Traces of Canine Inflammatory Bowel Disease Reflected by Intestinal Organoids.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that affects humans and several domestic animal species, including cats and dogs. In this study, we have analyzed duodenal organoids derived from canine IBD patients using quantitative proteomics. Our objective was to investigate whether these organoids show phenotypic traits of the disease compared with control organoids obtained from healthy donors. To this aim, IBD and control organoids were subjected to quantitative proteomics analysis via liquid chromatography-mass spectrometry. The obtained data revealed notable differences between the two groups. The IBD organoids exhibited several alterations at the levels of multiple proteins that are consistent with some known IBD alterations. The observed phenotype in the IBD organoids to some degree mirrors the corresponding intestinal condition, rendering them a compelling approach for investigating the disease and advancing drug exploration. Additionally, our study revealed similarities to some human IBD biomarkers, further emphasizing the translational and comparative value of dogs for future investigations related to the causes and treatment of IBD. Relevant proteins such as CALU, FLNA, MSN and HMGA2, which are related to intestinal diseases, were all upregulated in the IBD duodenal organoids. At the same time, other proteins such as intestinal keratins and the mucosal immunity PIGR were depleted in these IBD organoids. Based on these findings, we propose that these organoids could serve as a valuable tool for evaluating the efficacy of therapeutic interventions against canine IBD.

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach.

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.

Cancer-associated fibroblast-derived WNT2 increases tumor angiogenesis in colon cancer.

WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.

Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses.

Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo These 3D models displayed augmented anti-tumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.

Synthesis of Electron-Rich, Planarized Silicon(IV) Species and a Theoretical Analysis of Dimerizing Aminosilanes.

Equipping silicon(IV) with electron-rich, geometrically constrained NNN- and ONO-tridentate substituents leads to aminosilanes with increased Lewis acidity-expressed through the formation of Si2 N2 rings by head-to-tail dimerization. Depending on the substituents, the dimerization can be controlled for the first time, yielding monomeric, structurally reversible and dimeric states. The monomeric species display substantial distortions from tetrahedral towards planar geometry at silicon. The dimerization and the Lewis acidity of aminosilanes are rationalized by (conceptual) DFT, NBO, ETS-NOCV and QTAIM methods. The preorganization at silicon, London dispersion between the substituents and resonance phenomena inside the formed Si2 N2 tetracycles are identified as driving forces for the dimerization. Comparison with selected aminosilanes permits general conclusions to be reached on the Lewis acidity of silicon species and on the aggregation of amphiphilic compounds.

Increased complexity in carcinomas: Analyzing and modeling the interaction of human cancer cells with their microenvironment.

Solid cancers are not simple accumulations of malignant tumor cells but rather represent complex organ-like structures. Despite a more chaotic general appearance as compared to the highly organized setup of healthy tissues, cancers still show highly differentiated structures and a close interaction with and dependency on the interwoven connective tissue. This complexity within cancers is not known in detail at the molecular level so far. The first part of this article will shortly describe the technology and strategies to quantify and dissect the heterogeneity in human solid cancers. Moreover, there is urgent need to better understand human cancer biology since the development of novel anti-cancer drugs is far from being efficient, predominantly due to the scarcity of predictive preclinical models. Hence, in vivo and in vitro models were developed, which better recapitulate the complexity of human cancers, by their intrinsic three-dimensional nature and the cellular heterogeneity and allow functional intervention for hypothesis testing. Therefore, in the second part 3D in vitro cancer models are presented that analyze and depict the heterogeneity in human cancers. Advantages and drawbacks of each model are highlighted and their suitability to preclinical drug testing is discussed.

Full biological characterization of human pluripotent stem cells will open the door to translational research.

