DR-70 (fibrinogen-fibrin degradation products) as a prognostic biomarker in dogs with neoplasms.

Fibrinogen-fibrin degradation products (DR-70) are derived from tumor cells or metastases. Our previous study reported the diagnostic values in dogs with tumors, but no research has yet to be conducted to establish DR-70 as a prognostic marker. Herein, we investigated changes in DR-70 concentrations and disease courses in dogs with tumors. Overall survival time (OST) analysis was performed in 195 dogs with tumors, stratified with a recommended cut-off (1.514 µg/mL). Continual DR-70 measurements were performed during the medical interventions of 27 dogs with neoplasms. Clinical conditions and medical records were retrospectively reviewed. According to a cut-off value, dogs with plasma DR-70 concentrations above 1.514 µg/mL had shorter survival rates than those with concentrations below this threshold. In cases with complete or partial remission in response to treatment, the DR-70 concentration was decreased compared with that at the first visit, whereas it was increased in patients with disease progression. Our study suggested that changes in DR-70 concentration can be used as a prognostic biomarker for canine neoplasms. Furthermore, increased plasma DR-70 levels might be associated with shorter survival, and DR-70 concentrations may reflect responses to medical intervention.

Increased plasma DR-70 (fibrinogen-fibrin degradation products) concentrations as a diagnostic biomarker in dogs with neoplasms.

BACKGROUND: Tumor biomarkers have used widely in clinical oncology in human medicine. Only a few studies have evaluated the clinical utility of tumor biomarkers for veterinary medicine. A test for fibrinogen and fibrin degradation products (DR-70) has been proposed as an ideal biomarker for tumors in humans. The clinical value of DR-70 for veterinary medicine however has yet to be determined. OBJECTIVES: Investigate the diagnostic value of DR-70 concentrations by comparing them between healthy dogs and dogs with tumors. ANIMALS: Two hundred sixty-three dogs with different types of tumors were included. Sixty healthy dogs also were recruited for comparison. METHODS: The DR-70 concentrations were measured in all recruited individuals by ELISA. Clinical conditions were categorized based on histopathology, cytology, ultrasound examination, radiology, clinical findings, and a combination of these tests. RESULTS: The median concentration of DR-70 was 2.130 ± 0.868 µg/mL in dogs with tumors, which was significantly higher than in healthy dogs (1.202 ± 0.610 µg/mL; P < .0001). With a cut-off of 1.514 µg/mL, the sensitivity and specificity of DR-70 were 84.03% and 78.33%, respectively. The area under curve was 0.883. The DR-70 concentration can be an effective tumor biomarker in veterinary medicine. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased DR-70 concentrations were not affected by tumor type, sex, age, or body weight. However, in dogs with metastatic mast cell tumors and oral malignant melanoma, DR-70 concentrations were significantly increased. Additional studies, including more dogs with nonneoplastic diseases, are needed to further evaluate the usefulness of DR-70 as a tumor biomarker.

Paired Analysis of D-Dimer and Its Correlated Hemostatic Parameters in 30 Dogs with Neoplasms after Tumorectomy.

Previous studies have reported that dogs with neoplasms had elevated D-dimer levels. However, few studies have addressed whether D-dimer could be an indicator of tumor burden. The clinical significance of paired analysis of pre- and post-operation of D-dimer levels in dogs has rarely been described. The present study investigated the values of D-dimer levels and their correlated hemostatic alterations in dogs with surgically removable benign and malignant tumors. This study analyzed 30 clinically healthy and 30 tumor-bearing dogs and evaluated the hemostatic functions including D-dimer, thromboelastography G (TEG G), fibrinogen, activated partial thromboplastin time (aPTT), prothrombin time, and platelet count. The median level of pre-treatment D-dimer was 0.8 µg/mL (range: 0.1-6.3 µg/mL), whereas the control dogs exhibited a median value of 0.1 µg/mL (range: 0.1-0.1 µg/mL, p < 0.0001). After tumorectomy, the median levels of D-dimer (p < 0.0001), fibrinogen (p < 0.0001), TEG G value (p < 0.01), and aPTT (p < 0.05) were significantly lower than those of the pre-treatment samples. However, further studies are needed to clarify the values of other hemostatic evaluations. The study revealed the clinical significance of D-dimer and its correlated hemostatic parameters by paired analysis in dogs with tumors. Though more cases are needed for solid confirmation, these values could be potential tumor biomarkers for dogs.

Surface Expression of Kynurenine 3-Monooxygenase Promotes Proliferation and Metastasis in Triple-Negative Breast Cancers.

