Canine Cells Research Articles

Canine Adipose-Derived Mesenchymal Stromal Cells Reduce Cell Viability and Migration of Metastatic Canine Oral Melanoma Cell Lines In Vitro

Canine oral melanoma (COM) is a promising target for immunomodulatory therapies aimed at enhancing the immune system’s antitumor response. Given that adipose-derived mesenchymal stem cells (Ad-MSCs) possess immunomodulatory properties through cytokine release, we hypothesized that co-culturing Ad-MSCs and canine peripheral blood mononuclear cells (PBMCs) could stimulate interleukin (IL) production against melanoma cell lines (MCCLs) and help identify therapeutic targets. This study evaluated IL-2, IL-8, and IL-12 expressions in co-culture with MCCL, Ad-MSCs, and PBMCs and assessed the relationship between gene expression, cell viability, and migration. Using four experimental groups in a Transwell insert system to separate cell types, we found that Ad-MSCs can reduce MCCL migration and viability, though the effect may vary depending on each cell line’s susceptibility. Furthermore, Ad-MSCs modified IL expression profiles in co-cultured cells. Our findings suggest that Ad-MSCs could have therapeutic potential for COM by inhibiting cell migration and reducing viability. However, deeper insights into Ad-MSC interactions with the tumor microenvironment and melanoma-specific factors will be essential to optimize therapeutic efficacy.

Knockout of B2M in combination with PD-L1 overexpression protects MSC-derived new islet β cells from graft rejection in the treatment of canine diabetes mellitus

BackgroundThe immunogenicity of allogeneic mesenchymal stem cells (MSCs) is significantly enhanced after transplantation or differentiation, and these cells can be recognized and cleared by recipient immune cells. Graft rejection has become a major obstacle to improving the therapeutic effect of allogeneic MSCs or, after their differentiation, transplantation in the treatment of diabetes and other diseases. Solving this problem is helpful for prolonging the time that cells play a role in the recipient body and for significantly improving the clinical therapeutic effect.MethodsIn this study, canine adipose-derived mesenchymal stem cells (ADSCs) were used as seed cells, and gene editing technology was used to knock out the B2M gene in these cells and cooperate with the overexpression of the PD-L1 gene. Gene-edited ADSCs (GeADSCs), whose biological characteristics and safety are not different from those of normal canine ADSCs, have been obtained.ResultsThe immunogenicity of GeADSCs is reduced, the immune escape ability of GeADSCs is enhanced, and GeADSCs can remain in the body for a longer time. Using the optimized induction program, the efficiency of the differentiation of GeADSCs into new islet β-cells was increased, and the maturity of the new islet β-cells was increased. The immunogenicity of new islet β-cells decreased, and their immune escape ability was enhanced after the cells were transplanted into diabetic dogs (the graft site was prevascularized by the implantation of a scaffold to form a vascularized pouch). The number of infiltrating immune cells and the content of immune factors were decreased at the graft site.ConclusionsNew islet β-cell transplantation, which has low immunogenicity, can reverse diabetes in dogs, and the therapeutic effect of cell transplantation is significantly enhanced. This study provides a new method for prolonging the survival and functional time of cells in transplant recipients and significantly improving the clinical therapeutic effect.

Prognostic significance of YKL-40 expression in canine cutaneous mast cell tumors

