Invasive Front Grading and Epithelial-Mesenchymal Transition in Canine Oral and Cutaneous Squamous Cell Carcinomas.

Oral and cutaneous tissues are the most frequent origin in canine squamous cell carcinoma (SSC). In SCC, changes in adhesion molecule expression and transition from epithelial to mesenchymal phenotype are thought to be important in development of invasive behavior of neoplastic cells at the leading front of the tumor. We therefore investigated histological invasive front grading and epithelial-mesenchymal transition (EMT) in both oral SCCs and cutaneous SCCs. EMT was assessed by evaluating immunohistochemical expression of E-cadherin, ß-catenin, desmoglein, vimentin, and N-cadherin. Regardless of the anatomic location, invasive front grading resulted in higher histological grades than grading of the surface. Most oral SCCs were of significantly higher histologic grade than cutaneous SCCs ( P < .01). Expression of E-cadherin, ß-catenin, and desmoglein was significantly lower in oral SCC compared with cutaneous SCC ( P < .01). A significant association was found between invasive front grading and loss of E-cadherin, ß-catenin, and desmoglein ( P < .01). Also, vimentin-positive neoplastic cells had low immunoreactivity of these adhesion molecules, and a few of these neoplastic cells were positive for N-cadherin. These results suggest not only E-cadherin and ß-catenin but also desmoglein as markers for predicting biological behavior of canine SCC. Depending on their primary sites, EMT correlates with biological behavior and therefore histological grade of canine SCC. We suggest that combining invasive front grading with assessment of immunohistochemical expression of E-cadherin, ß-catenin, and desmoglein may allow more accurate prediction of biological behavior of canine SCCs.

Immunohistochemical expression of p63, Ki67 and ß-catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder.

Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or ß-catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and ß-catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.

Significance of caveolin-1 and matrix metalloproteinase 14 gene expression in canine mammary tumours.

Canine mammary tumours (CMTs) are the most common neoplasms affecting female dogs. There is an urgent need for molecular biomarkers that can detect early stages of the disease in order to improve accuracy of CMT diagnosis. The aim of this study was to examine whether caveolin-1 (Cav-1) and matrix metalloproteinase 14 (MMP14) are associated with CMT histological malignancy and invasion. Sixty-five benign and malignant CMT samples and six normal canine mammary glands were analysed using quantitative reverse transcription-polymerase chain reaction. Cav-1 and MMP14 genes were highly expressed in CMT tissues compared to normal tissues. Cav-1 especially was overexpressed in malignant and invasive CMT tissues. When a CMT cell line was cultured on fluorescent gelatin-coated coverslips, localisation of Cav-1 was observed at invadopodia-mediated degradation sites of the gelatin matrix. These findings suggest that Cav-1 may be involved in CMT invasion and that the markers may be useful for estimating CMT malignancy.

Diversity of Histologic Patterns and Expression of Cytoskeletal Proteins in Canine Skeletal Osteosarcoma.

Osteosarcoma (OS), the most common bone tumor, includes OS of the head (OSH) and appendicular OS (OSA). In dogs, it is classified into 6 histologic subtypes: osteoblastic, chondroblastic, fibroblastic, telangiectatic, giant cell, and poorly differentiated. This study investigated the significance of the histologic classification relevant to clinical outcome and the histologic and immunohistochemical relationships between pleomorphism and expression of cytoskeletal proteins in 60 cases each of OSH and OSA. Most neoplasms exhibited histologic diversity, and 64% of OS contained multiple subtypes. In addition to the above 6 subtypes, myxoid, round cell, and epithelioid subtypes were observed. Although the epithelioid subtypes were observed in only OSH, no significant difference in the frequency of other subtypes was observed. Also, no significant relevance was observed between the clinical outcome and histologic subtypes. Cytokeratin (CK) was expressed in both epithelioid and sarcomatoid tumor cells in various subtypes, and all CK-positive tumor cells also expressed vimentin. Vimentin and α-smooth muscle actin (SMA) were expressed in all subtypes. A few SMA-positive spindle-shaped tumor cells exhibited desmin expression. Glial fibrillary acidic protein-positive tumor cells were observed in many subtypes, and some of these cells showed neurofilament expression. Although OSH exhibited significantly stronger immunoreactivity for SMA than OSA, no significant difference in other cytoskeletal proteins was observed. Some tumor cells had cytoskeletal protein expression compatible with the corresponding histologic subtypes, such as CK in the epithelioid subtype and SMA in the fibroblastic subtype. Thus, canine skeletal OS is composed of pleomorphic and heterogenous tumor cells as is reflected in the diversity of histologic patterns and expression of cytoskeletal proteins.

