling. However, due to induction of feedback rescue mechanisms some cancers do not respond well to targeted inhibitor treatments. In this study we show that the APC and KRAS mutant human colorectal cancer cell line HCT-15 induces canonical WNT signaling through YAP in a MEK dependent mechanism. This inductive loop is disrupted with combined tankyrase (TNKS) and MEK inhibition. RNA sequencing analysis suggests that combined TNKS/MEK inhibition induces metabolic stress responses in HCT-15 cells promoting a positive FOXO3/FOXM1 ratio to reduce antioxidative and cryoprotective systems.

• Lin, Ann; Skottvoll, Frøydis Sved; Rayner, Simon; Pedersen-Bjergaard, Stig; Sullivan, Gareth & Krauss, Stefan [Show all 8 contributors for this article] (2019). 3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry. Electrophoresis. ISSN 0173-0835. 41, p. 56–64. doi: 10.1002/elps.201900170. Full text in Research Archive Show summary

  In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, self‐organizing in vitro developed “organoids” and tissue mimicking chips. Microfluidic devices that allow long‐term maintenance and combination with other tissues, cells or organoids are commonly referred to as “microphysiological” or “organ‐on‐a‐chip” systems. Organ‐on‐a‐chip technology allows a broad range of “on‐chip” and “off‐chip” analytical techniques, whereby “on‐chip” techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with “on‐chip” electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field.

• Brennecke, Philip; Rasina, Dace; Catevilla, Oscar Aubi; Herzog, Katja; Landskron, Johannes & Cautain, Bastien [Show all 42 contributors for this article] (2019). EU-OPENSCREEN: A novel collaborative approach to facilitate chemical biology. SLAS Discovery. ISSN 2472-5552. 24(3), p. 398–413. doi: 10.1177/2472555218816276. Full text in Research Archive Show summary

  Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN’s compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN.

• Solberg, Nina Therese; Waaler, Jo; Iversen, Kaja Lund; Mygland, Line; Olsen, Peter Angell & Krauss, Stefan (2018). TANKYRASE Inhibition Enhances the Antiproliferative Effect of PI3K and EGFR Inhibition, Mutually Affecting β-CATENIN and AKT Signaling in Colorectal Cancer. Molecular Cancer Research. ISSN 1541-7786. 16(3), p. 1541–1541. doi: 10.1158/1541-7786.MCR-17-0362. Full text in Research Archive
• Norum, Jens Henrik; Skarpen, Ellen; Brech, Andreas; Kuiper, Raoul; Waaler, Jo & Krauss, Stefan [Show all 7 contributors for this article] (2018). The tankyrase inhibitor G007-LK inhibits small intestine LGR5+ stem cell proliferation without altering tissue morphology. Biological Research (Print). ISSN 0716-9760. 51(1). doi: 10.1186/s40659-017-0151-6. Full text in Research Archive Show summary

  Background: The WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages. Results: We treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible. Conclusion: We show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.

• Solberg, Nina T.; Waaler, Jo; Lund, Kaja; Mygland, Line; Olsen, Petter A. & Krauss, Stefan (2017). TANKYRASE inhibition enhances the antiproliferative effect of PI3K and EGFR inhibition, mutually affecting β-CATENIN and AKT signaling in colorectal cancer. Molecular Cancer Research. ISSN 1541-7786. 16(3), p. 543–553. doi: 10.1158/1541-7786.MCR-17-0362. Full text in Research Archive
• Anumala, Upendra Rao; Waaler, Jo; Nkizinkiko, Yves; Ignatev, Alexander; Lazarow, Katina & Lindemann, Peter [Show all 15 contributors for this article] (2017). Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach. Journal of Medicinal Chemistry. ISSN 0022-2623. 60(24), p. 10013–10025. doi: 10.1021/acs.jmedchem.7b00883. Full text in Research Archive
• Lund, Kaja; Olsen, Cathrine Elisabeth; Wong, Judith Jing Wen; Olsen, Petter Angell; Solberg, Nina Therese & Høgset, Anders [Show all 8 contributors for this article] (2017). 5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin. Journal of Experimental & Clinical Cancer Research. 36, p. 1–18. doi: 10.1186/s13046-017-0662-6. Full text in Research Archive Show summary