Kynurenine 3-monooxygenase (KMO) is the pivotal enzyme in the kynurenine pathway and is located on the mitochondrial outer membrane. The dysregulation of KMO leads to various neurodegenerative diseases; however, it is rarely mentioned in cancer progression. Our previous study showed that KMO overexpression in canine mammary gland tumors (cMGT) is associated with poor prognosis in cMGT patients. Surprisingly, it was also found that KMO can be located on the cell membranes of cMGT cells, unlike its location in normal cells, where KMO is expressed only within the cytosol. Since cMGT and human breast cancer share similar morphologies and pathogenesis, this study investigated the possibility of detecting surface KMO in human breast cancers and the role of surface KMO in tumorigenesis. Using immunohistochemistry (IHC), flow cytometry (FC), immunofluorescence assay (IFA), and transmission electron microscopy (TEM), we demonstrated that KMO can be aberrantly and highly expressed on the cell membranes of breast cancer tissues and in an array of cell lines. Masking surface KMO with anti-KMO antibody reduced the cell viability and inhibited the migration and invasion of the triple-negative breast cancer cell line, MDA-MB-231. These results indicated that aberrant surface expression of KMO may be a potential therapeutic target for human breast cancers.

Kynurenine 3-monooxygenase (KMO), and signal transducer and activator of transcription 3 (STAT3) expression is involved in tumour proliferation and predicts poor survival in canine melanoma.

Canine melanoma is a malignant tumour that exhibits aggressive behaviour, and frequently metastasizes to regional lymph nodes and distant sites. Currently, there are no effective treatments or practical prognostic biomarkers for canine melanoma. The enzyme kynurenine 3-monooxygenase (KMO), which plays a central role in the tryptophan metabolism, has previously been identified as the main pathogenic factor in neurodegenerative diseases; however, it has recently been found to be positively associated with tumour malignancy in human hepatocellular carcinoma and canine mammary tumours. Signal transducer and activator of transcription 3 (STAT3) is a well-known oncoprotein contributing to the proliferation, survival, invasiveness and metastasis of a variety of cancers. Although whether STAT3 and KMO collaborate in tumorigenesis needs to be further verified, our previous findings showed that inhibition of KMO activity reduced activation of STAT3. This study investigated the expressions of KMO and STAT3/phosphorylated (pSTAT3) by immunohistochemical analysis in 85 cases of canine melanoma, showing their expression levels were high within highly mitotic melanoma cells. KMO Overexpression was significantly associated with increased STAT3 and pSTAT3 expressions. Melanoma tissues with higher KMO, STAT3 and pSTAT3 protein expressions were correlated with reduced survival rates of the canine patients. Moreover, inhibition of KMO activity in canine melanoma cells resulted in reduced cell viability, in addition to decreased expressions of STAT3 and pSTAT3. Our results indicated the significance of KMO and the potential role of KMO/STAT3 interaction in enhancing tumour development. Additionally, KMO and STAT3/pSTAT3 may be viewed as useful biomarkers for the prediction of prognosis of canine melanoma.

Inhibiting autophagy potentiates the antitumor efficacy of Euphorbia royleana for canine mammary gland tumors.

BACKGROUND: Canine mammary gland tumors (cMGTs) are the most common neoplasms in intact female canines and viewed as a suitable model for studying human breast cancers. Euphorbia royleana has been reported to have a variety of antitumor efficacies. We have prepared the crude extracts of E. royleana in ethanol and hexane solvents to evaluate the anti-tumor effects for cMGT in vitro and in vivo. RESULTS: The results showed that E. royleana could inhibit cell proliferation and colony formation in cMGT cells. The suppression of tumor cell growth resulted from necrosis and cell cycle arrest. Moreover, autophagy appears to play a critical role in E. royleana-mediated cell death by triggering cell apoptosis. The in vivo results also revealed that E. royleana treatment could reduce the size of solid tumors while exhibiting low toxicity in cMGT-bearing nude mice. CONCLUSIONS: The anti-tumor mechanisms of E. royleana were firstly verified to show it would cause autophagic cell death, apoptosis, and cell cycle arrest in canine mammary tumor cells. The in vitro and in vivo findings in the present study revealed E. royleana has potential anticancer effects for the treatment of canine mammary gland tumors.

Dovitinib Triggers Apoptosis and Autophagic Cell Death by Targeting SHP-1/p-STAT3 Signaling in Human Breast Cancers.

