Big Insulin-like Growth Factor 2-Producing Tumor in a Hypoglycemic Dog.

A 10-year-old female Papillon dog that had previously developed a mammary tumor was admitted for treatment of a hypoglycemic attack. Blood examination showed severe hypoglycemia and decreased blood insulin concentration. Computed tomography indicated multiple tumors in the cranial and caudal lobes of the right lung. These tumors were resected surgically and diagnosed as pulmonary adenocarcinomas by histopathologic examination. Hypoglycemia was temporarily improved after the resection, but a hypoglycemic event occurred 2 months after the surgery. Immunohistochemistry of the tumor demonstrated the expression of insulin-like growth factor 2 in tumor cells. Western blot analysis revealed the expression of high-molecular-weight (big)-insulin-like growth factor 2 in the tumor region. Insulin-like growth factor 2 mRNA expression was also confirmed in the tumor using reverse transcription-polymerase chain reaction. These findings indicate the diagnosis of non-islet cell tumor-induced hypoglycemia caused by big-insulin-like growth factor 2 produced by the tumor in the dog. This report provides information on differentiating tumors that cause paraneoplastic hypoglycemia.

Nimustine Treatment of 11 Cases of Canine Histiocytic Sarcoma.

The objective of this retrospective study was to report treatment outcomes in dogs with histiocytic sarcoma (HS) that were treated with nimustine (ACNU). This study evaluated data from 11 dogs including 5 with macroscopic tumors that were treated in the primary setting and 6 that underwent aggressive local therapy while being treated in the adjuvant setting. The median ACNU starting dose was 25 mg/m2 (range, 20-30 mg/m2; 3- to 5-wk intervals, 1-8 administrations). The median overall survival in the primary and adjuvant settings was 120 days (median progression-free survival [PFS], 63 days) and 400 days (median PFS, 212 days), respectively. Neutropenia was observed in eight cases (grade 1, n = 1; grade 2, n = 2; grade 3, n = 2; grade 4, n = 3) with nadir neutrophil count at 1 wk after ACNU administration. Mild gastrointestinal toxicity (grade 1-2) was observed in three cases. ACNU was well tolerated and showed a similar outcome to that seen for lomustine, which is a drug commonly used to treat canine HS, in terms of overall survival and PFS in the current study population. Further investigations will need to be undertaken to definitively determine if ACNU is an appropriate alternative to lomustine for the treatment of HS.

Expression and Roles of S100A4 in Anaplastic Cells of Canine Mammary Carcinomas.

S100A4 (metastasin), a member of the S100 protein family, was initially identified in metastatic cells and is well established as a marker of aggressive human cancer. However, expression and roles of S100A4 in canine mammary tumors have not been clarified. In this study, expression of S100A4 was examined immunohistochemically in normal, hyperplastic, and neoplastic mammary glands of dogs. In all normal and benign lesions, S100A4 was restricted to a few stromal fibroblasts and inflammatory cells. However, in 7 of 57 (12%) of the malignant tumors examined, cytoplasmic and nuclear expression of S100A4 was observed in epithelial tumor cells and stromal cells. Particularly, the frequency of S100A4-positive anaplastic carcinomas was high (4/8 cases, 50%). Next, we established a novel cell line, named NV-CML, from a S100A4-positive canine mammary carcinoma. The cultured NV-CML cells and the tumors that developed in the immunodeficient mice after subcutaneous injection of the cells maintained the immunophenotype of the original tumor, including S100A4 expression. Using this cell line, we examined the cellular functions of S100A4 using RNA interference. S100A4 expression level in NV-CML cells transfected with small interfering RNA (siRNA) targeting canine S100A4 (siS100A4) was reduced to about one-fifth of those with negative-control siRNA (siNeg). Cell proliferation in WST-8 assay and cell migration in Boyden chamber assay were significantly decreased in siS100A4-transfected cells compared with siNeg-transfected cells. These findings suggest that S100A4 may be related to progression of canine mammary carcinomas via its influence on cell growth and motility.

