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

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 BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS 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 NRAS 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.

The TiHoCL panel for canine lymphoma: a feasibility study integrating functional genomics and network biology approaches for comparative oncology targeted NGS panel design.

Targeted next-generation sequencing (NGS) enables the identification of genomic variants in cancer patients with high sensitivity at relatively low costs, and has thus opened the era to personalized human oncology. Veterinary medicine tends to adopt new technologies at a slower pace compared to human medicine due to lower funding, nonetheless it embraces technological advancements over time. Hence, it is reasonable to assume that targeted NGS will be incorporated into routine veterinary practice in the foreseeable future. Many animal diseases have well-researched human counterparts and hence, insights gained from the latter might, in principle, be harnessed to elucidate the former. Here, we present the TiHoCL targeted NGS panel as a proof of concept, exemplifying how functional genomics and network approaches can be effectively used to leverage the wealth of information available for human diseases in the development of targeted sequencing panels for veterinary medicine. Specifically, the TiHoCL targeted NGS panel is a molecular tool for characterizing and stratifying canine lymphoma (CL) patients designed based on human non-Hodgkin lymphoma (NHL) research outputs. While various single nucleotide polymorphisms (SNPs) have been associated with high risk of developing NHL, poor prognosis and resistance to treatment in NHL patients, little is known about the genetics of CL. Thus, the ~100 SNPs featured in the TiHoCL targeted NGS panel were selected using functional genomics and network approaches following a literature and database search that shielded ~500 SNPs associated with, in nearly all cases, human hematologic malignancies. The TiHoCL targeted NGS panel underwent technical validation and preliminary functional assessment by sequencing DNA samples isolated from blood of 29 lymphoma dogs using an Ion Torrent™ PGM System achieving good sequencing run metrics. Our design framework holds new possibilities for the design of similar molecular tools applied to other diseases for which limited knowledge is available and will improve drug target discovery and patient care.

Evaluation of the Submaximal Treadmill-Based Fitness Test in Six Brachycephalic Breeds-A Follow-Up Study.

Brachycephalic obstructive airway syndrome (BOAS) in dogs challenges veterinary surgeons both with a complex clinical picture as well as wide-ranging ways to diagnose the disease, often not easily implemented nor standardised in clinical practice. The assessment of a combination of exercise testing, the occurrence of breathing noises, recovery time, and respiratory effort proved to be an appropriate method to identify Pugs with BOAS. The purpose of this study was to apply an established standardised, submaximal, treadmill-based fitness test for Pugs to other brachycephalic dog breeds. A total of 79 participants, belonging to 6 different brachycephalic breeds, trotted 15 min with an individual comfort speed of 3-7 km/h on a treadmill. Additionally, functional BOAS grading based on respiratory clinical signs before and after exercise was applied. The test was passed if the dogs presented with a BOAS grade of 0 or 1 and their vital parameters recovered to baseline within 15 min after exercise. A total of 68% showed a BOAS grade of 0 or 1 and passed the fitness test. Of the failed participants, 65% failed due to BOAS affectedness, 9% were categorised as not affected by BOAS and failed due to not passing the fitness test only, and 26% showed both failure criteria. The fitness test can be a useful method to identify BOAS-affected dogs in other brachycephalic breeds and to diagnose BOAS in dogs that only show clinical signs under exercise.

Impact of body weight and sex in selected dog breeds on the canine adrenal gland dimensions measured by computed tomographic imaging.

BACKGROUND: The present study aimed to investigate possible influences of body weight and sex on adrenal gland size in endocrinologically healthy dogs. Possible factors influencing the adrenal size are discussed in relation to a universal upper reference value from the literature of 7.4 mm as the thickness in the caudal pole of the adrenal gland. The adrenal size was measured by computed tomography (CT) from 66 normal dogs of six different breeds (Labrador Retriever (n = 16), German Shepherd (n = 10), Boxer (n = 8), Beagle (n = 14), Dachshund (n = 6) and Jack Russell Terrier (n = 12); male n = 38 (thereof neutered n = 23), female n = 28 (thereof neutered n = 17)) based on volume quantification and linear measurements using the data processing software Amira. For interbreed comparability, a ratio consisting of the third root of adrenal volume to aortic diameter (Ratio volume-aorta, RVA) was introduced. Additionally, breed-related attenuation values in contrast-enhanced CT data sets were measured. RESULTS: The measured volumes ranged from 0.34 to 1.93 cm3 for the right and from 0.39 to 2.23 cm3 for the left adrenal gland. The present study was able to demonstrate a body weight effect on the adrenal volume as well as on length and height. In terms of adrenal size, no significant differences between male and female, nor between intact and neutered dogs were obtained due to the RVA. In addition, for the weight classes, a breed independent threshold for dogs less (left 1.4; right gland 1.5) or more than 20 kg body weight (left 1.1; right gland 1.2) based on RVA was defined. Breed-related significant differences with respect to attenuation were determined only for the left adrenal gland, with lower attenuation values in large dog breeds. CONCLUSION: The present study points out the importance of weight-related data when assessing CT data of the canine adrenal gland regarding volume, size and attenuation. The use of a universal reference value for the assessment of adrenal size appears unsuitable considering weight-related volume and linear measurements. Sex seems not to affect adrenal gland size.