BackgroundYKL-40, a secretory glycoprotein, is involved in tumor cell proliferation, metastasis, and angiogenesis in human cancers. Its overexpression has been correlated with unfavorable prognosis in many human cancers. In veterinary medicine, elevated YKL-40 levels in the serum of canine cutaneous mast cell tumors (cMCTs) were observed in our previous study. However, the expression pattern of YKL-40 in canine cMCT tissues, along with its association with clinical and pathological features, is still unknown. This study aims to retrospectively investigate the expression level of YKL-40 in the tissues of canine cMCTs and its correlation with clinical features, pathological characteristics, and clinical outcomes. Forty formalin-fixed paraffin-embedded cMCT tissues collected from forty dogs were diagnosed as low-grade (n = 20) or high-grade s(n = 20) MCT according to the Kiupel grading system. The expression level of YKL-40 in cMCT tissues was investigated using immunohistochemical staining and immunoreactivity score (IRS).ResultsYKL-40 was expressed in all cMCTs at different levels, with significantly stronger expression in low-grade cMCTs compared to high-grade cMCTs. The expression level was also associated with tumor diameter, histological grade, mitotic counts, vessel density, and survival of cMCTs. The overall survival of cMCT dogs showed significant differences (p < 0.01) among mild (n = 15, MST 219 days), moderate (n = 19, MST not reached), and high (n = 6, MST not reached) YKL-40 expression groups. Among low-grade cMCTs, overall survival was significantly different between mild YKL-40 expression (MST 319 days) and moderate to high YKL-40 (MST not reached) expression (p < 0.01). In high-grade cMCTs, overall survival was not correlated with YKL-40 expression (p = 0.6589).ConclusionsThis study found that the YKL-40 expression level was significantly stronger in low-grade than in high-grade canine cutaneous mast cell tumors and was associated with various clinical and pathological features. Stronger YKL-40 expression level correlated with longer survival time, especially in low-grade cMCTs. Therefore, YKL-40 could serve as a prognostic marker for cMCTs.

Development of Vincristine‐Loaded Nano Graphene Oxides for Dual Therapies Against Canine Anaplastic Carcinoma Cells Obtained From a Dog with Spontaneous Mammary Tumor

AbstractAmong pet species, mammary tumors are most commonly encountered in dogs. In the traditional treatment of mammary tumors in dogs; surgical operation, chemotherapy, immunotherapy, cryotherapy, radiotherapy and homeopathy are used. Herein, we reported vincristine loaded nano graphene oxide (Vin@nGO) for simultaneous photothermal therapy (PTT) and chemotherapy under near infrared laser (NIR, 808 nm), whereas GO was used both as a platform for vincristine and as a photothermal (PT) agent, and vincristine was used as a chemotherapeutic agent. Spontaneous mammary tumor masses of a dog were removed and primary tumor cells were obtained and propagated by tissue culture from tumor tissue samples under appropriate conditions. Then, these cells were incubated with Vin@nGO and exposed to NIR laser. NIR laser irradiation at 808 nm was converted to heat by nGO, then the target cancer cells were destroyed by both hyperthermia and chemotherapy. MTS analysis was performed to test the effectiveness of this dual therapy on cell proliferation and a statistically significant (p &lt; 0.05) decrease in cell proliferation/viability was found (90% reduction compared to the control group (p &lt; 0.0001)). Our study data also shed light on usability of Vin@nGO in the in vivo model for future work.

Retrospective Safety Evaluation of Combined Chlorambucil and Toceranib for the Treatment of Different Solid Tumours in Dogs.

Chlorambucil is used in veterinary medicine for various cancers, while Toceranib, which was licenced for treating canine mast cell tumours, is now used against other solid tumours. Both drugs are generally safe, but their combined use has not been studied. This study aimed to investigate retrospectively the safety profile of the Chlorambucil-Toceranib combination against canine solid tumours. Thirty-eight dogs received this combination. Chlorambucil was administered at a median dose intensity of 15.1 mg/m2 per week, while Toceranib was given at the median dosage of 2.5 mg/kg on a Monday-Wednesday-Friday schedule. Dosages were individually adjusted according to commercially available tablet formulation, co-morbidities, and adverse events (AEs). The resulting clinical benefit rate (CBR) and overall response rate (ORR) were 55.3% and 10.5%, respectively. The median progressive free survival (PFS) and median survival time (MST) were 45.5 (12-537) days and 259 (42-1178) days, respectively. Gastrointestinal AEs occurred in 39.5% of cases (n = 15), 15.8% (n = 6) experienced UPC elevation, while hematological and biochemistry AEs affected 13.2% (n = 5) each. Most of these AEs were grades 1-2 (G1-2). None of the dogs interrupted treatment due to AEs, and the combination appeared safe. Larger prospective clinical trials are required to confirm our findings and investigate its efficacy across various cancers.

