Evaluation serum soluble interleukin 2 receptor with diagnosis and prognosis in canine solid tumour: 34 cases.

BACKGROUND/AIM: The soluble interleukin-2 receptor (sIL-2R) serve as a valuable biomarker for tumors in human patients, as its levels increase during the activation of T lymphocytes in clinical states such as inflammation, infection, and tumor. This study aimed to demonstrate that sIL-2R levels can be also elevated in dogs with tumors and evaluate its applicability as a diagnostic and prognostic factor in canine cancer patients. PATIENTS AND METHODS: Serum was collected from 6 healthy dogs and 34 dogs with solid tumors. The concentration of sIL-2R was measured using a commercial enzyme-linked immunosorbent assay kit. RESULTS: The median sIL-2R concentration was significantly higher in dogs with solid masses than in healthy dogs (117.3 vs 68.33 pg/ml, p = 0.016). The highest median sIL-2R concentration was found in dogs with malignant tumors, followed by those with benign tumors, and healthy dogs (119.6 vs 93.74 vs 68.33 pg/ml, respectively). In dogs with malignant tumors, the mortality rate was significantly higher in the group with high sIL-2R levels than in the group with low sIL-2R levels. Dogs with solid tumors, particularly those with malignant tumors, had higher concentrations of sIL-2R than healthy dogs. Among dogs with malignant tumors, a correlation between sIL-2R concentration and mortality rate was confirmed. CONCLUSION: Serum sIL-2R levels may be used to detect malignant tumors and serve as a prognostic factor in dogs with malignant tumors.

Deferoxamine preconditioning of canine stem cell derived extracellular vesicles alleviates inflammation in an EAE mouse model through STAT3 regulation.

Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), specifically those preconditioned with deferoxamine (DFO) in canine adipose tissue-derived MSCs (cAT-MSCs), were explored for treating autoimmune diseases. This study assessed the effects of DFO-preconditioned EVs (EVDFO) in an experimental autoimmune encephalomyelitis (EAE) mouse model. cAT-MSCs were treated with DFO for 48 h, after which EVs were isolated. EAE mice received intranasal EV or EVDFO treatments and were euthanized following histopathologic analysis; RNA and protein expression levels were measured. Histologically, EV and EVDFO groups showed a significant reduction in inflammatory cell infiltration and demyelination. Immunofluorescence revealed increased CD206 and Foxp3 expression, indicating elevated M2 macrophages and regulatory T (Treg) cells, particularly in the EVDFO group. Treg cells also notably increased in the spleen of EVDFO -treated mice. STAT3 and pSTAT3 proteins were upregulated in the EAE groups compared to the naïve group. However, following EV treatment, STAT3 expression decreased compared to the EAE group, whereas pSTAT3 expression was similar in both the EV and EAE groups. In conclusion, EVDFO treatment resulted in reduced STAT3 expression, suggesting its role in T cell regulation and the potential of EVDFO in modulating the STAT3 pathway for reducing inflammation more effectively than non-preconditioned EVs.

Intravenous injection of allogenic canine mesenchymal stem cells in 40 client-owned dogs: a safety assessment in veterinary clinical trials.

BACKGROUND: The aim of this study was to evaluate the adverse effects of allogeneic mesenchymal stem cells (MSCs) transplanted via intravenous infusion in dogs and examine their safety. We performed a retrospective analysis of various clinical assessments, including physical examination, blood tests, and radiographs, and monitored the formation of neoplasms during a 6-month follow-up period in 40 client-owned dogs that received intravenous infusion of adipose tissue-derived MSCs (AT-MSCs) for the treatment of various underlying diseases between 2012 and 2018. RESULTS: No significant adverse effects of MSC therapy were detected by clinical assessment, blood tests, or radiographic examination in the 6-month follow-up period after the first MSC treatment. Additionally no new neoplasms were observed during this period. CONCLUSIONS: To our knowledge, this study is the first to evaluate the safety aspects (≥ 6 months) associated with intravenous allogeneic AT-MSC infusion. These results suggest that allogenic AT-MSC infusion could be a useful and relatively safe therapeutic approach in canines.

Benign Endocervical Polyp with Coexisting Nabothian Cysts and Endocervical Hyperplasia Mimicking Malignancy: A Case Report.