MDR1 Inhibition Reverses Doxorubicin-Resistance in Six Doxorubicin-Resistant Canine Prostate and Bladder Cancer Cell Lines.

Acquired chemoresistance during chemotherapy, often accompanied by cross- and multi-resistance, limits therapeutic outcomes and leads to recurrence. In order to create in vitro model systems to understand acquired doxorubicin-resistance, we generated doxorubicin-resistant sublines of canine prostate adenocarcinoma and urothelial cell carcinoma cell lines. Chemoresistance to doxorubicin, cross-resistance to carboplatin, and the reversibility of the acquired resistance by the specific MDR1-inhibitor tariquidar were quantified in metabolic assays. Resistance mechanisms were characterized by expression of the efflux transporters MDR1 and RALBP1, as well as the molecular target of doxorubicin, TOP2A, with qPCR and Western blotting. Six out of nine cell lines established stable resistance to 2 µM doxorubicin. Drug efflux via massive MDR1 overexpression was identified as common, driving resistance mechanism in all sublines. MDR1 inhibition with tariquidar extensively reduced or reversed the acquired, and also partly the parental resistance. Three cell lines developed additional, non-MDR1-dependent resistance. RALBP1 was upregulated in one resistant subline at the protein level, while TOP2A expression was not altered. Combination therapies aiming to inhibit MDR1 activity can now be screened for synergistic effects using our resistant sublines. Nevertheless, detailed resistance mechanisms and maintained molecular target expression in the resistant sublines are still to be examined.

20-year Results of a 3D Titanium Mesh Coating Stability of 31 Artificial Cups. / 20-Jahres-Ergebnisse der Verbundfestigkeit einer aufgesinterten 3-D-Titannetzbeschichtung von 31 Hüftpfannen.

BACKGROUND: The aim of this work was to demonstrate the bony bond strength and resilience of a three-dimensional titanium mesh coating of an artificial acetabulum produced using the diffusion bonding technique. Under the extreme conditions ranging from abrasion-related osteolysis to acetabular perforation, the degree of residual bone and the integrity of the coating were determined. The remaining zones of the (still) stable bone connection are inevitably exposed to a greater load of the layer adhesion between the titanium mesh and the core shell. The investigation was intended to provide information about the stages of damage according to Paprosky in which it was still justifiable to leave the implant in place and simply change the inlay from the purely material-technical point of view of a stable coating. The bond between bone and implant was examined with regard to a possible retention of the implant for its adaptive remodeling up to 27 years. MATERIALS AND METHODS: In a retrospective study, 31 explanted human acetabular cups of the Harris-Galante II type, with an average lifetime of 19.7 years (11-27 years), were examined by means of digital area measurement to determine both the bone areas remaining on the coating and the damaged areas of the titanium mesh. Periacetabular bone loss was recorded in a modified Paprosky (PAP) damage classification. Full hemispherical sections of 4 acetabular cups with a life time of 16, 20, 22 and 27 years were examined histopathologically using the diamond cut technique. RESULTS: The periacetabular bone loss resulted in damage class PAP I in 8 cases, PAP IIa in 7 cases, PAP IIb in 2 cases, PAP IIc in 9 cases, PAP IIIa in 3 cases and PAP IIIa in 2 cases PAP IIIb. The average amount of bone that was still firmly attached to the coating after explantation was 17% (0-70%) of the total cup surface. Paprosky I accounted for 44.1%, and PAP IIa and IIb stadiums together a total of 17.1%. The average bone fraction of the implants no longer anchored in the host bed at stages IIc, IIIa and IIIb was 2%. The average coating damage was 11% (0-100%) and was exclusively attributable to the unstable implants of stages IIc, IIIa and IIIb. The histopathological findings showed adaptive bone remodeling, that was detectable for up to 27 years through the titanium mesh down to the interface with the solid acetabular core. The titanium wire mesh was mostly surrounded by lamellar, mature bone. CONCLUSION: The results show that the connection between the Tivanium cup and the previously oldest and unchanged sintered coating - in the form of a three-dimensional titanium mesh applied in point and line contact - is very load-resistant even under the extreme loads of periacetabular osteolysis and cup perforations. Since there was no damage to the coating in periacetabular damage stages Paprosky I, IIa and IIb, it is justifiable in these damage stages to leave the implant in situ and to continue to use it with sole replacement of the inlay, but leaving the socket shell. The third-generation acetabular cup (Trilogy) with unchanged three-dimensional titanium mesh coating has been implanted in over 1.2 million cases for 26 years. After a long service life, an increasing number of wear and tear conditions can be expected in today's mostly elderly and vulnerable patient clientele. In view of the results presented here, the early detection of damage would make it possible to avoid costly and stressful explantation of the entire acetabular cup in favor of replacing the sole inlay in Paprosky stages I, IIa and IIb.