Since the discovery of human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), great hopes were held for their therapeutic application including disease modeling, drug discovery screenings, toxicological screenings and regenerative therapy. hESC and hiPSC have the advantage of indefinite self-renewal, thereby generating an inexhaustible pool of cells with, e.g., specific genotype for developing putative treatments; they can differentiate into derivatives of all three germ layers enabling autologous transplantation, and via donor-selection they can express various genotypes of interest for better disease modeling. Furthermore, drug screenings and toxicological screenings in hESC and hiPSC are more pertinent to identify drugs or chemical compounds that are harmful for human, than a mouse model could predict. Despite continuing research in the wide field of therapeutic applications, further understanding of the underlying basic mechanisms of stem cell function is necessary. Here, we summarize current knowledge concerning pluripotency, self-renewal, apoptosis, motility, epithelial-to-mesenchymal transition and differentiation of pluripotent stem cells.

Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

In vitro cell migration and invasion assays.

Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.

A Recurrent STAT5BN642H Driver Mutation in Feline Alimentary T Cell Lymphoma.

Alimentary lymphomas arising from T cells are rare and aggressive malignancies in humans. In comparison, they represent the most common anatomical form of lymphoma in cats. Due to the low prevalence in humans, the underlying pathomechanism for these diseases is poorly characterised, limiting experimental analysis and therapeutic exploration. To date, activating mutations of the JAK/STAT core cancer pathway and particularly the STAT5B oncoprotein have been identified in human enteropathy-associated T cell lymphoma. Here, we describe a high homology of human and feline STAT3 and STAT5B proteins and strong conservation at the genomic level. Analysis of 42 samples of feline T cell alimentary lymphoma reveals broad activation of STAT3 and STAT5B. Screening for known activating mutations in STAT3 or STAT5B identifies the presence of the STAT5BN642H driver mutation in feline enteropathy-associated T cell lymphoma in 7 out of 42 (16.67%) samples in total. Regarding lymphoma subtypes, the majority of mutations with 5 out of 17 (29.41%) cases were found in feline enteropathy-associated lymphoma type II (EATL II). This identification of an oncogenic STAT5B driver mutation in felines recapitulates the genetic situation in the corresponding human disease, thereby establishing the cat as a potential new model for a rare and incurable human T cell disease.

Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.

Tris(dimethylamino)silylium ion: structure and reactivity of a dimeric silaguanidinium.

Although several strategies for the stabilization of silylium ions have been established, "π-stabilization" with directly attached π-donor heteroatoms at silicon has not been developed yet. Hydride abstraction from (Me2N)3SiH generates dicationic [(Me2N)3Si+]2 in solution and in the solid state - constituting the dimer of an elusive silaguanidinium ion. This compound can be synthesized on a gram scale and is compatible with common organic solvents. However, it readily undergoes spontaneous electrophilic silylation of electron-rich aromatic compounds or initiates a catalytic hydro-defluorination reaction.

DRUGPATH - a novel bioinformatic approach identifies DNA-damage pathway as a regulator of size maintenance in human ESCs and iPSCs.

Genetic and biochemical screening approaches often fail to identify functionally relevant pathway networks because many signaling proteins contribute to multiple gene ontology pathways. We developed a DRUGPATH-approach to predict pathway-interactomes from high-content drug screen data. DRUGPATH is based upon combining z-scores of effective inhibitors with their corresponding and validated targets. We test DRUGPATH by comparing homeostatic pathways in human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) and human amniotic fluid stem cells (hAFSCs). We show that hAFSCs utilize distinct interactomes compared to hESCs/hiPSCs and that pathways orchestrating cell cycle and apoptosis are strongly interconnected, while pathways regulating survival and size are not. Interestingly, hESCs/hiPSCs regulate their size by growing exact additional sizes during each cell cycle. Chemical and genetic perturbation studies show that this "adder-model" is dependent on the DNA-damage pathway. In the future, the DRUGPATH-approach may help to predict novel pathway interactomes from high-content drug screens.

Companion Animals as Models for Inhibition of STAT3 and STAT5.