This report presents a unique case of an endocervical polyp-mimicking malignancy on pelvic MRI in a 45-year-old female. The MRI depicted a multilocular cystic lesion with an enhancing solid component, raising suspicion for malignancy. However, histopathological examination definitively revealed a benign endocervical polyp. This case highlights the limitations of diagnosing cervical lesions solely on MRI features, emphasizing the potential for benign conditions to mimic malignancy.

Interfacial Engineering for Controlled Crystal Mosaicity in Single-Crystalline Perovskite Films.

Organic-inorganic hybrid perovskites have attracted significant attention for optoelectronic applications due to their efficient photoconversion properties. However, grain boundaries and irregular crystal orientations in polycrystalline films remain issues. This study presents a method for producing crystalline-orientation-controlled perovskite single-crystal films using retarded solvent evaporation. It is shown that single-crystal films, grown via inverse temperature crystallization within a confined space, exhibit enhanced optoelectronic property. Using interfacial polymer layer, this method produces high-quality perovskite single-crystalline films with varying crystal orientations. Density functional theory calculations confirm favorable adsorption energies for (110) surfaces with methylammonium iodide and PbI2 terminations on poly(3-hexylthiophene), and stronger adsorption for (224) surfaces with I and methylammonium terminations on polystyrene, influenced by repulsive forces between the thiophene group and the perovskite surface. The correlation between charge transport characteristics and perovskite single-crystalline properties highlights potential advancements in perovskite optoelectronics, improving device performance and reliability.

Association between living alone and generalized anxiety disorder in Korean adults.

BACKGROUND: Globally, the rise in single-person households poses a potential risk to mental health, with generalized anxiety disorder (GAD) being a prominent concern. The proliferation of single-person households may exacerbate social isolation and foster loneliness and anxiety. Notably, research investigating the association between single-person households and GAD remains limited. Therefore, this study aimed to investigate the association between single-person households and GAD across sexes in Korea. METHODS: We utilized data from the Korea National Health and Nutrition Examination Survey conducted in 2021 and 2022, comprising a sample of 9936 participants aged 19 or older. The Generalized Anxiety Disorder Screening Tool (GAD-7) was employed to assess anxiety levels in adults. Multiple logistic regression analysis was conducted to investigate the correlation between single-person households and GAD. RESULTS: The reference variable used in the analysis was multi-person households (consisting of two or more individuals). The association between single-person households and GAD was statistically significant across sexes (male: odds ratio [OR]: 1.92, 95 % CI: 1.15-3.20; female: OR: 1.56, 95 % CI: 1.03-2.36). Participants in single-person households exhibited higher scores on the GAD-7 compared with those in multi-person households. Notably, marital status and education level displayed disparate effects based on sex, whereas physical activity demonstrated consistent effects irrespective of sex. LIMITATIONS: Given the use of cross-sectional data, only correlations could be established. CONCLUSION: The findings indicate an elevated risk of GAD in single-person households compared with multi-person households. Furthermore, promoting physical activity emerged as a potential strategy for mitigating GAD in single-person households.

Influence of Gelatin on Adhesion, Proliferation, and Adipogenic Differentiation of Adipose Tissue-Derived Stem Cells Cultured on Soy Protein-Agarose Scaffolds.

Scaffolds play a key role in cultured meat production by providing an optimal environment for efficient cell attachment, growth, and development. This study investigated the effects of gelatin coating on the adhesion, proliferation, and adipogenic differentiation of adipose tissue-derived stem cells (ADSCs) cultured on soy protein-agarose scaffolds. Gelatin-coated scaffolds were prepared using 0.5% and 1.0% (w/v) gelatin solutions. The microstructure, water absorption rate, mechanical strength, cytotoxicity, cell adhesion, proliferation, and differentiation capabilities of the scaffolds were analyzed. Field emission scanning electron microscopy revealed the porous microstructure of the scaffolds, which was suitable for cell growth. Gelatin-coated scaffolds exhibited a significantly higher water absorption rate than that of non-coated scaffolds, indicating increased hydrophilicity. In addition, gelatin coating increased the mechanical strength of the scaffolds. Gelatin coating did not show cytotoxicity but significantly enhanced cell adhesion and proliferation. The gene expression levels of peroxisome proliferator-activated receptor gamma, CCAT/enhancer-binding protein alpha, and fatty acid-binding protein 4 were upregulated, and lipid accumulation was increased by gelatin coating. These findings suggest that gelatin-coated scaffolds provide a supportive microenvironment for ADSC growth and differentiation, highlighting their potential as a strategy for the improvement of cultured meat production and adipose tissue engineering.