  Background Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. Methods 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. Results The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population. Conclusion Our findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

• Røberg-Larsen, Hanne; Lund, Kaja; Sæterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore & Solberg, Nina [Show all 9 contributors for this article] (2017). Mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes. Journal of Steroid Biochemistry and Molecular Biology. ISSN 0960-0760. 169, p. 22–28. doi: 10.1016/j.jsbmb.2016.02.006. Full text in Research Archive
• Vehus, Tore; Waaler, Jo; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Combining HIC, SEC, and IEX with Fluorescence Polarization for Drug Target Discovery. LC GC North America. ISSN 1527-5949. 30(5), p. 232–239. Full text in Research Archive Show summary

  Fluorescence polarization (FP) is a highly regarded technique for studying drug–protein interactions, but has limited value regarding protein mixtures. As a novel approach to drug target discovery, the possibility of combining FP with liquid chromatography (LC) was explored. Nondenaturing protein LC principles such as size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and ion exchange chromatography (IEX) were found to be orthogonal and compatible with FP because the mobile phases used do not negatively affect detection. For simple protein mixtures, the SEC/HIC/IEX–FP approach was able to identify tankyrase as the target of a triazole-based inhibitor of the Wnt signaling pathway, which is heavily associated with colon cancer. However, the total peak capacity of the three LC dimensions was not sufficient to resolve at cell-proteome level, calling for higher resolution of intact proteins to enable stand-alone drug target discovery with LC and FP.

• Vehus, Tore; Røberg-Larsen, Hanne; Waaler, Jo; Aslaksen, Sigrid; Krauss, Stefan & Wilson, Steven Ray Haakon [Show all 7 contributors for this article] (2016). Versatile, sensitive liquid chromatography mass spectrometry–Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins. Scientific Reports. 6. doi: 10.1038/srep37507. Full text in Research Archive
• Vehus, Tore; Sæterdal, Kristina Erikstad; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Comparison of commercial nanoliquid chromatography columns for fast, targeted mass spectrometry-based proteomics. Future science OA. 2(2). doi: 10.4155/fsoa-2016-0014. Full text in Research Archive
• Haikarainen, Teemu; Waaler, Jo; Ignatev, Alexander; Nkizinkiko, Yves; Venkannagaria, Harikanth & Obaji, Ezeogo [Show all 8 contributors for this article] (2016). Development and structural analysis of adenosine site binding tankyrase inhibitors. Bioorganic & Medicinal Chemistry Letters. ISSN 0960-894X. 26(2), p. 328–333. doi: 10.1016/j.bmcl.2015.12.018. Full text in Research Archive
• Machon, Ondrej; Masek, Jan; Machonova, Olga; Krauss, Stefan & Kozmik, Zbynek (2015). Meis2 is essential for cranial and cardiac neural crest development. BMC Developmental Biology. 15. doi: 10.1186/s12861-015-0093-6. Full text in Research Archive
• Zhong, Linlin; Ding, Yun; Bandyopadhyay, Gautam; Waaler, Jo; Börgeson, Emma & Smith, Susan [Show all 13 contributors for this article] (2015). The PARsylation activity of tankyrase in adipose tissue modulates systemic glucose metabolism in mice. Diabetologia. ISSN 0012-186X. 59(3), p. 582–591. doi: 10.1007/s00125-015-3815-1. Full text in Research Archive
• Thorvaldsen, Tor Espen; Pedersen, Nina Marie; Wenzel, Eva; Schultz, Sebastian; Brech, Andreas & Liestøl, Knut [Show all 9 contributors for this article] (2015). Structure, dynamics, and functionality of tankyrase inhibitor-induced degradasomes. Molecular Cancer Research. ISSN 1541-7786. 13(11), p. 1487–1501. doi: 10.1158/1541-7786.MCR-15-0125. Full text in Research Archive
• Olsen, Peter Angell; Lund, Kaja & Krauss, Stefan (2015). Expression profiling of wild type and β-catenin gene disrupted human BxPC-3 pancreatic adenocarcinoma cells. Genomics Data. 4, p. 150–152. doi: 10.1016/j.gdata.2015.04.010. Full text in Research Archive
• Lund, Kaja; Dembinski, Jennifer Lynn; Solberg, Nina; Urbanucci, Alfonso; Mills, Ian Geoffrey & Krauss, Stefan (2015). Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment. PLOS ONE. 10(4). doi: 10.1371/journal.pone.0123684. Full text in Research Archive Show summary

  BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU. RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

• Haikarainen, Teemu; Krauss, Stefan & Lehtiö, Lari (2014). Tankyrases: Structure, function and therapeutic implications in cancer. Current pharmaceutical design. ISSN 1381-6128. 20(41), p. 6472–6488. doi: 10.2174/1381612820666140630101525. Full text in Research Archive
• Olsen, Peter Angell; Solberg, Nina; Lund, Kaja; Vehus, Tore; Gelazauskaite, Monika & Wilson, Steven Ray Haakon [Show all 7 contributors for this article] (2014). Implications of targeted genomic disruption of β-catenin in B×PC-3 pancreatic adenocarcinoma cells. PLOS ONE. 9(12). doi: 10.1371/journal.pone.0115496. Full text in Research Archive
• Hustoft, Hanne Kolsrud; Vehus, Tore; Brandtzæg, Ole Kristian Merkesvik; Krauss, Stefan; Greibrokk, Tyge & Wilson, Steven Ray Haakon [Show all 7 contributors for this article] (2014). Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts. PLOS ONE. 9(9). doi: 10.1371/journal.pone.0106881. Full text in Research Archive Show summary

  A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ~1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.

• Stratford, Eva Wessel; Daffinrud, Jeanette L.; Munthe, Else; Castro, Russell; Waaler, Jo & Krauss, Stefan [Show all 7 contributors for this article] (2014). The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines. Cancer Medicine. 3(1), p. 36–46. doi: 10.1002/cam4.170. Full text in Research Archive
• Røberg-Larsen, Hanne; Lund, Kaja; Vehus, Tore; Solberg, Nina; Vesterdal, Caroline & Misaghian, Dorna [Show all 10 contributors for this article] (2014). Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols. Journal of Lipid Research. ISSN 0022-2275. 55(7), p. 1531–1536. doi: 10.1194/jlr.D048801. Full text in Research Archive Show summary

  Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000–80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1–21% and 2–20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics. This research was originally published in the Journal of Lipid Research. Roberg-Larsen H, Lund K, Vehus T, Solberg N, Vesterdal C, Misaghian D, Olsen PA, Krauss S, Wilson SR, Lundanes E.. Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols. J. Lipid Res. 2014 Jul;55(7):1531-6. © the American Society for Biochemistry and Molecular Biology.

• Lund, Kaja; Bostad, Monica; Skarpen, Ellen; Braunagel, Michael; Kiprijanov, Sergej M. & Krauss, Stefan [Show all 9 contributors for this article] (2014). The novel EpCAM-targeting monoclonal antibody 3-17I linked to saporin is highly cytotoxic after photochemical internalization in breast, pancreas and colon cancer cell lines. mAbs. ISSN 1942-0862. 6(4), p. 1038–1050. doi: 10.4161/mabs.28207. Full text in Research Archive
• Røberg-Larsen, Hanne; Strand, Martin Frank; Krauss, Stefan & Wilson, Steven Ray Haakon (2014). Metabolites in vertebrate Hedgehog signaling. Biochemical and Biophysical Research Communications - BBRC. ISSN 0006-291X. 446(3), p. 669–674. doi: 10.1016/j.bbrc.2014.01.087. Full text in Research Archive