Mutational Landscape of KIT Proto-Oncogene Coding Sequence in 62 Canine Cutaneous and Subcutaneous Mast Cell Tumors

Canine mast cell tumors (MCTs) are common skin neoplasms with varying biological behaviors. The KIT proto-oncogene plays a key role in the development of these tumors, and internal tandem duplications on exon 11 are usually associated with more aggressive behavior, increased local recurrence, and decreased survival time. However, apart from exons 8–11 and 17, there is limited understanding of the overall KIT mutational landscape in canine MCTs. This work aims to analyze the entire KIT coding sequence (21 exons) in a cohort of 62 MCTs, which included 38 cutaneous and 24 subcutaneous tumors, and potentially identify new variants. In addition to confirming previously reported activating KIT mutations in exons 8, 9, and 11, we identified new variants in exons 2, 3, 5, 16, and the 3′ untranslated region (UTR). Notably, these last variants include an amino acid change (Asp/His) in exon 16. Additionally, we confirmed a differential prevalence of KIT variants in cutaneous and subcutaneous MCTs. These findings enhance our understanding of the KIT proto-oncogene coding sequence and provide valuable information for future confirmatory studies.

Expression of Mutated BRAFV595E Kinase in Canine Carcinomas-An Immunohistochemical Study.

Alterations of the BRAF gene and the resulting changes in the BRAF protein are one example of molecular cancer profiling in humans and dogs. We tested 227 samples of canine carcinomas from different anatomical sites (anal sac (n = 23), intestine (n = 21), liver (n = 21), lungs (n = 19), mammary gland (n = 20), nasal cavity (n = 21), oral epithelium (n = 18), ovary (n = 20), prostate (n = 21), thyroid gland (n = 21), urinary bladder (n = 22)) with two commercially available primary anti-BRAFV600E antibodies (VE1 Ventana, VE1 Abcam). The immunohistochemical results were confirmed with droplet digital PCR (ddPCR). BRAFV595E-mutated cases were found in canine prostatic (16/21), urothelial (17/22), and oral squamous cell carcinomas (4/18), while other carcinoma types tested negative. Both antibodies showed consistent results, with intracytoplasmic immunolabeling of tumour cells, making them reliable tools for detecting the BRAFV595E mutation in canine carcinomas. In conclusion, identifying BRAF mutations from biopsy material offers a valuable opportunity to enhance cancer treatment strategies (BRAF inhibitors) in canine urothelial carcinomas, prostatic carcinomas, and oral squamous cell carcinomas.

Nuclear pleomorphism in canine cutaneous mast cell tumors: Comparison of reproducibility and prognostic relevance between estimates, manual morphometry, and algorithmic morphometry.

Variation in nuclear size and shape is an important criterion of malignancy for many tumor types; however, categorical estimates by pathologists have poor reproducibility. Measurements of nuclear characteristics can improve reproducibility, but current manual methods are time-consuming. The aim of this study was to explore the limitations of estimates and develop alternative morphometric solutions for canine cutaneous mast cell tumors (ccMCTs). We assessed the following nuclear evaluation methods for accuracy, reproducibility, and prognostic utility: (1) anisokaryosis estimates by 11 pathologists; (2) gold standard manual morphometry of at least 100 nuclei; (3) practicable manual morphometry with stratified sampling of 12 nuclei by 9 pathologists; and (4) automated morphometry using deep learning-based segmentation. The study included 96 ccMCTs with available outcome information. Inter-rater reproducibility of anisokaryosis estimates was low (k = 0.226), whereas it was good (intraclass correlation = 0.654) for practicable morphometry of the standard deviation (SD) of nuclear size. As compared with gold standard manual morphometry (area under the ROC curve [AUC] = 0.839, 95% confidence interval [CI] = 0.701-0.977), the prognostic value (tumor-specific survival) of SDs of nuclear area for practicable manual morphometry and automated morphometry were high with an AUC of 0.868 (95% CI = 0.737-0.991) and 0.943 (95% CI = 0.889-0.996), respectively. This study supports the use of manual morphometry with stratified sampling of 12 nuclei and algorithmic morphometry to overcome the poor reproducibility of estimates. Further studies are needed to validate our findings, determine inter-algorithmic reproducibility and algorithmic robustness, and explore tumor heterogeneity of nuclear features in entire tumor sections.

Biomimetic In Vitro Model of Canine Periodontal Ligament.