Breast cancer is the most common cancer and the leading cause of cancer deaths in women worldwide. The rising incidence rate and female mortality make it a significant public health concern in recent years. Dovitinib is a novel multitarget receptor tyrosine kinase inhibitor, which has been enrolled in several clinical trials in different cancers. However, its antitumor efficacy has not been well determined in breast cancers. Our results demonstrated that dovitinib showed significant antitumor activity in human breast cancer cell lines with dose- and time-dependent manners. Downregulation of phosphor-(p)-STAT3 and its subsequent effectors Mcl-1 and cyclin D1 was responsible for this drug effect. Ectopic expression of STAT3 rescued the breast cancer cells from cell apoptosis induced by dovitinib. Moreover, SHP-1 inhibitor reversed the downregulation of p-STAT3 induced by dovitinib, indicating that SHP-1 mediated the STAT3 inhibition effect of dovitinib. In addition to apoptosis, we found for the first time that dovitinib also activated autophagy to promote cell death in breast cancer cells. In conclusion, dovitinib induced both apoptosis and autophagy to block the growth of breast cancer cells by regulating the SHP-1-dependent STAT3 inhibition.

Transient downregulation of monocyte-derived dendritic-cell differentiation, function, and survival during tumoral progression and regression in an in vivo canine model of transmissible venereal tumor.

Tumors often target dendritic cells (DCs) to evade host immune surveillance. DC injury is reported in many rodent and human tumors but seldom in tumors of other mammals. Canine transmissible venereal tumor (CTVT), a unique and spontaneous cancer transmitted by means of viable tumor cells. CTVT causes manifold damage to monocyte-derived DCs. This cancer provides an in vivo model of cancer to study the role of monocyte-derived DCs during spontaneous regression. Using flow cytometry and real-time reverse-transcription polymerase chain reactions, we compared the expression of surface molecules on monocyte-derived DCs between normal dogs and dogs with CTVT. These markers were CD1a, CD83, costimulatory factors (CD40, CD80, and CD86), and major histocompatability complex classes I and II. In immature DCs (iDCs) and lipopolysaccharide-treated mature DCs (mDCs), the surface markers were mostly downregulated during tumoral progression and regression. The tumor lowered endocytic activity of iDCs, as reflected in dextran uptake, and decreased allogeneic mixed lymphocyte reactions of mDCs. In addition, it decreased the number of monocytes in the peripheral blood by 40%. The tumor substantially impaired the efficiency with which DCs were generated from monocytes and with which mDCs were generated from iDCs. We also found that progression-phase CTVT supernatants that were cultured for 48 h and that contained protein components killed both monocytes and DCs. Additionally, DC numbers were significantly lower in the draining lymph nodes in CTVT dogs than in normal dogs. In conclusion, CTVT caused devastating damage to monocyte-derived DCs; this might be one of its mechanisms for evading host immunity. Reestablishment of monocyte-derived DC activity by the host potentially might contribute to spontaneous tumoral regression. These findings provide insight into the extent of tumoral effects on host immune systems and responses. This information is useful for developing cancer immunotherapies.

Characterization of canine monocyte-derived dendritic cells with phenotypic and functional differentiation.

For therapeutic purposes, large numbers of dendritic cells (DCs) are essential. In this study, we used 2% autologous canine plasma, granulocyte/macrophage colony-stimulating factor (GM-CSF), fms-like tyrosine kinase 3 ligand (Flt3L), and interleukin 4 (IL-4) in generating monocyte-derived DCs from peripheral blood mononuclear cells of dogs. The plasma enriched the population of CD14-positive monocytes by greatly enhancing the efficiency of monocyte adherence, the proportion of adherent cells increasing from 6.6% with 10% fetal bovine serum to 15.3% with 2% autologous canine plasma. Culturing the adherent monocytes for 6 d with human GM-CSF, canine IL-4, and human Flt3L significantly increased the yield of DCs, more than 90% of which were CD14-negative. Because, in the presence of lipopolysaccharide (LPS), monocytes that were CD14-positive expressed tumor necrosis factor ac much more than DCs with low levels of CD14, it is important to decrease the numbers of CD14-positive cells in generating monocyte-derived DCs. With flow cytometry and real-time reverse-transcriptase-mediated polymerase chain reaction assays, we found that in canine immature DCs (iDCs) the expression of DLA class II molecules, CD1a, CD11c, CD40, and CD86 was high and the expression of CD80, CD83, and CD14 either low or negative. During maturation (stimulated by LPS), the expression of CDla, CD40, CD83, and CD80 was upregulated. However, the expression of DLA class II molecules, CD11c, and CD86 was not increased in mature DCs. Incubating the iDCs with LPS decreased antigen uptake and increased the cells' immunostimulatory capacity (assessed by the allogeneic mixed-lymphocyte reaction), indicating that LPS accelerates the functional maturation of DCs. This protocol may facilitate the use of DCs in cellular immunotherapy.