Expression of monocarboxylate transporter 1 in oral and ocular canine melanocytic tumors.

Solid tumors are composed of a heterogeneous population of cells surviving in various concentrations of oxygen. In a hypoxic environment, tumor cells generally up-regulate glycolysis and, therefore, generate more lactate that must be expelled from the cell through proton transporters to prevent intracellular acidosis. Monocarboxylate transporter 1 (MCT1) is a major proton transporter in mammalian cells that transports monocarboxylates, such as lactate and pyruvate, together with a proton across the plasma membrane. Melanocytic neoplasia occurs frequently in dogs, but the prognosis is highly site-dependent. In this study, 50 oral canine melanomas, which were subdivided into 3 histologic subtypes, and 17 ocular canine melanocytic neoplasms (14 melanocytomas and 3 melanomas) were used to examine and compare MCT1 expression. Immunohistochemistry using a polyclonal chicken anti-rat MCT1 antibody showed that most oral melanoma exhibited cell membrane staining, although there were no significant differences observed among the 3 histologic subtypes. In contrast, the majority of ocular melanocytic tumors were not immunoreactive. Additionally, we documented the presence of a 45-kDa band in cell membrane protein Western blots, and sequencing of a reverse transcriptase polymerase chain reaction band of expected size confirmed its identity as a partial canine MCT1 transcript in 3 oral tumors. Increased MCT1 expression in oral melanomas compared with ocular melanocytic tumors may reflect the very different biology between these tumors in dogs. These results are the first to document canine MCT1 expression in canine tumors and suggest that increased MCT1 expression may provide a potential therapeutic target for oral melanoma.

Clinical pharmacokinetics of anti-angiogenic photodynamic therapy with benzoporphyrin derivative monoacid ring-A in dogs having naturally occurring neoplasms.

The aim of this study was to examine the pharmacokinetics of clinically applied benzoporphyrin derivative monoacid ring-A (BPD-MA; Verteporfin), a second-generation photosensitizer, during a trial of photodynamic therapy (PDT) in nine dogs having naturally occurring neoplasms. After injecting BPD-MA at 0.5 mg/kg intravenously, its mean half-life (t1/2) was found to be 8.14 +/- 5.34 h, mean clearance (Cl) 35.13 +/- 9.62 ml/(h kg), the mean value of the volume of distribution (Vc) 0.08 +/- 0.01 l/kg and the mean steady state volume of distribution (Vss) 0.38 +/- 0.31 l/kg respectively. With the exception of a transitional increase in serum alkaline phosphatase activity, no other clinical abnormalities were observed. The t1/2 in dogs with naturally occurring tumours was longer than that in humans, but similar to that in rats. The values of Cl and Vss in dogs having naturally occurring neoplasms were lower than those in humans. It is suggested that the pharmacokinetics of BPD-MA in tumour-bearing dogs would be helpful in determining the protocol of a short drug-light interval PDT with BPD-MA that mainly targets the tumour vasculature.

Urethral transitional cell carcinoma in a cat.

A 15-year-old, male neutered cat was referred for investigation of dysuria. A retrograde urethrography was performed which showed two space-occupying masses within the lumen of the mid-to-proximal urethra. Exploratory coeliotomy revealed two urethral masses. Segmental urethrectomy was performed to resect the mass, and the lower urinary tract was reconstructed by vesico-urethral anastomosis. Histopathology showed the mass to be a transitional cell carcinoma with incomplete surgical margins. Tumour regrowth was suspected when dysuria was found approximately 318 days after surgery. Clinical signs were palliated by radiation using weekly fractions of 6 Gy for three weeks. The cat died of unknown causes 386 days postoperatively.

Neuroendocrine carcinoma in the nasal cavity of ten dogs.