Effect of Oligo-Fucoidan, Fucoxanthin, and L-Carnitine on Chronic Kidney Disease in Dogs: A Retrospective Study.

Chronic kidney disease (CKD) commonly occurs in old dogs and cats. Oligo-fucoidan, fucoxanthin, and L-carnitine (OFL) compounds have a variety of reno-protective properties, including anti-inflammatory, anti-oxidative, and anti-fibrotic effects. Because their effects have not been investigated in naturally occurring canine CKD, we examined their reno-protective activities in dog patients with CKD. A total of 50 patients (OFL, n = 28; control, n = 22) were included in the analysis. A significant difference was identified in serum blood urea nitrogen and creatinine concentrations between the control and OFL groups at 6 months. No significant difference in electrolytes was found between the groups. A significant difference was identified in serum creatinine concentration between the control and OFL groups in azotemic (CKD IRIS stage 2-4) at 6 months. The OFL compounds showed a reno-protective effect, consistent with previous animal studies. The OFL combination can potentially delay the progression of canine CKD and be used as an adjuvant therapy.

Pamidronate-induced irreversible symptomatic hypocalcemia in a dog with hypercalcemia after glucocorticoid withdrawal: a case report.

BACKGROUND: Pamidronate is used for the treatment of hypercalcemia. However, a rare but potential adverse event of pamidronate treatment is hypocalcemia. This report describes an unusual case of severe, irreversible hypocalcemia after a single injection of pamidronate for the treatment of hypercalcemia due to glucocorticoid withdrawal in a dog. CASE PRESENTATION: An 11-year-old castrated male Maltese dog presented with anorexia, vomiting, and diarrhea (day 0). The patient had calcinosis cutis throughout the body, calcification of intraabdominal organs, mild azotemia, and severe hypercalcemia. The severe calcification was attributed to long-term glucocorticoid administration, which was discontinued 1 month before presentation. Fluid therapy, diuretics, calcitonin, and a single intravenous injection of pamidronate were used for the treatment of hypercalcemia. On day 14, normocalcemia was achieved, but renal failure occurred. On day 20, severe and irreversible hypocalcemia occurred, and on day 42, the patient was euthanized at the owner's request because of worsened hypocalcemia and renal failure. CONCLUSIONS: Although hypocalcemia is an extremely rare adverse event of bisphosphonate treatment, bisphosphonates like pamidronate can result in potentially life-threatening conditions according to the patient's underlying conditions. Therefore, the patient's condition should be closely monitored and any underlying conditions should be carefully evaluated before initiating the treatment for hypercalcemia using pamidronate.

Neuroprotective and immunomodulatory effects of superoxide dismutase on SH-SY5Y neuroblastoma cells and RAW264.7 macrophages.

Superoxide dismutase (SOD) is an antioxidant enzyme that protects the body from free radicals. It has both antioxidant and immunomodulatory properties, inducing macrophage polarization from M1 to M2. Macrophages, key mediators of the innate immune response, are divided into the M1 (pro-inflammatory) and M2 (anti-inflammatory) subtypes. In this study, we aimed to assess the antioxidant and neuroprotective effects of SOD on nerve cells and its immunomodulatory effects on macrophages. We observed that SOD inhibited the accumulation of reactive oxygen species and enhanced the viability of H2O2-treated nerve cells. Furthermore, SOD reduced the degree of necrosis in nerve cells treated with the conditioned medium from macrophages, which induced inflammation. In addition, SOD promoted the M1 to M2 transition of macrophages. Our findings suggest that SOD protects nerve cells and regulates immune responses.

Assessment of MMP-9 and clinical characteristics in dogs with tracheal collapse based on cough severity and fluoroscopic findings: a cross-sectional study.