The use of transgenic mouse models has revolutionized the study of many human diseases. However, murine models are limited in their representation of spontaneously arising tumors and often lack key clinical signs and pathological changes. Thus, a closer representation of complex human diseases is of high therapeutic relevance. Given the high failure rate of drugs at the clinical trial phase (i.e., around 90%), there is a critical need for additional clinically relevant animal models. Companion animals like cats and dogs display chronic inflammatory or neoplastic diseases that closely resemble the human counterpart. Cat and dog patients can also be treated with clinically approved inhibitors or, if ethics and drug safety studies allow, pilot studies can be conducted using, e.g., inhibitors of the evolutionary conserved JAK-STAT pathway. The incidence by which different types of cancers occur in companion animals as well as mechanisms of disease are unique between humans and companion animals, where one can learn from each other. Taking advantage of this situation, existing inhibitors of known oncogenic STAT3/5 or JAK kinase signaling pathways can be studied in the context of rare human diseases, benefitting both, the development of drugs for human use and their application in veterinary medicine.

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells.

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost α-catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.

Correction: The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth.

[This corrects the article DOI: 10.18632/oncotarget.9315.].

An Optimized 3D Coculture Assay for Preclinical Testing of Pro- and Antiangiogenic Drugs.

Angiogenesis is a promising target for anticancer therapies, but also for treating other diseases with pathologic vessel development. Targeting the vascular endothelial growth factor (VEGF) pathway did not proof as effective as expected due to emerging intrinsic resistance mechanisms, as well as stromal contributions leading to drug insensitivity. Therefore, alternative strategies affecting the interaction of endothelial cells (ECs) with other stromal cells seem to be more promising. Human preclinical in vitro angiogenesis models successfully recapitulating these interactions are rare, and two-dimensional (2D) cell cultures cannot mimic tissue architecture in vivo. Consequently, models combining three-dimensionality with heterotypic cell interaction seem to be better suited. Here, we report on an improved human fibroblast-EC coculture assay mimicking sprouting angiogenesis from EC-covered microbeads resembling existing endothelial structures. Culture conditions were optimized to assess pro- and antiangiogenic compounds. Important characteristics of angiogenesis, that is, the number of sprouts and branch points, sprout length protrusion, and overall vessel structure areas, were quantified. Notably, the endothelial sprouts display lumen formation and basal membrane establishment. In this model, angiogenesis can be inhibited by genetic interference of pro-angiogenic factors expressed in the fibroblasts. Moreover, bona fide antiangiogenic drugs decreased, whereas pro-angiogenic factors increased vessel formation in 24-well and 96-well settings, demonstrating the applicability for screening approaches.

The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth.

The role of STAT1 and STAT3 for colorectal carcinoma (CRC) development and progression is controversial. We evaluated 414 CRC patient samples on tissue microarrays for differential expression of STAT1 and STAT3 protein levels and correlated ratios with clinical parameters. Concomitant absence of nuclear STAT1 and STAT3 expression was associated with significantly reduced median survival by ≥33 months (p=0.003). To gain insight into underlying mechanisms, we generated four CRC cell lines with STAT3 knockdown. The cell lines harbor different known mutational drivers and were xenografted into SCID mice to analyze the influence of STAT3 on their tumor growth behavior. Experimental downregulation of STAT3 expression had differential, cell-line specific effects on STAT1 expression levels. STAT1 consistently showed nuclear localization irrespective of its tyrosine phosphorylation status. Two characteristic STAT1/3 expression patterns with opposite growth behavior could be distinguished: cell lines with a low STAT1/high STAT3 ratio showed faster tumor growth in xenografts. In contrast, xenografts of cell lines showing high STAT1 and low STAT3 levels grew slower. Importantly, these ratios reflected clinical outcome in CRC patients as well. We conclude that the ratio of STAT1 to STAT3 expression is a key determinant of CRC progression and that STAT1 counteracts pro-tumorigenic STAT3 signaling. Thus, we suggest that the STAT3/STAT1 ratios are better clinical predictors in CRC as compared to STAT3 or STAT1 levels alone.

High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts.

We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.

Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rß, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this early step of metastatic progression.