Left Ventricular Wall Reconstruction with Autologous Vascularized Tunica Muscularis of Stomach in a Porcine Pilot Model.

INTRODUCTION: Surgical replacement of dysfunctional cardiac muscle with regenerative tissue is an important option to combat heart failure. But, current available myocardial prostheses like a Dacron or a pericardium patch neither have a regenerative capacity nor do they actively contribute to the heart's pump function. This study aimed to show the feasibility of utilizing a vascularized stomach patch for transmural left ventricular wall reconstruction. METHODS: A left ventricular transmural myocardial defect was reconstructed by performing transdiaphragmatic autologous transplantation of a vascularized stomach segment in six Lewe minipigs. Three further animals received a conventional Dacron patch as a control treatment. The first 3 animals were followed up for 3 months until planned euthanasia, whereas the observation period for the remaining 3 animals was scheduled 6 months following surgery. Functional assessment of the grafts was carried out via cardiac magnetic resonance tomography and angiography. Physiological remodeling was evaluated histologically and immunohistochemically after heart explantation. RESULTS: Five out of six test animals and all control animals survived the complex surgery and completed the follow-up without clinical complications. One animal died intraoperatively due to excessive bleeding. No animal experienced rupture of the stomach graft. Functional integration of the heterotopically transplanted stomach into the surrounding myocardium was observed. Angiography showed development of connections between the gastric graft vasculature and the coronary system of the host cardiac tissue. CONCLUSIONS: The clinical results and the observed physiological integration of gastric grafts into the cardiac structure demonstrate the feasibility of vascularized stomach tissue as myocardial prosthesis. The physiological remodeling indicates a regenerative potential of the graft. Above all, the connection of the gastric vessels with the coronary system constitutes a rationale for the use of vascularized and, therefore, viable stomach tissue for versatile tissue engineering applications.

Transcription profiling of feline mammary carcinomas and derived cell lines reveals biomarkers and drug targets associated with metabolic and cell cycle pathways.

The molecular heterogeneity of feline mammary carcinomas (FMCs) represents a prognostic and therapeutic challenge. RNA-Seq-based comparative transcriptomic profiling serves to identify recurrent and exclusive differentially expressed genes (DEGs) across sample types and molecular subtypes. Using mass-parallel RNA-Seq, we identified DEGs and performed comparative function-based analysis across 15 tumours (four basal-like triple-negative [TN], eight normal-like TN, and three luminal B fHER2 negative [LB fHER2-]), two cell lines (CL, TiHo-0906, and TiHo-1403) isolated from the primary tumours (LB fHER2-) of two cats included in this study, and 13 healthy mammary tissue controls. DEGs in tumours were predominantly upregulated; dysregulation of CLs transcriptome was more extensive, including mostly downregulated genes. Cell-cycle and metabolic-related DEGs were upregulated in both tumours and CLs, including therapeutically-targetable cell cycle regulators (e.g. CCNB1, CCNB2, CDK1, CDK4, GTSE1, MCM4, and MCM5), metabolic-related genes (e.g. FADS2 and SLC16A3), heat-shock proteins (e.g. HSPH1, HSP90B1, and HSPA5), genes controlling centrosome disjunction (e.g. RACGAP1 and NEK2), and collagen molecules (e.g. COL2A1). DEGs specifically upregulated in basal-like TN tumours were involved in antigen processing and presentation, in normal-like TN tumours encoded G protein-coupled receptors (GPCRs), and in LB fHER2- tumours were associated with lysosomes, phagosomes, and endosomes formation. Downregulated DEGs in CLs were associated with structural and signalling cell surface components. Hence, our results suggest that upregulation of genes enhancing proliferation and metabolism is a common feature among FMCs and derived CLs. In contrast, the dissimilarities observed in dysregulation of membrane components highlight CLs' disconnection with the tumour microenvironment. Furthermore, recurrent and exclusive DEGs associated with dysregulated pathways might be useful for the development of prognostically and therapeutically-relevant targeted panels.