Periodontal disease affects about 80% of dogs, highlighting the importance of addressing periodontitis in veterinary dental care. The periodontal ligament (PDL) is a key structure holding the potential to regenerate the entire periodontal complex. This work presents an in vitro model of canine PDL-derived cell cultures that mimic the PDL's regenerative capacity for both mineralised and soft tissues. Explant outgrowth-derived PDL cells were cultured under standard conditions in osteoinductive medium and with hydroxyapatite nanoparticles (Hap NPs). Cell behaviour was assessed for viability/proliferation, morphology, growth patterns, and the expression of osteogenic and periodontal markers. Osteogenic conditions, either achieved with osteoinducers or an osteoconductive biomaterial, strongly promoted PDL-derived cells' commitment towards the osteogenic phenotype and significantly increased the expression of periodontal markers. These findings suggest that cultured PDL cells replicate the biological function of the PDL, supporting the regeneration of both soft and hard periodontal tissues under normal and demanding healing conditions. This in vitro model will offer a platform for testing new regenerative treatments and materials, ultimately contributing to canine dental care and better outcomes.

Effect of Subconjunctival Injection of Canine Adipose-Derived Mesenchymal Stem Cells on Canine Spontaneous Corneal Epithelial Defects.

Spontaneous chronic corneal epithelial defects (SCCEDs) are characterized by nonadherent corneal epithelium leading to poor attachment to the corneal stroma. The objective of this study was to characterize corneal outcomes concurrently with the quantification of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor-A (VEGF-A) in tear fluid after the subconjunctival injection of canine adipose-derived mesenchymal stem cells (cAD-MSCs) in canine SCCEDs. Ten eyes with SCCEDs, which were nonresponsive to two rounds of diamond burr debridement, were included in this study. All eyes received a single subconjunctival injection of 1 × 106 cAD-MSCs. Ophthalmic examinations were performed before treatment and on day 7, 14, and 21 after treatment. Tear samples were collected for the quantification of TNF-α and VEGF-A concentrations by a canine multiplex immunoassay. Nine out of ten eyes revealed complete healing by day 21. The mean healing time was 10.89 ± 1.7 days. All eyes showed a decreased degree of ocular discomfort, in accordance with the degree of corneal characteristics. The concentrations of VEGF-A significantly reduced from pre-treatment (4334.91 ± 1275.92 pg/mL) to day 21 post-treatment (3064.61 ± 1028.66 pg/mL). No significant difference in TNF-α concentration was observed before/after treatment. In conclusion, the single use of a subconjunctival injection of cAD-MSCs could be used as an alternative treatment for canine SCCEDs.

Characterisation and Sensitivity of a Canine Mast Cell Tumour Line to Oncolytic Viruses.

Canine mast cell tumours (MCTs) are one of the most common skin cancers of dogs. Surgical removal is the primary treatment, but recurrence and metastasis can occur even with low-grade tumours. As a result, new treatment strategies are being sought. We tested the potential of several oncolytic viruses (OVs) to infect and kill a cell line isolated from a canine MCT. Employing a resazurin-based metabolic assay and flow cytometry technology, we used recombinant vesicular stomatitis virus (rVSV-Δm51), avian orthoavulavirus-1 (AOaV-1), and Orf viruses in our assessment. Our study aimed to evaluate the potential of oncolytic virotherapy in treating canine cancers. We found that MCT-1 cells showed different sensitivities to the OVs, with rVSV-Δm51 showing the most promising results invitro. These findings suggest that further investigation into using OVs for treating canine MCTs is needed, although clinical efficacy is yet to be determined.

Assessment of Biofilm Formation and Anti-Inflammatory Response of a Probiotic Blend in a Cultured Canine Cell Model.