BACKGROUND: Tracheal collapse (TC), a common disease in dogs, is characterized by cough; however, little is known about the serum biomarkers that can objectively evaluate the severity of cough in canine TC. Furthermore, studies elucidating the relationship of fluoroscopic characteristics with the severity of cough are lacking. Therefore, this study aimed to evaluate the relationship between cough severity and clinical characteristics, fluoroscopic images, and new serum biomarkers in canine TC. RESULTS: Fifty-one client-owned dogs diagnosed with TC based on fluoroscopic and clinical signs were enrolled in this study and divided into three groups according to the severity of cough (grade of cough: 0, 1, and 2). Signalments, comorbidities, and fluoroscopic characteristics were compared among the groups retrospectively. The serum matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), surfactant protein-A (SP-A), and syndecan-1 (SDC-1) levels were measured in all groups. No significant differences in age, breed, sex, or clinical history were observed among the groups. Concomitant pharyngeal collapse increased significantly with the severity of cough (p = .031). Based on the fluoroscopic characteristics, the TC grade of the carinal region increased significantly and consistently with the grade of cough (p = .03). The serum MMP-9 level was significantly higher in the grade 2 group than that in the grade 0 group (p = .014). The serum IL-6 level was significantly lower in the grade 1 group than that in the grade 0 group (p = .020). The serum SP-A and SDC-1 levels did not differ significantly among the groups. CONCLUSIONS: The severity of cough with the progression of TC can be predicted with the fluoroscopic TC grade at the carinal region. MMP-9 may be used as an objective serum biomarker that represents cough severity to understand the pathogenesis.

In vitro evaluation of the antitumor activity of axitinib in canine mammary gland tumor cell lines.

BACKGROUND: Axitinib, a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor (VEGFR) tyrosine kinase 1,2 and 3, is used in chemotherapy because it inhibits tumor angiogenesis by blocking the VEGF/VEGFR pathway. In veterinary medicine, attempts have been made to apply tyrosine kinase inhibitors with anti-angiogenic effects to tumor patients, but there are no studies on axitinib in canine mammary gland tumors (MGTs). OBJECTIVES: This study aimed to confirm the antitumor activity of axitinib in canine mammary gland cell lines. METHODS: We treated canine MGT cell lines (CIPp and CIPm) with axitinib and conducted CCK, wound healing, apoptosis, and cell cycle assays. Additionally, we evaluated the expression levels of angiogenesis-associated factors, including VEGFs, PDGF-A, FGF-2, and TGF-ß1, using quantitative real-time polymerase chain reaction. Furthermore, we collected canine peripheral blood mononuclear cells (PBMCs), activated them with concanavalin A (ConA) and lipopolysaccharide (LPS), and then treated them with axitinib to investigate changes in viability. RESULTS: When axitinib was administered to CIPp and CIPm, cell viability significantly decreased at 24, 48, and 72 h (p < 0.001), and migration was markedly reduced (6 h, p < 0.05; 12 h, p < 0.005). The apoptosis rate significantly increased (p < 0.01), and the G2/M phase ratio showed a significant increase (p < 0.001). Additionally, there was no significant change in the viability of canine PBMCs treated with LPS and ConA. CONCLUSION: In this study, we confirmed the antitumor activity of axitinib against canine MGT cell lines. Accordingly, we suggest that axitinib can be applied as a new treatment for patients with canine MGTs.

Anti-cancer effects of DHP107 on canine mammary gland cancer examined through in-vitro and in-vivo mouse xenograft models.

BACKGROUND: Canine mammary gland cancer (CMGC) is a common neoplasm in intact bitches. However, the benefit of adjuvant chemotherapy is unclear. The aim of this study was to investigate the anti-proliferative effects of paclitaxel on CMGC in in-vitro and in-vivo settings. RESULTS: Paclitaxel dose-dependently inhibited viability and induced G2/M phase cell cycle arrest and apoptosis in both primary and metastatic CMGC cell lines (CIPp and CIPm). In animal experiments, the average tumour volume decreased significantly in proportion to the administered oral paclitaxel dose. By examining tumour tissue using a TUNEL assay and immunohistochemical staining with anti-CD31 as a marker of endothelial differentiation, respectively, it was confirmed that oral paclitaxel induced apoptosis and exerted an anti-angiogenetic effect in tumour tissues. Further, downregulation of cyclin D1 in tumour tissues suggested that oral paclitaxel induced cell cycle arrest in tumour tissues in-vivo. CONCLUSIONS: Our results suggest that paclitaxel may have anti-cancer effects on CMGC through cell cycle arrest, induction of apoptosis, and anti-angiogenesis. This study could provide a novel approach to treat CMGC.