Methods of Radiographic Measurements of Heart and Left Atrial Size in Dogs with and without Myxomatous Mitral Valve Disease: Intra- and Interobserver Agreement and Practicability of Different Methods.

Dogs suffering from Myxomatous Mitral Valve Disease (MMVD) show a potential heart enlargement, especially in the left atrium, detectable by radiography. Due to digital radiography, different radiographic measurements estimate cardiac size quite uncomplicatedly. The Vertebral Heart Size (VHS), Radiographic Left Atrial Dimension (RLAD), Left Atrial Width (LAWidth), and the Vertebral Left Atrial Size (VLAS) used anatomical landmarks for measuring cardiac size in relation to the vertebral column. This study aimed to compare VHS, RLAD, LAWidth, and VLAS measured in conventional and inverted radiographs by veterinarians with different levels of experience in healthy dogs and dogs with MMVD. The reliability and user-friendliness of these measurements were evaluated, and the staging was compared to the echocardiography staging. A total of 50 unaffected dogs and 150 dogs with MMVD in stages B1, B2, and C were assessed. Three veterinarians with different levels of experience examined 200 conventional radiographs and their corresponding inverted radiographs blinded to the echocardiographic and clinical examination results. Analyses were performed to compare the measurements' grading and determine anatomical landmarks with measurement difficulties. Additionally, inter- and intraobserver agreement was assessed using intraclass correlation coefficient, and the agreement between radiographic and echocardiographic staging was compared using the kappa coefficient. The VHS, LAWidth, and VLAS were easier to define than the RLAD. The interobserver agreement was almost perfect for VHS (0.962) and good for the other radiographic measurements (RLAD: 0.778, LAWidth: 0.772, VLAS: 0.858). The VHS assigned the most dogs to the correct stage. However, VHS, RLAD, LAWidth, and VLAS presented an almost perfect intraobserver agreement. The dorsal left atrial margin of the RLAD was the most difficult measurement point to identify. The VHS is the most reproducible radiographic method for measuring the canine heart size and shows the highest agreement with echocardiography. An observer-related influence could be detected for RLAD, LAWidth, and VLAS.

Breed-specific values for vertebral heart score (VHS), vertebral left atrial size (VLAS), and radiographic left atrial dimension (RLAD) in pugs without cardiac disease, and their relationship to Brachycephalic Obstructive Airway Syndrome (BOAS).

This prospective study aims to establish reference ranges for vertebral heart score (VHS), vertebral left atrial size (VLAS), and radiographic left atrial dimension (RLAD) in pugs. The impact of clinical severity of Brachycephalic Obstructive Airway Syndrome (BOAS), gender, body condition score, and body weight on VHS, VLAS, and RLAD were investigated. Intra- and interobserver correlation was determined. Correlation of radiographic scores to echocardiographic left atrial dimension was inspected. Additionally, for VLAS and RLAD, correlation to VHS was examined. Additionally, an assessment of thoracic and vertebral malformations was performed. Forty-seven privately owned pugs underwent physical examination, echocardiography, and thoracic radiography to determine cardiac health. Thirty-two pugs were eligible for establishing reference ranges for VHS in right lateral radiographs, which was 11.25 ± 0.62 (95% range, 10.1-12.8). Reference ranges for VHS in left lateral, and for VLAS and RLAD in right lateral radiograph were determined in 30 pugs. The VHS in left lateral radiograph was 11.01 ± 0.70 (95% range, 9.4-12.6), VLAS was 1.96 ± 0.38 (95% range, 1.1-2.8), and RLAD was 1.59 ± 0.34 (95% range, 0.7-2.4). Clinical severity of BOAS did not show any impact on radiographic measurements. For VLAS, a significant correlation to VHS was detected by all observers. No other variables had a consistent influence on the radiographic scores given by all observers. Interobserver agreement was almost perfect for VHS (0.89 on right lateral and 0.91 on left lateral image), moderate for VLAS (0.49), and fair for RLAD (0.22). More than one third of the entire study population (18 of 47 pugs) showed at least one thoracic cavity or spine abnormality, often leading to considerable changes in vertebral body shape and size.