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• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean Philip; Sullivan, Gareth John; Lundanes, Elsa & Bogen, Inger Lise [Show all 9 contributors for this article] (2023). Organ-in-a-column – automated on-line incubation, sampling, and LC-MS analysis for measurement of drug metabolites. Full text in Research Archive
• Vogt, Yngve; Krauss, Stefan Johannes Karl; Mossige, Joachim; Dysthe, Dag Kristian; Angheluta, Luiza & Jensenius, Alexander Refsum (2023). Bereder grunnen for kunstige organer. [Journal]. Apollon. Full text in Research Archive
• Røberg-Larsen, Hanne; Kømurcu, Kristina Sæterdal; Kogler, Stian; Olsen, Christine; Skottvoll, Frøydis Sved & Aizenshtadt, Aleksandra [Show all 8 contributors for this article] (2023). Organoids, Organ-on-a-chip and Mass Spectrometry. Full text in Research Archive
• Haavaag, Jon Erlend; Kogler, Stian; Zawadzka, Malgorzata Elzbieta; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2023). Building a high-resolution nano DESI-MS for organoid imaging: Analysis using a built and optimized imaging system. Full text in Research Archive
• Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne; Krauss, Stefan Johannes Karl & Aizenshtadt, Aleksandra (2022). Organ-in-a-column - An automated on-line incubation and analysis system for drug metabolism studies. Full text in Research Archive
• Skottvoll, Frøydis Sved; Aizenshtadt, Aleksandra; Harrison, Sean; Krauss, Stefan Johannes Karl; Sullivan, Gareth & Bogen, Inger Lise [Show all 9 contributors for this article] (2022). Organ-in-a-column - On-line incubation and LC-MS-analysis for drug metabolism studies. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Oxysterols are secreted from Non-alcoholic Fatty Liver disease induced organoids. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L.; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Oxysterols are secreted from Non-Alcoholic Fatty Liver Disease (NAFLD) induced organoids. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease. Full text in Research Archive
• Kogler, Stian; Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne; Krauss, Stefan Johannes Karl; Lundanes, Elsa & Skottvoll, Frøydis Sved (2021). Organ-in-a-column. Full text in Research Archive
• Abadpour, Shadab; Niemi, Essi Maria; Orrhult, Linnea Strid; Nogueira, Liebert Parreiras; Haugen, Håvard Jostein & Josefsen, Dag [Show all 10 contributors for this article] (2021). 307.7: 3D Bioprinting of Functional Islets With Adipose-derived Stromal Cells in an Alginate/Nanocellulose Scaffold. Transplantation. ISSN 0041-1337. p. S25–S25. doi: 10.1097/01.tp.0000804424.50335.7e. Full text in Research Archive
• Nordli, Henriette; Aizenshtadt, Aleksandra; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2021). Oxysterols secreted from a Non-Alcoholic Fatty Liver Disease (NAFLD) induced liver organoids. Full text in Research Archive
• Lien, Vegard Torp; Kristiansen, Margrethe Konstanse; Pettersen, Solveig; Haugen, Mads Haugland; Krauss, Stefan Johannes Karl & Olberg, Dag Erlend [Show all 8 contributors for this article] (2020). Erratum: Towards dual inhibitors of the MET kinase and WNT signaling pathway; Design, synthesis and biological evaluation (RSC Advances (2019) 9 (37092-37100) DOI: 10.1039/D0RA90007A). RSC Advances. 10(9), p. 4927–4927. doi: 10.1039/d0ra90007a. Full text in Research Archive
• Skottvoll, Frøydis Sved; Nilsen, Ola; Krauss, Stefan; Sullivan, Gareth & Wilson, Steven Ray Haakon (2018). Integrating highly miniaturized LC-MS systems with “Organ on a Chip”; the future of preclinical modelling. Full text in Research Archive
• Sæterdal, Kristina E.; Vehus, Tore; Røberg-Larsen, Hanne; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). CYP27A1 – its relation to oxysterols and cancer, and analytical challenges. Full text in Research Archive
• Røberg-Larsen, Hanne; Lund, Kaja; Sæterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore & Solberg, Nina [Show all 9 contributors for this article] (2016). Capillary LC mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes. Full text in Research Archive
• Brandtzæg, Ole Kristian Merkesvik; Hustoft, Hanne Kolsrud; Vehus, Tore; Greibrokk, Tyge; Krauss, Stefan & Reubsaet, Leon [Show all 8 contributors for this article] (2015). On-line protein digestion and peptide separation using “ultra-nano” LC-MS. Full text in Research Archive
• Røberg-Larsen, Hanne; Lund, Kaja; skottvoll, Frøydis S; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2015). Oxysterols in exosomes. Full text in Research Archive
• Røberg-Larsen, Hanne; Krauss, Stefan; Greibrokk, Tyge; Lundanes, Elsa & Wilson, Steven Ray Haakon (2014). Investigating the Role of Endogenous Small Molecules on the Hedgehog Signal Pathway: Very Sensitive, Highly Automated Nano-LC Based Determination of Oxysterols in Cancer Cells. Full text in Research Archive
• Røberg-Larsen, Hanne; Solberg, Nina; Olsen, Petter Angell; Lund, Kaja; Vesterdal, Caroline & Krauss, Stefan [Show all 8 contributors for this article] (2014). High sensitive, automated quantification of hedgehog active oxysterols in 10 000- 80 000 pancreatic cancer cells using nanoLC-MS. Full text in Research Archive
• Brandtzaeg, Ole Kristian; Wilson, Steven Ray Haakon; Lundanes, Elsa; Reubsaet, Leon; Krauss, Stefan & Martinsen, Ørjan Grøttem (2018). Online sample Preparation Platforms for Targeted Nano Liquid Chromatography Mass Spectrometry Bioanalysis. Universitetet i Oslo. Full text in Research Archive
• Vehus, Tore; Wilson, Steven Ray Haakon; Lundanes, Elsa; Krauss, Stefan; Halvorsen, Trine Grønhaug & Morth, Jens Preben (2018). Nano Liquid Chromatography-Tandem Mass Spectrometry Platforms for Determination of Low Abundant Wnt/Beta-Catenin Proteins in Cancer Research. Universitetet i Oslo. Full text in Research Archive
• Sæterdal, Kristina Erikstad; Vehus, Tore; Røberg-Larsen, Hanne; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Determination of CYP27a1 in biological samples using nano liquid chromatography mass spectrometry. Universitetet i Oslo. Full text in Research Archive Show summary