Accumulation of Laforin and Other Related Proteins in Canine Lafora Disease With EPM2B Repeat Expansion.

Canine Lafora disease (LD) is an autosomal recessive genetic disorder causing nonfatal structural epilepsy, mainly affecting miniature wirehaired dachshunds. Repeat expansion in the EPM2B gene causes a functional impairment of the ubiquitin ligase malin which regulates glycogen metabolism. Abnormally structured glycogen accumulates and develop polyglucosan bodies predominantly in the central nervous system. The authors performed a comprehensive clinical, genetic, and pathological study of 4 LD cases affecting miniature wirehaired dachshund dogs with EPM2B repeat expansions, with systemic distribution of polyglucosan bodies and accumulation of laforin and other functionally associated proteins in the polyglucosan bodies. Myoclonic seizures first appeared at 7-9 years of age, and the dogs died at 14-16 years of age. Immunohistochemistry for calbindin revealed that the polyglucosan bodies were located in the cell bodies and dendritic processes of Purkinje cells. Polyglucosan bodies were also positive for laforin, hsp70, α/ß-synuclein, ubiquitin, LC3, and p62. Laforin-positive polyglucosan bodies were located in neurofilament-positive neurons but not in GFAP-positive astrocytes. In nonneural tissues, periodic acid-Schiff (PAS)-positive polyglucosan bodies were observed in the heart, skeletal muscle, liver, apocrine sweat gland, and smooth muscle layer of the urinary bladder. In the skeletal muscle, polyglucosan bodies were observed only in type 1 fibers and not in type 2 fibers. The results indicate that although the repeat expansion of the EPM2B gene is specific to dogs, the immunohistochemical properties of polyglucosan body in canine LD are comparable to human LD. However, important phenotypic variations exist between the 2 species including the affected skeletal muscle fiber type.

Canine REIC/Dkk-3 interacts with SGTA and restores androgen receptor signalling in androgen-independent prostate cancer cell lines.

BACKGROUND: The pathological condition of canine prostate cancer resembles that of human androgen-independent prostate cancer. Both canine and human androgen receptor (AR) signalling are inhibited by overexpression of the dimerized co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), which is considered to cause the development of androgen-independency. Reduced expression in immortalised cells (REIC/Dkk-3) interferes with SGTA dimerization and rescues AR signalling. This study aimed to assess the effects of REIC/Dkk-3 and SGTA interactions on AR signalling in the canine androgen-independent prostate cancer cell line CHP-1. RESULTS: Mammalian two-hybrid and Halo-tagged pull-down assays showed that canine REIC/Dkk-3 interacted with SGTA and interfered with SGTA dimerization. Additionally, reporter assays revealed that canine REIC/Dkk-3 restored AR signalling in both human and canine androgen-independent prostate cancer cells. Therefore, we confirmed the interaction between canine SGTA and REIC/Dkk-3, as well as their role in AR signalling. CONCLUSIONS: Our results suggest that this interaction might contribute to the development of a novel strategy for androgen-independent prostate cancer treatment. Moreover, we established the canine androgen-independent prostate cancer model as a suitable animal model for the study of this type of treatment-refractory human cancer.

Measurement of transepidermal water loss (TEWL) in cats with experimental skin barrier dysfunction using a closed chamber system.

BACKGROUND: A closed chamber evaporimeter is suitable for measuring transepidermal water loss (TEWL) in cats because of the compact device size, tolerance to sudden movement and short measuring time. TEWL is a representative parameter for skin barrier dysfunction, which is one of the clinical signs of atopic dermatitis in humans and dogs. Measurement of feline TEWL has been reported, but applicability of this parameter has not been validated. HYPOTHESIS/OBJECTIVES: The aims of this study were to determine if tape stripping is a valid experimental model in cats for studying TEWL and to determine if a closed chambered system is a suitable measurement tool for cats. ANIMALS: Ten clinically normal cats. METHODS: In order to evaluate variation of the measured values, TEWL was measured at the right and left side of the three clipped regions (axillae, lateral thigh and groin). Subsequently, TEWL was measured using sequential tape stripping of the stratum corneum as a model of acute barrier disruption. RESULTS: The variations between both sides of the three regions showed no significant difference. Sequential tape stripping was associated with increasing values for TEWL. CONCLUSIONS AND CLINICAL IMPORTANCE: Feline TEWL was shown to reflect changes in the skin barrier in an experimental model using a closed chamber system and has the potential for evaluating skin barrier function in cats with skin diseases.