Reference ranges for standard-echocardiography in pugs and impact of clinical severity of Brachycephalic Obstructive Airway Syndrome (BOAS) on echocardiographic parameters.

BACKGROUND: Echocardiographic measurements may be influenced by breed-specific characteristics. Therefore, this study aims to establish reference values for standard echocardiographic measurements in pugs by investigating the influence of age, sex, heart rate, body weight and Brachycephalic Obstructive Airway Syndrome (BOAS). Sixty-two privately owned pugs underwent physical examination, blood sample collection, non-invasive blood pressure measurements and echocardiography. Influences of independent variables on echocardiographic measurements were examined using a multiple linear regression analysis model. For the entire study population, 95% prediction intervals were generated. Further, reference ranges for subcategories of clinical severities of BOAS were provided. Selected echocardiographic measurements of pugs were compared to reference values of previous studies generated from various breeds. RESULTS: In the study, a total of fifty-one privately owned pugs aged between two and 10 years were included for establishing reference ranges. Mainly body weight, but also age, sex and heart rate had influence on several echocardiographic parameters. The clinical grading of BOAS was conducted in 42 pugs. Except for pulmonic peak velocity (Pvel), which declined with increasing severity of BOAS, clinical symptoms of upper airway disease did not have significant impact on echocardiographic measurement results. Significant deviations, however, of left ventricular (LV) internal dimension (LVID), interventricular septum (IVS), LV posterior wall (LVPW), and tricuspid annular plane systolic motion excursion (TAPSE) compared to interbreed reference values were observed. CONCLUSIONS: Breed-specific reference ranges for echocardiographic values with special regard to BOAS are provided to enable a more accurate assessment of cardiac health in pugs.

Evaluation of new and old biomarkers in dogs with degenerative mitral valve disease.

BACKGROUND: Dogs with degenerative mitral valve disease are commonly presented to small animal clinicians. Diagnosis, clinical staging, and therapeutic design are based on a combination of clinical examination, radiography, and echocardiography. To support diagnosis and clinical monitoring, a multi-marker-based approach would be conceivable. The aim of this study was to investigate the suitability of Galectin-3 and interleukin-1 receptor-like 1 protein (ST2) in dogs with degenerative mitral valve disease in accordance with N-terminal-prohormone-B-type natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI). For this purpose, serum concentrations of Galectin-3 and ST2 of 64 dogs with different stages of mitral valve disease and 21 dogs without cardiac disease were analyzed at the first examination and six months later. Echocardiography, blood cell count and clinical chemistry were performed and established biomarkers NT-proBNP and cTnI were measured additionally. Differences in the biomarker concentrations between all groups at both timepoints and the change in biomarker concentrations from first to second evaluation was investigated. Furthermore, correlations of each biomarker, between biomarkers and echocardiographic measurements, were calculated. Finally, the receiver-operating characteristic curve and the area under the curve analysis were performed to differentiate between disease stages and controls. RESULTS: Serum concentrations of Galectin-3 and ST2 were not statistically different between canine patients in the respective stages of mitral valve disease or in comparison to dogs in the control group at any timepoint. A significant increase in ST2 concentrations from the baseline to the follow-up examination was observed in dogs classified as stage B1 and the control group. The concentrations of NT-proBNP and cTnI in stage C dogs were significantly increased in comparison to the other groups. CONCLUSIONS: In this study, no relation between Galectin-3 and ST2 levels to the presence or stage of mitral valve disease could be detected. Nevertheless, considering the increase in ST2 concentrations from the first to second measurement, its value on monitoring disease progress could be feasible. In agreement with previous studies, NT-proBNP and cTnI have once more proven their utility in assessing disease severity. The approach of examining new cardiac biomarkers in dogs is still worth pursuing.

Evaluation of a Treadmill-Based Submaximal Fitness Test in Pugs, and Collecting Breed-Specific Information on Brachycephalic Obstructive Airway Syndrome.

