generation-apical-out-intestinal-organoids-assessment-organoid

Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Here, we describe the generation of floating cultures of apical-out intestinal organoids from hydrogel-embedded intestinal organoid cultures. ...

University of Veterinary Medicine

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

We have previously shown that immunization of horses with bovine papillomavirus type 1 (BPV1) L1 virus-like particles (VLPs) is safe and highly immunogenic and that BPV1 and bovine papillomavirus type 2 (BPV2) are closely related serotypes. Here we evaluated the protective potential of a BPV1 L1 VLP vaccine against experimental BPV1 and BPV2 challenge and studied the safety and immunogenicity of a bivalent equine papillomavirus type 2 (EcPV2)/BPV1 L1 VLP vaccine. Fourteen healthy horses were immunized with BPV1 L1 VLPs (100 µg per injection) plus adjuvant on days 0 and 28, while seven remained unvaccinated. On day 42, all 21 horses were challenged intradermally at 10 sites of the neck with 107 BPV1 virions per injection. In analogy, 14 horses immunized twice with EcPV2 plus BPV1 L1 VLPs (50 µg each) and seven control animals were challenged with 107 BPV2 virions per injection. Immunization with BPV1 L1 VLPs alone induced a robust antibody response (day 42 median titre: 12 800), and BPV1-inoculated skin remained unchanged in 13/14 vaccinated horses. Immunization with the bivalent vaccine was safe, resulted in lower median day 42 antibody titres of 400 for BPV1 and 1600 for EcPV2 and conferred significant yet incomplete cross-protection from BPV2-induced tumour formation, with 11/14 horses developing small, short-lived papules. Control horses developed pseudo-sarcoids at all inoculation sites. The monovalent BPV1 L1 VLP vaccine proved highly effective in protecting horses from BPV1-induced pseudo-sarcoid formation. Incomplete protection from BPV2-induced tumour development conferred by the bivalent vaccine is due to the poorer immune response by immune interference or lower cross-neutralization titres to heterologous BPV2 virions.

Potential of a BPV1 L1 VLP vaccine to prevent BPV1- or BPV2-induced pseudo-sarcoid formation and safety and immunogenicity of EcPV2 L1 VLPs in horse.

Canine oral malignant melanoma (COMM) is a potentially lethal cancer disease. We established primary cell lines from mostly amelanotic primary COMM and metastases and assessed lesions and derived cells for Melan A, PNL2 and CD146 expression. Then, migration and invasion of CD146-enriched vs -depleted COMM cells were analysed. Epithelial-to-mesenchymal transition (EMT) was addressed by Vimentin-staining and MMP2/MMP9 zymography. Phagocytic behaviour was analysed by histopathological examination and phagocytosis assay. While Melan A- and PNL2-staining yielded inconsistent data, 100% of COMM sections and primary cells showed CD146 expression, suggesting that this protein may serve as a prognostic marker. An overall correlation between CD146-expression and migration/invasion was not observed. All primary cell lines consistently expressed Vimentin and secreted biologically active MMP2, indicating that they had undergone EMT. Importantly, COMM sections exhibited cell-in-cell structures, and all primary cell lines exhibited phagocytic activity, supporting the concept that cell cannibalism may have a role in COMM progression.

Canine oral primary melanoma cells exhibit shift to mesenchymal phenotype and phagocytic behaviour.

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.

Companion Animals as Models for Inhibition of STAT3 and STAT5.

Equine malignant melanoma (EMM) is a frequently occurring dermoepidermal tumor in grey horses. Currently available therapies are either challenging or inefficient. Betulinic acid (BA), a naturally occurring triterpenoid, is a promising compound for cancer treatment. To evaluate the potential of BA as a topical therapy for EMM, its anticancer effects on primary equine melanoma cells and dermal fibroblasts and its percutaneous permeation through isolated equine skin were assessed in vitro.

Betulinic acid shows anticancer activity against equine melanoma cells and permeates isolated equine skin in vitro.

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.

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

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  or  identifies the presence of the  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  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.

A Recurrent  Driver Mutation in Feline Alimentary T Cell Lymphoma.

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day -55 and -27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day -70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation.