The modified corneocyte surface area measurement as an index of epidermal barrier properties: inverse correlation with transepidermal water loss.

BACKGROUND: Corneocyte surface area (CSA) is as established parameter for skin barrier function in humans. Measurement of canine CSA has been previously reported but has not been validated. HYPOTHESIS/OBJECTIVES: The aim of this study was to evaluate the validity of CSA as a barrier function parameter in dogs. ANIMALS: Six clinically normal beagle dogs. METHODS: CSA was measured and compared with transepidermal water loss (TEWL) using sequential tape stripping of the stratum corneum as a model of acute barrier disruption. Then, CSA and TEWL were measured at four anatomical sites (groin, lower back, nasal bridge and pinna). The correlation between the two indices was also evaluated. RESULTS: From the results of sequential tape stripping, CSA values gradually decreased with increasing number of tape strippings. The CSA values were inversely correlated with the TEWL ones. The two indices at different sites were variable and were strongly correlated. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine CSA was demonstrated to be a useful parameter for the canine skin barrier function. The results from the anatomical sites imply that the cephalic sites (nasal bridge and pinna) were lower than others in skin barrier function.

Meningoencephalitis with malacia caused by Sarcocystis calchasi in a rock pigeon in Japan.

Sarcocystis spp. cause pigeon protozoan encephalitis, a neuronal disease. A female pigeon exhibiting torticollis had a necrotic area in the cerebral hemisphere surrounded by lesions with perivascular cuffing, gliosis, granulomatous foci, and meningitis. Non-necrotic lesions were also observed in the brainstem. Intact and degenerative schizonts were observed within the neuropils and neurons in the lesions. Deoxyribonucleic acid (DNA) was extracted from paraffin-embedded brain tissues and genetically analyzed after gel electrophoresis to determine Sarcocystis spp. using specific primer sets for 28S ribosomal ribonucleic acid and internal transcribed spacer region-1. DNA sequencing confirmed a significant homology with S. calchasi. This is the first report of meningoencephalitis with malacia caused by S. calchasi in a rock pigeon in Japan.

Antitumor effect of bevacizumab in a xenograft model of canine hemangiopericytoma.

Canine hemangiopericytoma (CHP) is characterized by frequent local recurrence and increased invasiveness. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in tumors. The aim of the present study was to investigate the effect of a single dose of bevacizumab on a xenograft model of CHP. VEGF protein was secreted from cultured CHP cells and interacted with bevacizumab. Bevacizumab treatment suppressed tumor growth by inhibiting tumor angiogenesis, whereas no significant differences were observed in the proliferation index and apoptosis rates of treated and untreated mice. Thus, bevacizumab had antitumor effects in a xenograft model of CHP.

Transepidermal water loss in cats: comparison of three differently clipped sites to assess the influence of hair coat on transepidermal water loss values.