Gut dysbiosis and an inflamed bowel are growing concerns in mammals, including dogs. Probiotic supplements have been used to restore the natural microbial community and improve gastrointestinal health. Biofilm formation, antimicrobial activities, and immunological responses of probiotics are crucial to improving gut health. Thus, we tested a commercial probiotic blend (LabMAX-3), a canine kibble additive comprising Lactobacillus acidophilus, Lacticaseibacillus casei, and Enterococcus faecium for their ability to inactivate common enteric pathogens; their ability to form biofilms; epithelial cell adhesion; and their anti-inflammatory response in the Madin-Darby Canine Kidney (MDCK) cell line. Probiotic LabMAX-3 blend or individual isolates showed a strong inhibitory effect against Salmonella enterica, Listeria monocytogenes, enterotoxigenic Escherichia coli, and Campylobacter jejuni. LabMAX-3 formed biofilms comparable to Staphylococcus aureus. LabMAX-3 adhesion to the MDCK cell line (with or without lipopolysaccharide (LPS) pretreatment) showed comparable adhesion and biofilm formation (p < 0.05) to L. casei ATCC 334 used as a control. LabMAX-3 had no cytotoxic effects on the MDCK cell line during 1 h exposure. The interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFα) ratio of LabMAX-3, compared to the L. casei control, showed a significant increase (p < 0.05), indicating a more pronounced anti-inflammatory response. The data show that LabMAX-3, a canine kibble supplement, can improve gastrointestinal health.

Antibacterial Effects of Essential Oils on P. aeruginosa, Methicillin-Resistant S. aureus, and Staphylococcus spp. Isolated from Dog Wounds.

Background: Essential oils exhibit several biological activities such as antimicrobial, antioxidant, proliferative, and anti-inflammatory. This study was aimed at investigating the antimicrobial effects and cytotoxic activities of niaouli, palmarosa, and clove essential oils. Methods: Content analyses of these essential oils were carried out by gas chromatography-mass spectrometry. The antibacterial activity was screened against methicillin-resistant S. aureus ATCC 43300, P. aeruginosa ATCC 27853, P. aeruginosa PAO1, S. aureus ATCC 25923, and 44 isolates (22 P. aeruginosa isolates, 4 S. aureus isolates, and 18 Staphylococcus spp. isolates) obtained from dogs with previous wound infections who were included in the current study. The antimicrobial effects of essential oils were investigated using disk diffusion and minimum inhibition/bactericidal concentration methods. Additionally, the antibiofilm, protease, elastase, and gelatinase activities of the essential oils were evaluated. Different concentrations of each essential oil ranging from 10 to 1000 µg/mL were also analyzed in terms of cell viability by WST-8 assay in primary canine fibroblast cells. Results: The fibroblast cell viabilities of palmarosa, niaouli, and clove oils at a 1000 µg/mL concentration were 75.4%, 96.39%, and 75.34%, respectively. All the EOs were found to have bactericidal effects with MBCs/MICs of 0.015 to 0.5 µL/mL against P. aeruginosa, Staphylococcus isolates (p < 0.001). Palmarosa was found to have the largest inhibition zone diameter (20.5 ± 6.6, 16.4 ± 2.3) compared to other essential oils in the disk diffusion test against Staphylococcus spp. and P. aeruginosa (p < 0.001). But none of the EOs reduced protease, elastase, and gelatinase activities, which are some of the virulence properties of the tested bacteria. Conclusions: These results showed that palmarosa, niaouli, and clove essential oils act as potential antibacterial agents for dogs against P. aeruginosa, methicillin-resistant S. aureus, and Staphylococcus spp., without damaging the skin.

Longitudinal Study of Transcriptomic Changes Occurring over Six Weeks of CHOP Treatment in Canine Lymphoma Identifies Prognostic Subtypes.

The majority of canine lymphoma patients treated with the standard of care, the CHOP chemotherapy protocol, initially achieve remission but eventually relapse with a multi-drug-resistant phenotype. This study assesses gene expression profiles of canine lymphoma tumor cell populations using RNA-Seq data from 15 matched patient samples taken prior to treatment and again six weeks into treatment with CHOP. Two distinct clusters were present in the t-SNE dimensionality reduction of the gene expression profiles. There was a significant difference in progression-free survival (PFS) between the cluster groups, with a median of 43.5 days in a group of six patients and 185 days in another group of nine patients. Comparing the group with shorter PFS to the group with longer PFS, we identified 265 significantly enriched GO:BP terms in 3874 significantly up-regulated genes and 740 significantly enriched GO:BP terms in 3236 significantly down-regulated genes. Comparing the six-week timepoint against the initial timepoint, in the group with longer PFS, we identified 277 significantly enriched GO:BP terms in 413 significantly up-regulated genes and 222 significantly enriched GO:BP terms in 267 significantly down-regulated genes. In the group with shorter PFS, we only identified 27 significantly differentially expressed genes, for this comparison. We found DNA damage response genes to be enriched in the down-regulated genes in both comparisons. These results identify and characterize two transcriptionally distinct groups of canine lymphoma patients with significantly different responses to CHOP chemotherapy.

