Establishment and characterization of urothelial carcinoma cell lines with and without BRAF mutation (V595E) in dogs.

Each 5 urothelial carcinoma (UC) cell lines with and without the v-Raf murine sarcoma virus oncogene homolog B (BRAF) gene mutation (V595E) were established and examined V595E-related tumorigenic characteristics in dogs. No typical morphological features were observed in cloned cells with and without V595E. The cell proliferation of both cloned cells showed logarithmic growth curve and those doubling time were 24.9 ± 4.1 h in V595E ( +) and 29.3 ± 11.3 h in V595E ( -). On the growth curve of xenotransplanted tumor in severe combined immunodeficiency mice, 3 out of 5 V595E ( +) and 2 out of 5 V595E ( -) cloned cells revealed gradually and remarkably increasing curve, indicating clearly tumorigenicity. The xenotransplanted tumors with V595E ( +) showed typical features of UC, such as solid proliferation of pleomorphic tumor cells, formation of papillary structure, and glandular structure. Additionally, various vascular formation was observed, probably indicating an advanced growth phase of UC. In mitogen-activated protein kinase (MAPK) signaling pathway, cytoplasmic phosphorylated-BRAF (pBRAF) and cytoplasmic and nuclear phosphorylated-ERK1/2 (pERK1/2) were detected in all 4 tumors with V595E ( +), whereas only cytoplasmic and nuclear pERK1/2 was detected in tumors with V595E ( -). Since V595E can directly activate MAPK signaling pathway, coincidence of V595E with pBRAF (phosphor Thr598/Ser601) indicates acquired resistance to BRAF inhibitors. These established UC cell lines, especially V595E ( +) cell lines, are useful tool for understanding pathophysiological states and controlling therapeutic manners of UC in dogs.

Establishment and characterization of a novel cell line of medullary thyroid carcinoma from a dog.

A novel cell line of canine medullary thyroid carcinoma (MTC) was established from the neck mass, diagnosed histopathologically and immunohistochemically as ectopic MTC. The neoplastic cells arranging trabecular structures were characterized as pleomorphic cells with eosinophilic cytoplasm and nucleus, containing often clear nucleolus. These tumor cells were immuno-positive for calcitonin gene-related protein (CGRP), somatostatin, and chromogranin A. In addition, 8th passaged cultured cells were also immuno-positive for CGRP, somatostatin, and chromogranin A. The cloned tumor cells showed logarithmic cell growth with a doubling time of 33.3 h. From the results of DNA sequencing of rearranged during transfection (RET) proto-oncogene, the cloned tumor cells had four single base substitution, including exon 5 codon 82, exon 16 codon 750, exon 17 codon 777, and exon 24 codon 1085, all of which were single nucleotide polymorphism reported in RET gene of dogs. After the xenotransplantation into severe combined immunodeficiency (SCID) mice, the cloned cells showed tumorigenicity potentials. The morphological and immunohistochemical features of the xenotransplanted tumor were almost in conformity with those of the original tumor, including positive immunoreactivity for calcitonin, CGRP, and chromogranin A. To our knowledge, this is the first report of canine MTC cell line, which provides useful in vitro tool for understanding oncogenic mechanism and pathophysiological state of MTC in dogs.

Flow cytometric analysis of peripheral blood and tumor-infiltrating regulatory T cells in dogs with oral malignant melanoma.

It is well known that tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) from patients with advanced-stage cancer have a poor immune response. Regulatory T cells (Tregs), characterized by the expression of a cluster of differentiation 4 and intracellular FoxP3 markers, can inhibit antitumor immunoresponse. In the present study, the prevalence of Tregs in peripheral blood and tumor tissue from dogs with oral malignant melanoma was evaluated by triple-color flow cytometry. The percentage of Tregs in the peripheral blood of the dogs with malignancy was significantly increased compared with healthy control dogs, and the percentage of Tregs within tumors was significantly increased compared with Tregs in peripheral blood of dogs with oral malignant melanoma. This finding suggests that the presence of tumor cells induced either local proliferation or selective migration of Tregs to tumor-infiltrated sites. A better understanding of the underlying mechanisms of Treg regulation in patients with cancer may lead to an effective anticancer immunotherapy against canine malignant melanoma and possibly other tumors.

Relationship between regulatory and type 1 T cells in dogs with oral malignant melanoma.

Recent data suggest a decreased prevalence of IFN-gamma-producing T lymphocytes (Type 1 T cells) in tumor-bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti-tumor responses.

[Soluble fibrin is not excreted in urine and its plasma level is elevated in nephrotic syndrome].

Soluble fibrin (SF) is produced by activated blood coagulation reaction and is useful to diagnose thrombotic diseases. We measured plasma and urine SF levels in nephritic patients to assess the hypercoagulability state associated with the disease. Before they received anti-coagulation or anti-platelet therapies, 60 patients underwent measurement of plasma SF and D-dimer levels by Latex agglutination turbidimetric immnoassay (LA). Urinary SF levels were also measured by LA. Plasma and urinary thrombin antithrombin III complex (TAT) levels were measured by enzyme immunoassay (EIA). Plasma SF levels showed a good correlation with plasma TAT levels but only weak positive correlations were observed between plasma D-dimer and SF or TAT levels. Plasma SF and D-dimer levels were significantly higher in the Iatients with nephrotic-range hypoalbuminemia (< or =3 g/dL) than those without it. Contrarily there was no significant difference in plasma TAT levels between these two groups of patients. In almost all patients, urinary SF levels were under the detection limit. However, TAT was excreted into urine more frequently in patients showing the nephrotic range of hypoalbuminemia at 38.2% than in non-nephrotic patients at 8.0%. Thus, plasma SF levels more precisely indicate activated blood coagulation reaction than plasma TAT levels in nephrotic patients, probably because the plasma SF is not excreted into urine, while plasma TAT is.

Co-localization of chondromodulin-I (ChM-I) and bone morphogenetic protein-6 (BMP-6) in myoepithelial cells of canine mammary tumors.

To compare the roles of chondromodulin-I (ChM-I) and bone morphogenetic protein-6 (BMP-6) in ectopic mesenchymal tissue formation in canine mammary gland tumors, 33 tumors and 2 normal mammary glands were examined. Immunohistochemical analysis revealed co-expression of ChM-I and BMP-6 in canine mammary tumors. In mixed tumors, newly formed woven bone with ossified cartilage matrix was observed in 4/9 cases. The osteoblasts lining the woven bone showed moderate immunoreactivity to ChM-I and BMP-6. Almost all chondrocytes and proliferative myoepithelial cells within the basement membrane showed intense immunoreactivity to both, and the myoepithelial cells adjacent to the mature cartilage showed the most intense immunoreactivity. The immunoreactivity to ChM-I and BMP-6 of the interstitial myoepithelial cells in the myxomatous stroma varied in each focus of mixed tumors. Similar findings were found in complex adenomas. In simple adenomas, hyperplasic myoepithelial cells within the basement membrane showed moderate immunoreactivity to both markers. Western blot analysis detected a 25 kDa band of ChM-I in fresh tissue samples from three mixed tumors. Our results support the hypothesis that proliferating myoepithelial cells with ChM-I and BMP-6 expression play important roles in mesenchymal metaplasia in canine mammary tumors.