Despite efforts of veterinarians and breeders, brachycephalic obstructive airway syndrome (BOAS) is still a common problem in pugs, underlining the need for objective tests to identify and prevent breeding with affected dogs. In the current study, a submaximal, treadmill-based fitness test was evaluated as a tool to identify signs of airway obstruction not recognisable under rest conditions. In addition to this, different body conformation and measurements were assessed regarding their association with BOAS. A total of 62 pugs and 10 mesocephalic dogs trotted with an individual comfort speed on a treadmill for 15 min. Before and during the examination, dogs were examined for signs of respiratory distress, and a functional BOAS grading was applied. The influence of body conformation on BOAS grading was tested in a univariable and multivariable logistic regression model. During exercise, more respiratory noises were observed, and existing respiratory noises became more apparent in comparison to when at rest. In the multivariable logistic regression model, no factor had a statistically significant influence on BOAS classification. Submaximal fitness testing helped to identify signs of respiratory distress not apparent under resting conditions, and could be a valuable addition for identifying dogs with BOAS. Performing testing on a treadmill facilitates continuous observation of the patients, and enables standardisation of the test regarding the test environment, as well as provides an uninterrupted, steady workload.

Evaluation of the therapeutic potential of masitinib and expression of its specific targets c-Kit, PDGFR-α, PDGFR-ß, and Lyn in canine prostate cancer cell lines.

Canine prostate cancer is classified into adenocarcinoma, transitional cell carcinoma with prostatic involvement, and mixed forms. Early metastatic spread leads to poor prognosis and limited treatment options. Masitinib is approved for the treatment of canine mast cell tumours and inhibits tyrosine kinase c-Kit, tyrosine-protein kinase Lyn (Lyn), and platelet-derived growth factor receptors alpha and beta (PDGFR-α, PDGFR-ß), which are known to be expressed in canine prostate cancer. The aim of this study was to evaluate masitinib in an in vitro model consisting of cell lines from primary prostate adenocarcinoma, the associated lymph node metastasis of the same patient, and transitional cell carcinoma. To assess the suitability of the model system, the targets of masitinib were investigated by immunocytochemistry in the cell lines and by immunohistochemistry in the respective formalin-fixed, paraffin-embedded (FFPE) original neoplastic tissue. After exposure to masitinib, cell viability, cell count, apoptosis induction, and protein expression of c-Kit, Lyn, PDGFR-α, and PDGFR-ß were assessed. To hedge the efficacy, two application protocols of masitinib (single application or 12-h double-dose regimen) were compared. Immunocytochemical and immunohistochemical analysis revealed increased Lyn, PDGFR-α, and PDGFR-ß expression in cell lines and FFPE original neoplastic tissue compared to healthy prostate tissue. Masitinib exposure increased apoptosis, while the cell counts and cell viability decreased in a dose- and application interval-dependent manner, with increased impact in the 12-h double-dose regimen. These in vitro effects of masitinib in canine prostate cancer and associated metastasis support further in vivo research and modifications of the clinical treatment protocol in future studies.

RNA-seq of nine canine prostate cancer cell lines reveals diverse therapeutic target signatures.

BACKGROUND: Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount. METHODS: This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin. RESULTS: Independent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing. CONCLUSION: Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.

Establishment and Characterization of FusionRed Stable Transfected Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cells.

BACKGROUND/AIM: Cancer cell inoculation is routinely used to evaluate novel therapeutic approaches in vivo. However, without reporter genes enabling deep tissue imaging, study of early tumor progression and therapeutic responses is often limited. We describe the establishment and characterization of two canine cancer cell lines stably expressing red fluorescence proteins as tools for later in vivo imaging. MATERIALS AND METHODS: Two red fluorescence cell lines were generated by plasmid transfection. Fluorescence protein expression was confirmed by flow cytometry and microscopy. Deep tissue imaging was demonstrated in mice using a NightOWL LB 983. Gene expression changes after transfection were analyzed by RNAseq. RESULTS: Both cell lines were detectable in vivo by subcutaneous injection of 1×106 cells. RNAseq revealed up to 2005 transfection-induced differentially expressed genes but no significant changes in cellular key pathways. CONCLUSION: The fluorescent cell lines provide a solid basis for future in vivo studies on canine cancer.