Neuroendocrine (NE) carcinoma was diagnosed in 10 dogs. In six cases examined by cephalometric radiography and computerized tomography, a large mass was seen to fill the nasal cavity. Histopathologically, sheets, nests or ribbons of neoplastic cells were separated by delicate or thick fibrovascular stroma. The neoplastic cells were round, oval, or spindle-shaped; cytoplasmic granules and hyperchromatic nuclei with prominent nucleoli were present. Neoplastic cells were invariably immunohistochemically positive for cytokeratin (CK) AE1/AE3, neuron-specific enolase, chromogranin A and vasoactive intestinal polypeptide. Eight dogs were positive for S100 protein, seven for synaptophysin, five for protein gene product 9.5, two for somatostatin, and one for Leu-7. Immunolabelling gave negative results for CK 8, CK 19, calcitonin, calcitonin gene-related polypeptide, neurofilaments, serotonin, gastrin and glial fibrillary acidic protein. Ultrastructurally, the neoplastic cells contained a large number of round, membrane-bounded, densely-cored granules corresponding to neurosecretory granules. These observations were consistent with the neuroendocrine nature of the carcinomas.

Canine ganglioneuroblastoma in the oral mucosa.

A ganglioneuroblastoma of the oral cavity in a dog was examined histologically and immunohistochemically. This rare neoplasm was considered to be derived from ectopic neural crest cells. This is the first report of a canine ectopic ganglioneuroblastoma located in the oral mucosa.

Biological properties of allogenic articular chondrocytes on the surface of bovine cartilage explants in vitro.

Bovine cartilage explants were co-cultured with or without allogenic chondrocytes for 4 weeks. The attachment of the applied chondrocytes to cartilage after labelling with fluorescence was assessed using a confocal laser microscope. Morphological changes and the production of extracellular matrix (ECM) of co-cultured chondrocytes on intact and damaged surfaces of cartilage were evaluated by histological and immunohistochemical methods. Co-cultured chondrocytes attached to and proliferated on the intact and damaged areas of cartilage, and a new layer was created there. The defects were also filled with ECM produced by the co-cultured chondrocytes. Glycosaminoglycans and collagen type II were detected in the newly formed ECM, and large numbers of rounded chondrocytes were observed at primitive lacunae in this matrix at 4 weeks of culture. The results suggest that chondrocytes have the ability to attach to, to proliferate on and to establish a new matrix on the intact and damaged surfaces of cartilage explants.

Perianal rhabdomyosarcoma in a dog.

A perianal rhabdomyosarcoma was diagnosed in an 11-year-old neutered male Labrador retriever. Following two incomplete surgical excisions of the tumour, the dog was treated by means of surgery combined with local radiotherapy and systemic chemotherapy using one cycle of vincristine sulphate, doxorubicin and cyclophosphamide (VAC protocol). The dog died 252 days after the first combined therapy. Radiography at this time demonstrated enlargement of the iliac lymph nodes, suggesting metastasis of the tumour. To the authors' knowledge, this is the first report of treatment of canine perianal rhabdomyosarcoma.

Establishment and characterization of a new canine mast cell tumor cell line.

A new cell line (CoMS) was established from a 3-year-old male mongrel dog with mast cell tumor of the oral mucosa. CoMS cells grow in suspension with a doubling time of 27.0 +/- 0.7 hr. The cytoplasmic granules were formalin-sensitive, showed diverse appearances in their ultrastructural findings and contained heparin proteoglycan and neutral protease chymase. Calcium ionophore A23187, substance P and concanavalin A caused significant histamine release from CoMS cells, while compound 48/80 failed to release histamine. This cell line will make an available source for studies on canine mast cell tumors.

Efficacy of enamel matrix proteins on apical periodontal regeneration after experimental apicoectomy in dogs.

Adult dogs have a complex apical delta structure in all root apexes of teeth. This complex structure may affect the formation of apical lesions in the teeth such as apical abscesses. The purpose of this study was to evaluate the efficacy of enamel matrix protein (EMP) which was used for periodontal regeneration therapy after an experimental apicoectomy for an assumed apical lesions of the teeth in dogs. The maxillar canine roots and maxillar fourth premolar buccal mesial roots in five beagles were experimentally apicoectomized under general inhalation anesthesia. After the root apex was exposed and excised, EMP was applied on the surface of the exposed dentin. After 12 weeks, dogs were euthanized. and the experimental teeth together with the surrounding soft and hard periodontal tissues were collected for histological evaluation under a light microscope. In the EMP group, the size of the defect where the root apex was removed was smaller than that of the control group. New cementum was dominantly achieved in the EMP group compared to the control group. Furthermore, new collagen fibers that bridged area between the new cementum and new alveolar bone were detected only in the EMP group. The present results demonstrated marked apical periodontal regeneration after apicoectomy in the EMP group. These results, therefore, suggest that the application of EMP can effectively induce the regeneration of periodontal strUctures in apicoectomized dogs.