BACKGROUND: The measurement of transepidermal water loss (TEWL) is one of the parameters that can be used to assess skin barrier function. The variability and reliability of TEWL measurements in dogs have been controversial, and the hair coat has been considered as one of the factors that may cause variation of TEWL values. HYPOTHESIS/OBJECTIVES: The aims of the study were to establish a suitable procedure for measuring feline TEWL, to evaluate the influence of hair coat on TEWL measurements and to assess variations of TEWL at different anatomical sites. METHODS: Transepidermal water loss was measured using a closed-chamber evaporimeter, the VapoMeter(®). We compared three adjacent sites in the groin area of 10 clinically normal, domestic short hair cats. One site was unclipped, the second was trimmed with scissors and the third was shaved using electric clippers. Values of TEWL were obtained for 48 h after trimming with scissors and clippers. Five sites were clipped (upper back, lumbar back, lateral thigh, axillae and groin), and the TEWL was measured. RESULTS: The mean and SD of TEWL values of the clipper-trimmed site were the smallest, followed in order by the site trimmed with scissors and the unclipped site. The TEWL values were statistically constant in the clipper-trimmed site, while the values in the unclipped sites were not. There was no statistically significant difference in TEWL values between all of the anatomical sites except for the axillae. CONCLUSIONS AND CLINICAL IMPORTANCE: Hair clipping of sites with electric clippers is recommended for TEWL measurement in cats.

Multiple intra-abdominal fungal granulomas caused by Scedosporium apiospermum effectively treated with voriconazole in a Golden Retriever.

Scedosporium apiospermum is a saprophytic filamentous fungus that is pathogenic to dogs. This report describes a case of S. apiospermum infection that caused multiple large peritoneal fungal granulomas in a dog with a history of jejunojejunostomy. The lesions were firmly attached to multiple organs and could not be surgically removed. In such cases, no precedent for the response to the treatment of this disease exists, and all affected dogs have died. This is the first report of an effective medical treatment for multiple intra-abdominal fungal granulomas using voriconazole.

mTOR pathway as a potential therapeutic target for cancer stem cells in canine mammary carcinoma.

Mammary adenocarcinoma, the most common cancer in female dogs, often exhibits the lymph node and lung metastases and has a higher mortality rate. However, mammary adenocarcinoma has no established treatment, except early surgical excision. Canine mammary carcinoma has many common features with human mammary carcinoma, including clinical characteristics, heterogeneity, and genetic aberrations, making it an excellent spontaneous tumor model for human breast cancer. Diverse cancers comprised heterogeneous cell populations originating from cancer stem cells (CSCs) with self-renewal ability. Therefore, in addition to conventional therapy, therapeutic strategies targeting CSCs are essential for cancer eradication. The present study aimed to extract inhibitors of canine mammary CSCs that suppress their self-renewal ability. Sphere-formation assay, which evaluates self-renewal ability, was performed for the canine mammary cancer cell lines CTBp and CNMp. The spheres formed in this assay were used in inhibitor library screening, which identified various signaling pathways such as proteosome, stress inducer, and mammalian target of rapamycin (mTOR). The present study focused on the mTOR signaling pathway. Western blotting showed higher levels of phosphorylated mTOR in sphere-forming CTBp and CNMp cells than in adherent cells. Drug sensitivity examination using the mTOR inhibitors everolimus and temsirolimus revealed dose-dependent reductions in viability among both sphere-forming cells and adherent cells. Expression of phosphorylated mTOR in adherent and sphere-forming cells decreased by everolimus and temsirolimus treatment. In mice transplanted with CTBp-derived spheres, everolimus treatment significantly decreased tumor volume compared to control. These results reveal that the mTOR signaling pathway may be a potential to be a therapeutic target in both cancer cells and CSCs. Novel therapeutic strategies for canine mammary carcinoma are expected to benefit to human breast carcinoma as well.

Pulmonary solid adenocarcinoma in a dog.

A 12-year-old castrated male Beagle dog presented with a 1-month history of progressive loss of appetite and cough. One month after the initial visit, a detailed clinical examination was performed due to weight loss and persistent cough. Computed tomography demonstrated diffuse opacification of the entire right lung and cranial lobe of the left lung. Samples of the pulmonary lesions obtained by fine-needle aspiration (FNA) were highly cellular with scattered and clustered foci of large round cells, suggestive of a round cell tumour. Ten days after the FNA, the dog was euthanized due to decreased activity and severe respiratory symptoms. At necropsy, enlargement of the entire right lung and cranial lobe of the left lung was seen. The external and cut surfaces of the lungs were homogeneously grey-white. Histopathological examination of sections of the right lung and the cranial lobe of the left lung revealed proliferation of large round or polygonal neoplastic cells arranged in nests of variable size separated by a thin fibrous stroma. Neoplastic cells were immunopositive for cytokeratin and thyroid transcription factor-1 but negative for vimentin, CD204, chromogranin A and synaptophysin. On the basis of these findings, the tumour was diagnosed as pulmonary solid adenocarcinoma.