Anti-VEGF agents were found to have clinical implications for the successful treatment of vascular-driven diseases in humans. In this study, a detailed biological characterization of bevacizumab in a variety of in vitro assays was carried out to determine the effect of bevacizumab on equine umbilical vein endothelial cells (EqUVEC). EqUVECs were harvested from umbilical cords of clinically healthy horses and exposed to different concentrations (1, 2, 4, 6, 8 mg/mL) of bevacizumab (Avastin). Assays concerning the drug's safety (cell viability and proliferation assay) and efficacy (cell tube formation assay, cell migration assay, and Vascular endothelial growth factor (VEGF) expression) were carried out reflecting multiple cellular processes. Bevacizumab significantly decreased VEGF expression at all concentrations over a 72 h period. No cytotoxic effect of bevacizumab on EqUVECs was observed at concentrations of 4 mg/mL bevacizumab or lower. Incubated endothelial cells showed delayed tube formation and bevacizumab efficiently inhibited cell migration in a dose-dependent manner. Bevacizumab potently inhibits VEGF-induced cellular processes and could be a promising therapeutic approach in vascular-driven diseases in horses.

Bevacizumab Efficiently Inhibits VEGF-Associated Cellular Processes in Equine Umbilical Vein Endothelial Cells: An In Vitro Characterization.

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.

Traces of Canine Inflammatory Bowel Disease Reflected by Intestinal Organoids.

Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes  exon 11 and 15,  exon 2 and 3,  exon 2, and  exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the  p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry  p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the  p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Cross-Species Comparison of the Pan-RAF Inhibitor LY3009120's Anti-Tumor Effects in Equine, Canine, and Human Malignant Melanoma Cell Lines.

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

Angiogenesis plays a crucial role in various physiological and pathological conditions. However, research in equine angiogenesis is relative limited, necessitating the development of suitable  models. To effectively analyze angiogenesis in-vitro, it is essential to target the specific cells responsible for this process, namely endothelial cells. Human umbilical vein endothelial cells (HUVECs) are one of the most used  models for studying angiogenesis in humans. Serving as an equivalent to HUVECs, we present a comprehensive isolation protocol for equine umbilical vein endothelial cells (EqUVECs) with relatively minimal requirements, thereby enhancing accessibility for researchers. Umbilical cords obtained from five foals were used to isolate endothelial cells, followed by morphological and immunohistochemical identification. Performance of the cells in various assays commonly used in angiogenesis research was studied. Additionally, EqUVEC expression of vascular endothelial growth factor (VEGF) was assessed using ELISA. EqUVECs exhibited endothelial characteristics, forming a homogeneous monolayer with distinctive morphology. Immunohistochemical staining confirmed positive expression of key endothelial markers including von Willebrand factor (vWF), CD31, and vascular endothelial growth factor receptor-2 (VEGFR-2). Furthermore, performance assessments in  assays demonstrated the viability, proliferation, migration, tube formation and VEGF-expression capabilities of EqUVECs. The findings suggest that EqUVECs are a promising  model for studying equine angiogenesis, offering a foundation for further investigations into equine-specific vascular processes and therapeutic interventions.

Article	Year
SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients. PloS one\| PubMed ID: 26562020	2015
Potential of a BPV1 L1 VLP vaccine to prevent BPV1- or BPV2-induced pseudo-sarcoid formation and safety and immunogenicity of EcPV2 L1 VLPs in horse. The Journal of general virology\| PubMed ID: 28284277	2017
Canine oral primary melanoma cells exhibit shift to mesenchymal phenotype and phagocytic behaviour. Veterinary and comparative oncology\| PubMed ID: 30719836	2019
Companion Animals as Models for Inhibition of STAT3 and STAT5. Cancers\| PubMed ID: 31861073	2019
Betulinic acid shows anticancer activity against equine melanoma cells and permeates isolated equine skin in vitro. BMC veterinary research\| PubMed ID: 32024502	2020
Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine. Cells\| PubMed ID: 32231153	2020
A Recurrent Driver Mutation in Feline Alimentary T Cell Lymphoma. Cancers\| PubMed ID: 34680385	2021
An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation. Viruses\| PubMed ID: 35891381	2022
Bevacizumab Efficiently Inhibits VEGF-Associated Cellular Processes in Equine Umbilical Vein Endothelial Cells: An In Vitro Characterization. Veterinary sciences\| PubMed ID: 37999456	2023
Traces of Canine Inflammatory Bowel Disease Reflected by Intestinal Organoids. International journal of molecular sciences\| PubMed ID: 38203746	2024
Cross-Species Comparison of the Pan-RAF Inhibitor LY3009120's Anti-Tumor Effects in Equine, Canine, and Human Malignant Melanoma Cell Lines. Genes\| PubMed ID: 38397192	2024
A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium. Veterinary research\| PubMed ID: 38493107	2024
Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro. Cells\| PubMed ID: 38891088	2024
Isolation-protocol, characterization, and performance of equine umbilical vein endothelial cells. Frontiers in veterinary science\| PubMed ID: 39411390	2024

Barbara Pratscher

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