Engraftment Effects after Intra-Bone Marrow versus Intravenous Allogeneic Stem Cell Transplantation in a Reduced-Intensity Conditioning Dog Leukocyte Antigen-Identical Canine Model.

Following conventional i.v. hematopoietic stem cell transplantation (IV-HSCT), most of the hematopoietic stem cells get trapped in peripheral organs and do not reach the bone marrow niche. A promising approach to overcome this cell loss during the homing process seems to be the infusion of hematopoietic stem cells directly into the bone marrow cavity (intra-bone marrow [IBM]-HSCT). This study aimed to investigate the engraftment efficiency of IBM-HSCT compared with IV-HSCT following reduced-intensity conditioning in a canine HSCT model. Furthermore, the impact of 2 different graft infusion rates during IBM-HSCT on the engraftment was evaluated. Dogs received 4.5 Gy total body irradiation for conditioning at day -1 and 15 mg/kg cyclosporin A twice daily at days -1 to +35 as immunosuppression. The IV-HSCT group (n = 7) received unmodified bone marrow. The IBM-HSCT cohorts received buffy coat-enriched bone marrow that was applied into the humerus and femur simultaneously with an infusion time of either 10 minutes (IBM10; n = 8) or 60 minutes (IBM60; n = 7). Statistical analyses were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction for multiple comparisons. Statistical significance was declared at Bonferroni-adjusted P < .017. All dogs initially engrafted. One dog of the IBM10 cohort died at day +15 from infection. All 21 evaluable dogs developed a durable mixed donor chimerism over the course of 112 days. Engraftment kinetics did not differ significantly across the 3 groups. Leukocyte and platelet nadirs, as well as the durations of leukopenia and thrombocytopenia, were comparable in the 3 groups. Signs of toxicity for ingestion, body temperature, activity, and defecation did not show statistically significant differences among the 3 groups; only weight loss was greater in the IBM60 group compared with the IV group. IBM-HSCT following reduced-intensity conditioning resulted in an engraftment efficiency and hematopoietic recovery comparable to that seen with conventional IV-HSCT. In addition, modification of the graft infusion rate had no impact on engraftment and hematopoietic recovery in the canine IBM-HSCT model.

Evaluation of blood pressure and NT-proBNP in pugs with and without clinical signs of Brachycephalic Obstructive Airway Syndrome.

Brachycephalic Obstructive Airway Syndrome (BOAS) is a pathologic condition of the upper airways, frequently occurring in dogs of brachycephalic breeds including pugs. It has been suspected that BOAS may be associated with cardiovascular changes and an increased risk for hypertension. The cardiac biomarker NT-proBNP can help to differentiate cardiac from non-cardiac respiratory distress. A possible influence of BOAS on NT-proBNP values has not been investigated, however. The aim of the current study was to examine blood pressure and NT-proBNP levels in pugs with and without clinical signs of BOAS and compare them to values of mesocephalic dogs. For this purpose, NT-proBNP values of 42 pugs and six mesocephalic dogs and blood pressure measurements of 34 pugs and four mesocephalic dogs were explored in the present study. Pugs were examined for clinical signs of BOAS at rest and after a submaximal fitness test, and a functional BOAS grading was applied. Blood pressure (BP) was measured at the beginning and end of the study day and NT-proBNP values were obtained before and after exercise. Measured values of pugs with different degrees of clinical impairment due to BOAS were compared among each other as well as to the CG. In terms of systolic, mean, diastolic BP, and NT-pro BNP, there were no relevant differences between pugs and the CG and no obvious connection between the severity of BOAS symptoms and measured values. BP values of all groups were lower at the second measurement at the end of the study day. NT-proBNP measurements were higher after exercise. BP and NT-proBNP values in all groups were in agreement with commonly used reference ranges. In conclusion, the study adds evidence, that BP and NT-proBNP values did not differ between mesocephalic dogs and pugs with different levels of severity of BOAS but between the measurement times. Thus, in the present study, excitement and exercise seemed to have a greater influence on BP and NT-proBNP values than presence of BOAS symptoms or breed. Discovered values show that the commonly used reference ranges for BP and NT-proBNP are applicable in pugs. This indicates that NT-proBNP can be used to differentiate between cardiac and non-cardiac respiratory distress even in pugs with clinical symptoms of BOAS.

BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells.

Bruton's tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3ß, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.

Ablation of Red Stable Transfected Claudin Expressing Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cell Lines by C-CPE Gold-Nanoparticle-Mediated Laser Intervention.

Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.