A case of a dog with paragonimiasis after consumption of raw deer meat.

A 6-year-old castrated male Cavalier King Charles Spaniel was referred to the Animal Medical Center, Tokyo University of Agriculture and Technology, for examination and treatment of recurrent pneumothorax. Chest radiography and computed tomography showed multiple cavitary lesions in the caudal right posterior lobe. These lesions were surgically excised via thoracotomy. Subsequent histopathological examination revealed paragonimiasis. In the postoperative review, we found that the owner had fed raw deer meat to the dog four months earlier. Deer meat has attracted attention as a source of Paragonimus in humans. To our knowledge, this is the first report of Paragonimus infection in a dog due to deer meat consumption.

Establishment of an experimental model of canine malignant mesothelioma organoid culture using a three-dimensional culture method.

Canine malignant mesothelioma (cMM) is a rare and drug-resistant malignant tumor. Due to few patients and experimental models, there have not been enough studies to demonstrate the pathogenesis of the disease and novel effective treatment for cMM. Since cMM resembles human MM (hMM) in histopathological characteristics, it is also considered a promising research model of hMM. Compared with conventional 2-dimensional (2D) culture methods, 3-dimensional (3D) organoid culture can recapitulate the properties of original tumor tissues. However, cMM organoids have never been developed. In the present study, we for the first time generated cMM organoids using the pleural effusion samples. Organoids from individual MM dogs were successfully generated. They exhibited the characteristics of MM and expressed mesothelial cell markers, such as WT-1 and mesothelin. The sensitivity to anti-cancer drugs was different in each strain of cMM organoids. RNA sequencing analysis showed cell adhesion molecule pathways were specifically upregulated in cMM organoids compared with their corresponding 2D cultured cells. Among these genes, the expression level of E-cadherin was drastically higher in the organoids than that in the 2D cells. In conclusion, our established cMM organoids might become a new experimental tool to provide new insights into canine and human MM therapy.

Derivation of a new model of lung adenocarcinoma using canine lung cancer organoids for translational research in pulmonary medicine.

Canine primary lung cancer (cPLC) is a rare malignant tumor in dogs, and exhibits poor prognosis. Effective therapeutic drugs against cPLC have not been established yet. Also, cPLC resembles human lung cancer in histopathological characteristics and gene expression profiles and thus could be an important research model for this disease. Three-dimensional organoid culture is known to recapitulate the tissue dynamics in vivo. We, therefore, tried to generate cPLC organoids (cPLCO) for analyzing the profiles of cPLC. After samples from cPLC and the corresponding normal lung tissue were collected, cPLCO were successfully generated, which recapitulated the tissue architecture of cPLC, expressed lung adenocarcinoma marker (TTF1), and exhibited tumorigenesis in vivo. The sensitivity of cPLCO to anti-cancer drugs was different among strains. RNA-sequencing analysis showed significantly upregulated 11 genes in cPLCO compared with canine normal lung organoids (cNLO). Moreover, cPLCO were enriched with the MEK-signaling pathway compared with cNLO. The MEK inhibitor, trametinib decreased the viability of several strains of cPLCO and inhibited the growth of cPLC xenografts. Collectively, our established cPLCO model might be a useful tool for identifying novel biomarkers for cPLC and a new research model for dog and human lung cancer.

A canine case of splenic hemangiosarcoma complicated with infectious splenic abscess.