  The cytochrome P450 enzyme, CYP27a1, converts cholesterol into 27-hydroxycholesterol, and both the metabolite and the enzyme are associated with breast cancer, promoting proliferation and metastasis. CYP27A1 is enriched in cancer tissues, and the amount of CYP27a1 correlates to tumor grade. Hence, CYP27a1 is a potential biomarker, and a method for determination and quantification is needed. Antibody assays shows limited performance, and therefore the goal of this work was to develop a nano liquid chromatography mass spectrometry (LC-MS) method for determination of CYP27a1. A sample preparation workflow for CYP27a1 in cell samples was investigated. CYP27a1 was identified by nano LC-MS in MDA-MB-231 breast cancer cells by fractionating cell lysates using gel electrophoresis. Enrichment of CYP27a1 in cell samples using immunoprecipitation, an antibody based method for protein extraction, did not provide adequate identification. However the former method works when looking at other proteins, and this confirms that there are limitations in using antibody based methods for determination and quantification of CYP27a1.

• Røberg-Larsen, Hanne; Lundanes, Elsa; Wilson, Steven Ray Haakon & Krauss, Stefan (2016). High sensitivity measurements of side-chain oxysterols using liquid chromatography mass spectrometry. Universitetet i Oslo. Full text in Research Archive
• Strand, Martin Frank; Krauss, Stefan & Machon, Ondrej (2013). Modulating the Hedgehog pathway. Universitetet i Oslo. Full text in Research Archive

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