Acute respiratory failure caused by leukaemic infiltration of the lung of a dog.

A seven-year-old crossbred male dog with a suspected leukaemic condition was referred for investigation and treatment. A bone marrow aspirate revealed an acute myeloid leukaemia. Combination chemotherapy was administered and the dog initially improved, but 18 days after the initiation of therapy its body condition deteriorated and the animal developed acute respiratory distress. On postmortem examination, extensive leukaemic pulmonary infiltrates were evident.

The formation of apical delta of the permanent teeth in dogs.

To determine the process of formation of apical delta, a histological study on the permanent teeth was carried out in dogs. A litter of 7 clinically healthy beagle dogs and 33 adult dogs (4- to 15- year-old) of 12 breeds with periodontal disease were used for the experiments. Teeth extracted from 6-,7-,8- and 9-month-old beagles were sectioned and stained with HE solution. Tooth roots obtained from adult dogs with periodontal disease were ground. Each tooth was classified into the following root types under a light microscope: Type I (no apical delta = no apical closure), II (few apical delta), IIIA (low apical delta) and IIIB (high apical delta). In the 6-month-old beagles, more than half the tooth roots were classified as type I. In the 7-month-old beagles, type IIIB apical delta was the most predominant and types I, II and IIIA apical delta were occassionally seen. Apical closure and delta were observed in all beagles at 8 months of age histologically. In the 8- and 9-month-old beagles, all root apexes observed were type IIIB. Most of the 314 tooth roots extracted from 33 adult dogs were type IIIB, but a few were type IIIA.

Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages.

Chitosan is reported as an accelerator of wound healing. Histological findings of previous reports indicate that chitosan accelerates the reformation of connective tissue, however the details of the mechanism are not clear. In this study, firstly L929 mouse fibroblasts were cultured with chitosan and the production of extracellular matrix (ECM) was evaluated in vitro. Type I and III collagens and fibronectin were secreted by L929 with or without chitosan; however there was no significant difference in the amount of ECM between the control and the chitosan groups. Secondly, macrophages were stimulated with chitosan, and then transforming growth factor-beta 1 (TGF-beta1) and platelet-derived growth factor (PDGF) messenger ribonucleic acid (mRNA) expressions and production of their proteins were assayed in vitro. As a result, chitosan promoted the production of TGF-beta1 and PDGF. These results indicate that chitosan does not directly accelerate ECM production by fibroblast and the ECM production may increase by the growth factors.

Biomechanical and morphologic evaluation of a three-dimensional fabric sheep artificial intervertebral disc: in vitro and in vivo analysis.

STUDY DESIGN: We have developed a new artificial intervertebral disc consisting of triaxial three-dimensional fabric for the sheep lumbar spine. To clarify the characteristics of the new implant, a series of biomechanical tests and morphologic evaluations were conducted. OBJECTIVES: To investigate the static, viscoelastic, and fatigue properties of the three-dimensional fabric disc in comparison with natural sheep disc and to evaluate their biomechanical and morphologic alteration in vivo. SUMMARY OF BACKGROUND DATA: In its human dimensions the three-dimensional fabric disc revealed mechanical properties similar to a natural human disc. METHODS: The disc-body units from sheep spine and the sheep three-dimensional fabric discs underwent tensile-compressive (200 N), torsional (5 Nm), and creep-recovery tests (30 minutes-30 minutes, 200 N). After fatigue loading (2 million, compressive 200 N) the biomechanical changes and the debris were investigated. For in vivo evaluation after placing in the sheep psoas muscles for 6 months, the surface of the three-dimensional fabric disc was evaluated using macroscopy and scanning electron microscopy, followed by previous biomechanical tests. RESULTS: The behavior of the sheep three-dimensional fabric disc was similar to that of natural sheep disc in tensile-compressive and creep-recovery tests. In torsional testing the behavior of natural sheep disc was more rigid than that of the sheep three-dimensional fabric disc. After fatigue loading there was no biomechanical change and no debris detected. Six months after surgery no morphologic deterioration was observed nor were there changes in biomechanical parameters. CONCLUSIONS: The sheep three-dimensional fabric disc exhibited biomechanical and morphologic biostability, appropriate viscoelasticity, and excellent fatigue properties. The three-dimensional fabric disc has a potential for clinical application of human intervertebral disc replacement.