Antitumor effects of inhibitors of ERK and Akt pathways in canine histiocytic sarcoma cell lines

Canine histiocytic sarcoma (CHS) is characterized by aggressive biological behavior. In our previous study, ERK and Akt pathways were found to be activated in CHS tissues. Thus, the objective of this study was set to investigate the relationships between the activation status of these pathways and the proliferation of CHS cell lines by examining the effects of single and co-administrations of drugs targeting these pathways. First, we evaluated the changes in cell proliferations and the activations of ERK and Akt pathways after treatments with ERK and Akt-specific inhibitors in CHS cells. Then, these changes after treatments with dasatinib and trametinib were also examined in CHS cells. Inhibitors specific to ERK and Akt pathways successfully inhibited the respective pathways in CHS cell lines. It was also indicated that these pathways were associated with the regulations of proliferations of CHS cells, although the anti-proliferative effect was not necessarily observed by inhibition of Akt pathway alone. Dasatinib and trametinib also showed the inhibitions of Akt and ERK pathway activations, respectively, in CHS cells. However, the anti-proliferative effects of these drugs varied among CHS cell lines, and co-administration showed enhanced anti-proliferative effects in only a part of CHS cell lines. Further studies are needed to investigate the molecular mechanisms associated with the sensitivities to these molecular-targeted drugs in CHS cells.

Early apoptosis detection in canine granulosa cells through the analysis of BCL-2 and BAX proteins during the follicular development associated with oocyte maturation

The objective of this study was to investigate the early follicular apoptosis in canine ovarian follicles by examining the expression of anti-apoptotic BCL-2 and pro-apoptotic BAX proteins throughout the estrous cycle associated with oocyte maturation. Follicular cells from preantral and antral follicles of varying sizes were isolated and grouped based on follicle type and estrous phase. Antral follicles underwent flow cytometry analysis, whereas preantral follicles were subjected to Western blotting. The meiotic capacity of oocytes from these different follicle types was evaluated through in vitro maturation. Statistical analysis included ANOVA and Duncan’s test. Results showed fluctuations in BCL-2 and BAX levels across different follicular stages and estrous phases. BCL-2 levels increased (P<0.05) with follicular development in antral follicles, particularly during estrus, while BAX exhibited variations peaking (P<0.05) during estrus. The BCL-2/BAX ratio in antral follicles was higher (P<0.05) in estrus and diestrus compared to anestrus and proestrus. Additionally, BCL-2 and BAX proteins were detected in preantral follicles, with varying expression levels (P<0.05) across estrous phases. The BCL-2/BAX ratio in preantral follicles was highest (P<0.05) during anestrus and estrus and decreased (P<0.05) in proestrus and diestrus. Oocytes from preantral follicles did not reach the MII stage, regardless of the levels of BCL-2/BAX. Conversely, oocytes obtained from large follicles during estrus showed the highest (P<0.05) maturation percentages, which were associated with the highest BCL-2/BAX ratio. These findings provide insights into the dynamic patterns of BCL-2 /BAX in canine follicles across the estrous cycle, shedding light on their potential roles in oocyte development.

Human and canine osteosarcoma cell lines: How do they react upon incubation with calcium phosphate-coated lipid nanoparticles carrying doxorubicin and curcumin?