Anticancer effect of superoxide dismutase on canine mammary gland tumour in vitro.

BACKGROUND: Reactive oxygen species (ROS) have been shown to promote tumour growth and metastasis in human cell lines. The superoxide anion (•O2 - ) is produced during ROS formation and is involved in tumour cell signalling. OBJECTIVES: Superoxide dismutase (SOD) has been applied to canine mammary gland tumours to investigate its antitumour effects in vitro. METHODS: Cell proliferation, cell cycle cell migration assays, reverse transcription-quantitative polymerase chain reaction, and western blot analysis were performed to determine the effects of SOD on canine mammary tumour cell line. RESULTS: SOD treatment resulted in anti-proliferative effects and mediated cell cycle arrest in the canine mammary gland tumour cell lines (CIPp and CIPm). It also downregulated the expression of N-cadherin and Vimentin. CONCLUSIONS: The results confirmed that SOD inhibits tumour cell proliferation and migration, thus supporting the potential applications of SOD as a chemotherapeutic agent for canine mammary gland tumours.

Changes in Urinary Exosomal Nuclear Matrix Protein-22 in Dogs With Urothelial Carcinoma: A Pilot Study.

BACKGROUND/AIM: Nuclear matrix protein-22 (NMP-22) is widely used in human medicine as a prognostic and diagnostic tool for urothelial carcinoma (UC). In addition, the use of urinary exosomes as a liquid biopsy tool is emerging for the diagnosis of certain types of cancer in human medicine. This study aimed to investigate the change in urinary exosomal NMP-22 for the diagnosis of UC in dogs. PATIENTS AND METHODS: Among canine patients who visited the veterinary hospital, urine was collected from those whose owners provided consent. A total of 23 dogs (UC group, n=6; control group, n=17) were included in the analysis. After exosomes were isolated from the urine, NMP-22 was measured using enzyme-linked immunosorbent assay. RESULTS: In the UC group, the expression of NMP-22 in urinary exosomes was significantly higher than that in non-UC groups (p<0.0001). CONCLUSION: NMP-22 is significantly increased in exosomes in the urine of dogs diagnosed with UC, suggesting that urinary exosome NMP-22 can be considered as one of the liquid biopsy tools for diagnosing UC in dogs.

Retrospective evaluation of prognosis and survival with various immunosuppressants in 82 dogs diagnosed with meningoencephalitis of unknown etiology (2010-2021).

BACKGROUND: Meningoencephalomyelitis of unknown etiology (MUE) is a comprehensive term for non-infectious inflammatory brain diseases of the central nervous system (CNS) caused by abnormal autoimmune responses. This study aims to compare the differences in survival and clinical response of MUE according to the adjuvant immunosuppressant use. Medical records of 82 dogs diagnosed with MUE were reviewed retrospectively. RESULTS: The overall survival time was 769 days (range 14-2687 days). The median survival time for each adjunctive was: leflunomide 1035 days (range 126-2163 days), mycophenolate mofetil 865 days (range 39-2191 days), cyclosporin 441 days (range 11-2176 days), cytosine arabinoside 754 days (range 6-1898 days) and a combination of mycophenolate mofetil and cytosine arabinoside 132 days (range 23-1227 days). There was no significant difference in the incidence rate of adverse events according to the immunosuppressants, but moderate to severe anemia was confirmed in 3 patients (18.7%) in the leflunomide group. CONCLUSIONS: The survival time and response rate of MUE dogs differed depending on which adjunctive immunosuppressants were used. Leflunomide showed a long survival time and a relatively good response rate in dogs with MUE. However, a large-scale further study with standardized doses of immunosuppressants and supportive treatment and constant monitoring interval is needed.

Anti-tumor Effect of Activated Canine B Cells With Interleukin-21 and Anti-B Cell Receptor.