Chitosan accelerates the production of osteopontin from polymorphonuclear leukocytes.

Chitosan is a copolymer of beta(1 --> 4) glucosamine and N-acetyl-D-glucosamine, which accelerates the infiltration of polymorphonuclear leukocytes (PMN) in the early phase of wound healing. In the granulation tissue treated with chitosan in canine experimental wound, osteopontin (OPN) was strongly positive in PMN immunohistochemically. OPN is a glycosylated phosphoprotein and promotes the attachment or spread of a variety of cell types. In addition, OPN may play a role in granulomatous inflammation. Production of OPN in PMN was therefore investigated in vitro using human PMN in this study. PMN stimulated with granulocyte-colony stimulating factor (G-CSF) and chitosan accumulated OPN mRNA, and released OPN into their culture supernatants. These findings suggest that OPN is synthesized by migrating PMN which plays the novel role of regulating the evolution of wound healing with chitosan treatment at the early phase of healing.

Association between exogenous atrial natriuretic peptide and hemodynamics in dogs with congestive heart failure produced by experimental mitral regurgitation.

Association between exogenous atrial natriuretic peptide (ANP) and hemodynamic changes was ascertained in 3 dogs with overt congestive heart failure (CHF(+)) and 3 dogs without congestive heart failure (CHF(-)) caused by experimental mitral regurgitation (MR). The hemodynamic measurements were recorded in all dogs during and after 1 hr infusion of ANP at the rate of 0.1 (low dose), 0.5 (medium dose) and 1.0 (high dose) microg/kg/min, respectively. Heart rate, mean arterial pressure, pulmonary capillary wedge pressure (PCWP) and systemic vascular resistance decreased significantly during and after ANP infusion even with low dose in the CHF(+). Stroke volume, stroke volume index and cardiac output in the CHF(+) during and after ANP infusion showed an increasing trend as compared with the CHF(-). Double product, an indicator of myocardial oxygen consumption, significantly decreased during and after ANP administration at all doses in the CHF(+). These findings indicate that even at low dose, exogenous ANP improves cardiac performance and reduces myocardial oxygen consumption in the CHF(+), and suggest that ANP has beneficial effects in the treatment of dogs with overt congestive heart failure resulting from MR.

Effect of all-trans and 9-cis retinoic acid on growth and metastasis of xenotransplanted canine osteosarcoma cells in athymic mice.

OBJECTIVE: To determine effects of all-trans and 9-cis retinoic acid (RA) on tumor growth and metastatic ability of canine osteosarcoma cells transplanted into athymic (nude) mice. ANIMALS: Forty-five 5-week-old female BALB/c nude mice. PROCEDURE: 1 X 10(7) POS osteosarcoma cells were transplanted subcutaneously into the intrascapular region of mice. All-trans RA (3 or 30 microg/kg of body weight in 0.1 ml of sesame oil), 9-cis RA (3 or 30 mg/kg in 0.1 ml of sesame oil), or sesame oil (0.1 ml; control treatment) were administered intragastrically 5 d/wk for 4 weeks beginning 3 days after transplantation (n = 4 mice/group) or after formation of a palpable tumor (5 mice/group). Tumor weight was estimated weekly by measuring tumor length and width, and retinoid toxic effects were evaluated daily. Two weeks after the final treatment, mice were euthanatized, and number of mice with pulmonary metastases was determined. RESULTS: Adverse treatment effects were not detected. Tumor weight was less in mice treated with either dose of 9-cis RA than in control mice, although this difference was not significant. Treatment with 30 mg of 9-cis RA/kg initiated after tumor formation significantly reduced the incidence of pulmonary metastasis, compared with the control group. CONCLUSIONS AND CLINICAL RELEVANCE: 9-cis RA decreased the incidence of pulmonary metastasis in nude mice transplanted with canine osteosarcoma cells and may be a potential adjunct therapy for treatment of osteosarcoma in dogs.