Osteosarcoma (OSA) is a bone cancer that affects both humans and animals, with dogs being particularly vulnerable. Standard therapy is often limited by multidrug resistance (MDR), primarily due to the overexpression of P-glycoprotein (P-gp), which expels drugs from the cells, reducing their efficacy. To overcome this challenge, drug delivery systems (DDS) and P-gp modulators have been explored. However, developing DDS that selectively target cancer cells remains difficult, with many current options lacking efficiency. Our research group has recently developed an innovative type of nanoparticle with a lipid core and a calcium phosphate shell (CaP-NPs), which enhances the uptake of doxorubicin (DOXO) in OSA cells. In this study, we loaded a lipophilic ester of doxorubicin (C12DOXO) and curcumin (CURC), a natural P-gp modulator, into CaP-NPs and co-incubated them into human and canine OSA cell lines, including DOXO-resistant cells. The results demonstrated a significant reduction in viability in human OSA cells. Additionally, the combination treatment led to a further increase in C12DOXO retention when cells were also treated with the P-gp inhibitor verapamil, indicating enhanced efficacy against MDR mechanisms. Notably, canine OSA cells exhibited a distinct response pattern, suggesting the presence of species-specific differences that warrant further investigation.

Preclinical Safety Assessment of Deferoxamine-preconditioned Canine Adipose Tissue-derived Mesenchymal Stem Cells.

In the pursuit of translating stem cell therapy technology into clinical practice, ensuring the safety and efficacy of treatments is paramount. Despite advancements, the effectiveness of stem cell applications often falls short of clinical requirements. This study aimed to address the challenge of limited efficacy by investigating the safety and effectiveness of canine adipose tissue-derived mesenchymal stem cells (cATMSCs) preconditioned with deferoxamine (DFO). Different concentrations of DFO were used to evaluate its impact on cATMSC activity. The therapeutic potential of these preconditioned cells was validated using a mouse model of systemic inflammation. Comprehensive evaluations, including clinical hematological and radiological assessments before and after intravenous injection of preconditioned cells were conducted. The study showed a notable reduction in inflammatory markers and an overall decrease in the inflammatory response in the mouse model. The data collected from the clinical hematological and radiological assessments provided essential insights. This study lays the groundwork for the future clinical deployment of DFO-preconditioned cATMSCs, demonstrating their potential to improve the efficacy and safety of stem cell therapies.

Beta-Adrenergic Activation of the Inward Rectifier K+ Current Is Mediated by the CaMKII Pathway in Canine Ventricular Cardiomyocytes.

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K+ current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (IK1) and the rapid delayed rectifier (IKr) K+ currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. IK1 and IKr were dissected by 50 µM BaCl2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased IK1 under the plateau phase of the action potential (0-+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall IK1 integral. The ISO-induced enhancement of IK1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the IK1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the IKr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances IK1 in canine left ventricular cells through the CaMKII pathway, while IKr remains unaffected under the experimental conditions used.

Proteomic analysis of extracellular vesicles derived from canine mammary tumour cell lines identifies protein signatures specific for disease state

BackgroundCanine mammary tumours (CMT) are among the most common types of tumours in female dogs. Diagnosis currently requires invasive tissue biopsies and histological analysis. Tumour cells shed extracellular vesicles (EVs) containing RNAs and proteins with potential for liquid biopsy diagnostics. We aimed to identify CMT subtype-specific proteome profiles by comparing the proteomes of EVs isolated from epithelial cell lines derived from morphologically normal canine mammary tissue, adenomas, and carcinomas.MethodsWhole-cell protein lysates (WCLs) and EV-lysates were obtained from five canine mammary cell lines: MTH53A (non-neoplastic); ZMTH3 (adenoma); MTH52C (simple carcinoma); 1305, DT1406TB (complex carcinoma); and their proteins identified by LC-MS/MS analyses. Gene Ontology analysis was performed on differentially abundant proteins from each group to identify up- and down-regulated biological processes. To establish CMT subtype-specific proteomic profiles, weighted gene correlation network analysis (WGCNA) was carried out.ResultsWCL and EVs displayed distinct protein abundance signatures while still showing the same increase in adhesion, migration, and motility-related proteins in carcinoma-derived cell lines, and of RNA processing and RNA splicing factors in the adenoma cell line. WGCNA identified CMT stage-specific co-abundant EV proteins, allowing the identification of adenoma and carcinoma EV signatures not seen in WCLs.ConclusionsEVs from CMT cell lines exhibit distinct protein profiles reflecting malignancy state, allowing us to identify potential biomarkers for canine mammary carcinomas, such as biglycan. Our dataset could therefore potentially serve as a basis for the development of a less invasive clinical diagnostic tool for the characterisation of CMT.