Identification and evaluation of putative tumour-initiating cells in canine malignant melanoma cell lines.

Tumour-initiating cells (TICs) have been identified in many solid human tumours, including malignant melanoma. In this study, an enriched TIC population was identified in two canine malignant melanoma cell lines (CML1 and CML6M) using cell surface markers and functional assays, including the sphere forming assay, aldehyde dehydrogenase (ALDH) assay, reverse transcriptase-polymerase chain reaction and γH2AX staining for double-stranded DNA (dsDNA)break identification and repair. The CD34(-) population of cells in both cell lines expressed stem cell genes, such as Oct4, Nanog and Ptch1, were more efficient at making spheres in adherence-free media conditions and were able to repair dsDNA breaks faster than the CD34(+) population. A subpopulation of cells with high expression of ALDH was identified in both cell lines by flow cytometry. The findings indicate the presence of TICs in two canine malignant melanoma cell lines.

Expression of leptin and iNOS in oral melanomas in dogs.

BACKGROUND: Oral melanoma (OM) in dogs is an aggressive malignancy, with clinical behavior resembling cutaneous melanomas in humans. Melanoma in humans is promoted by an inflammatory environment that is contributed to by leptin and inducible nitric oxide synthase (iNOS). OBJECTIVE: To determine if the patterns of leptin and iNOS expression are similar in OM in dogs and cutaneous melanomas in humans. ANIMALS: Twenty client-owned dogs. METHODS: Retrospective case study. Immunostaining of the OM tumors from each dog was scored for percentage and intensity of leptin and iNOS expression. Mitotic index was used as an indicator of tumor aggression. RESULTS: Leptin was detected in ≥75% of the tumor cells in specimens from 11 dogs. One tumor expressed leptin in ≤25% of the cells. The intensity of leptin expression was variable with 6, 9, and 5 cases exhibiting low-, moderate-, and high-intensity staining, respectively. OM with the lowest percentage of iNOS positive cells displayed the highest mitotic indices (P = .006, ANOVA). CONCLUSIONS AND CLINICAL IMPORTANCE: The expression of leptin is a common finding in melanomas in dogs. These data suggest that the possibility of future clinical applications, such as measuring the concentrations of plasma leptin as a screening tool or leptin as a target for therapy. The relevance of iNOS is not as clear in dogs with OM, for which other directed therapeutics might be more appropriate.

Effect of tolfenamic acid on canine cancer cell proliferation, specificity protein (sp) transcription factors, and sp-regulated proteins in canine osteosarcoma, mammary carcinoma, and melanoma cells.

BACKGROUND: Tolfenamic acid (TA) is an NSAID currently under investigation as an anticancer agent in humans. TA induces proteosome-dependent degradation of transcription factors Sp 1, 3, and 4. These proteins are known to be overexpressed in many human cancers. HYPOTHESIS: To evaluate the protein expression of Sps in canine tissue, and efficacy of TA against several canine tumor cell lines. METHODS: Six canine cell lines (2 osteosarcoma, 2 mammary carcinoma, 2 melanoma) were evaluated. Protein levels of Sp 1-4 and their downstream targets were evaluated using Western Blots. Cell survival and TUNEL assays were performed on cell lines, and Sp1 expression was evaluated on histologic samples from archived canine cases. ANIMALS: Six immortalized canine cancer cell lines derived from dogs were used. Archived tissue samples were also used. RESULTS: Sps were highly expressed in all 6 cell lines and variably expressed in histologic tissues. TA decreased expression of Sps 1-4 in all cell lines. All of the downstream targets of Sps were inhibited in the cell lines. Variable Sp1 expression was identified in all histologic samples examined. TA significantly inhibited cell survival in all cell lines in a dose dependant fashion. The number of cells undergoing apoptosis was significantly increased (P < .05) in all cell lines after exposure to TA in a dose-dependent fashion. CONCLUSIONS, AND CLINICAL IMPORTANCE: Tolfenamic acid is a potential anticancer NSAID and further investigation is needed to determine its usefulness in a clinical setting.

Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples.

Differentiating between inflammatory bowel disease (IBD) and small intestinal lymphoma in cats is often difficult, especially when only endoscopic biopsy specimens are available for evaluation. However, a correct diagnosis is imperative for proper treatment and prognosis. A retrospective study was performed using surgical and endoscopic intestinal biopsy specimens from 63 cats with a history of chronic diarrhea or vomiting or weight loss. A diagnosis of lymphoma or inflammation was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone, HE-stained sections plus results of immunohistochemical labeling (IHC) for CD3e and CD79a, and HE staining, immunophenotyping, and polymerase chain reaction (PCR) results for B and/or T cell clonality. In addition, various histomorphologic parameters were evaluated for significant differences between lymphoma and IBD using Fisher's exact test. The sensitivity and specificity of each parameter in the diagnosis of lymphoma were also determined. Results of Bayesian statistical analysis demonstrated that combining histologic evaluation of small intestinal biopsy specimens with immunophenotyping and analysis of clonality of lymphoid infiltrates results in more accurate differentiation of neoplastic versus inflammatory lymphocytes. Important histologic features that differentiated intestinal lymphoma from IBD included lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. Based on the results of this study, a stepwise diagnostic algorithm that first uses histologic assessment, followed by immunophenotyping and then PCR to determine clonality of the lymphocytes, was developed to more accurately differentiate between intestinal lymphoma and IBD.