BACKGROUND/AIM: Recently, novel studies on the pivotal role of B cells in the tumor-microenvironment and anti-tumor immunity have been conducted. Additionally, Interleukin-21 (IL-21) and anti-B cell receptor (BCR) have been reported to stimulate B cells to secrete granzyme B, which exhibits cytotoxic effects on tumor cells. However, the direct anti-tumor effect of B cells is not yet fully understood in the veterinary field. This study is the first attempt in veterinary medicine to identify the immediate effect of B cells on tumor suppression and the underlying mechanisms involved. MATERIALS AND METHODS: Canine B cells were isolated from peripheral blood and activated with IL-21 and anti-B cell receptor (BCR). The canine leukemia cell line GL-1 was co-cultured with B cells, and the anti-tumor effect was confirmed by assessing the changes in cell viability and apoptotic ratio. RESULTS: When B cells were activated with IL-21 and anti-BCR, the secretion of granzyme B and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increased. Simultaneously, the viability of GL-1 cells decreased, and the apoptotic ratio increased, particularly when co-cultured with activated B cells. CONCLUSION: The results demonstrated the direct anti-tumor effect of granzyme B-and TRAIL and its enhanced potential of B cells to inhibit tumor cell growth after activation with IL-21 and anti-BCR. This study is the first study dealing with immunomodulation in the canine tumor micro-environment.

Amelioration of DSS-induced colitis in mice by TNF-α-stimulated mesenchymal stem cells derived from feline adipose tissue via COX-2/PGE2 activation.

BACKGROUND: Mesenchymal stem cells (MSCs) have been investigated as therapeutic agents for inflammatory bowel disease (IBD). Stimulation of MSCs with pro-inflammatory cytokines is an approach to enhance their immunomodulatory effects. However, further investigation is required to support their application in immune-mediated disorders and companion animals. OBJECTIVES: This study aimed to assess the therapeutic effect of tumor necrosis factor (TNF)-α-stimulated feline adipose tissue-derived MSCs (fAT-MSCs) in a dextran sulfate sodium (DSS)-induced colitis mouse model. METHODS: Colitis mice was made by drinking water with 3% DSS and fAT-MSCs were injected intraperitoneally. Colons were collected on day 10. The severity of the disease was evaluated and compared. Raw 264.7 cells were cultured with the conditioned medium to determine the mechanism, using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: TNF-α-stimulated fAT-MSCs more improved severity of DSS-induced colitis in disease activity, colon length, histologic score, and inflammatory cytokine. In sectionized colon tissues, the group comprising TNF-α-stimulated fAT-MSCs had higher proportion of CD11b+CD206+ macrophages than in the other groups. In vitro, TNF-α-stimulation increased cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) secretion from fAT-MSCs. The conditioned medium from TNF-α-stimulated fAT-MSCs enhanced the expression of interleukin-10 and arginase-1 in LPS-activated Raw 264.7 cells. CONCLUSIONS: These results represent that TNF-α-stimulated fat-mscs ameliorate the inflamed colon more effectively. Furthermore, we demonstrated that the effectiveness was interlinked with the COX-2/PGE2 pathway.

Macrophage induces anti-cancer drug resistance in canine mammary gland tumor spheroid.

Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment by producing cytokines and growth factors. Furthermore, TAMs play multifunctional roles in tumor progression, immune regulation, metastasis, angiogenesis, and chemoresistance. Hypoxia in the tumor microenvironment induces tumor-supporting transformation of TAMs, which enhances tumor malignancy through developing anti-cancer resistance, for example. In this study, a hybrid spheroid model of canine mammary gland tumor (MGT) cell lines (CIPp and CIPm) and canine macrophages (DH82) was established. The effects of hypoxia induced by the spheroid culture system on the anti-cancer drug resistance of canine MGT cells were investigated. A hybrid spheroid was created using an ultralow-adhesion plate. The interactions between canine MGT cells and DH82 were investigated using a co-culture method. When co-cultured with DH82, cell viability and expression levels of tumor growth factors and multi-drug resistance genes were increased in canine MGT cells under doxorubicin. Additionally, doxorubicin-induced apoptosis and G2/M cell cycle arrest were attenuated in canine MGT cells co-cultured with DH82. In conclusion, the hybrid spheroid model established in this study reflects the hypoxic TME, allowing DH82 to induce anti-cancer drug resistance in canine MGT cells.