A 12-year-old spayed female Yorkshire Terrier presented with anorexia, and an intra-abdominal mass was examined at the Animal Medical Center, Tokyo University of Agriculture and Technology, Tokyo, Japan. Abdominal ultrasonography revealed that the mass originated from the spleen. Total splenectomy was performed the following day, where evidence of past rupture of the splenic mass and a small amount of purulent ascites were found. No gastrointestinal perforation or abscess formation in other tissues was noted during the surgery. Intra-operative cytological examination of the ascites revealed a large number of degenerated neutrophils and macrophages phagocytosing cocci. The splenic mass was histopathologically diagnosed as a splenic abscess, concurrent with a splenic hemangiosarcoma. Bacterial culture examination of the ascites identified Staphylococcus schleiferi. Although surgical management had been successful, the dog eventually developed clinical signs suggestive of intra-abdominal hemorrhage attributable to hemangiosarcoma approximately five months after surgery. This clinical report describes that splenic hemangiosarcoma might have created a milieu favorable for the development of splenic abscess.

Primary pulmonary diffuse large B-cell lymphoma associated with feline leukaemia virus infection in a young cat.

CASE SUMMARY: A 4-year-old castrated male domestic shorthair cat with a continuous cough was brought to a private veterinary clinic for detailed examination. Radiography of the thoracic cavity revealed a severe radiopaque region in the caudal lobe of the right lung. At 108 days after the initial visit, CT showed a mass of 27 × 23 × 18 mm in the caudal lobe of the right lung. At that time, no abnormalities in other organs except for the lung were detected on CT and peripheral blood and blood biochemistry tests. The mass in the caudal lobe of the right lung was resected by lobectomy; it had a white surface and was firm. Histopathologically, the mass was non-encapsulated, showing an unclear boundary with surrounding tissues. The mass comprised large, round or polygonal neoplastic cells arranged in a diffuse pattern. Immunohistochemically, neoplastic cells were diffusely positive for CD20, feline leukaemia virus (FeLV) p27 and FeLV glycoprotein 70 but negative for CD3, CD204 and E-cadherin. Based on these findings, diffuse large B-cell lymphoma associated with FeLV infection was diagnosed. Although the cat showed no clinical signs of gastrointestinal or respiratory injury, a routine ultrasonography revealed thickening in the jejunum wall 196 days after lobectomy, and subsequent fine-needle aspiration examination confirmed high-grade lymphoma. RELEVANCE AND NOVEL INFORMATION: This is the first report of primary pulmonary diffuse large B-cell lymphoma associated with FeLV infection in a young cat.

In vitro anticancer effects of alpelisib against PIK3CA­mutated canine hemangiosarcoma cell lines.

Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome­wide analysis of canine cases of HSA. The present cloned the full­length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA­mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.

Establishment of an experimental model of normal dog bladder organoid using a three-dimensional culture method.

Dog bladder cancer (BC) is mostly muscle-invasive (MI) with poor prognosis, and its pathogenesis is close to human MIBC. Three-dimensional (3D) organoid culture ensures novel knowledge on cancer diseases including BC. Recently, we have established dog BC organoids (BCO) using their urine samples. BCO recapitulated the epithelial structures, characteristics, and drug sensitivity of BC-diseased dogs. However, organoids from dog normal bladder epithelium are not established yet. Therefore, the present study aimed to establish dog normal bladder organoids (NBO) for further understanding the pathogenesis of dog BC and human MIBC. The established NBO underwent various analyzes including cell marker expressions, histopathological structures, cancer-related gene expression patterns, and drug sensitivity. NBO could be produced non-invasively with a continuous culturing and recapitulated the structures and characteristics of the dog's normal bladder mucosal tissues. Different drug sensitivities were observed in each NBO. The analysis of RNA sequencing revealed that several novel genes were changed in NBO compared with BCO. NBO showed a higher expression of p53 and E-cadherin, but a lower expression of MDM2 and Twist1 compared with BCO. These results suggest that NBO could be a promising experimental 3D model for studying the developmental mechanisms of dog BC and human MIBC.