Cellular immunity to canine mammary tumor cells demonstrated by the leucocyte migration technique.

Cellular immunity to canine mammary tumor cells was studied by means of the leucocyte migration technique (LMT). Intact tumor cells, separated either by enzymatical or mechanical disruption, were used as antigen, and efforts were made to cultivate tumor cells in vitro. Fifteen female tumorous dogs were studied, and 12 non-tumorous mainly male dags were used as controls. Leucocytes from tumor-bearing females were mixed with own autologous or foreign homologous tumor cells, and control leucocytes were presented with cells from the same source. In addition, leucocytes from tumorous animals and controls were mixed.Animal group A comprised 8 tumor-bearing females. In this group mixtures of different cell numbers and different tumor cell/leucocyte ratios were tried. Animal group B comprised 7 tumor-bearing females, and 40 × 10⁶ leucocytes from these were mixed with 2 × 10⁶ antigencells, antigen-cell/leucocyte ratio 0.05. A great number of tumor cells (tumor cell/leucocyte ratio > 0.05) caused strong non-specific inhibition of leucocyte migration, but in spite of marked inhibition (< 61%) in the homologous system in animal group A, inhibition in the autologous system was found to be stronger (72.2–92.3%). In animal group B, dogs presented with own tumor cells showed marked inhibition (23.7–90.1%), while the controls showed a migration inhibition below 20%. Mixtures of homologous leucocytes showed inhibition of the same order as mixtures of control leucocytes and tumor cells. Thus evidence of cellular immunity against own canine mammary tumor cells was obtained. It proved difficult to cultivate the tumor cells for more than 2–3 passages. Some evidence of antigenic cross reactivity was obtained between 2 adenocarcinomas. Enzymatical separation of tumor cells did not seem to alter antigenic characteristics of the cell surface. Mechanical separation, however, proved to be simpler, more rapid and yielded cell suspensions largely free of debris, and is therefore recommended for further work.     

Effect of cyclooxygenase inhibitors in a xenograft model of canine mammary tumours

Inhibition of cyclooxygenase-2 (COX-2) represents a possible avenue for the prevention and/or treatment of some cancers. Our goal was to compare the effect of a selective inhibitor of COX-2, deracoxib, and a COX-1 and -2 inhibitor, piroxicam, on the growth of canine mammary tumours in a murine model. CMT-9 was used to induce xenografts in nude mice. Mice were treated with piroxicam (0.6 mg kg(-1)), deracoxib (6 mg kg(-1)) or a control solution. Tumour volumes between 0 and 24 days post-treatment showed no significant difference between all groups. A second series of experiments was performed with a higher dose of piroxicam (0.9 mg kg(-1)). Tumour volumes between 14 and 21 days post-treatment were significantly smaller in piroxicam-treated mice compared with controls. These results demonstrate that COX inhibition reduced the growth of canine mammary cancer xenografts in mice, suggesting that COX inhibitors could have a positive effect in dogs.     

Exploring new biomarkers in the tumour microenvironment of canine inflammatory mammary tumours

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (CIMC) are the most aggressive forms of mammary cancer. Current research aims to identify new therapeutic targets. Here, we investigated gene expression levels of biomarkers associated with the inflammatory microenvironment. A total of 32 formalin‐fixed paraffin‐embedded samples of canine mammary carcinoma (CIMC = 26; non‐CIMC = 6) were used and their cDNA subjected to quantitative polymerase chain reaction (qPCR) to establish gene expression levels for mediators commonly implicated in linking carcinogenesis with inflammation. Gene expression differences between CIMC and non‐CIMC types were obtained for cyclooxygenase 2 (COX‐2) (P = 0.004), synuclein gamma (SNCG) (P = 0.006), tribbles 1 (P = 0.025), vascular endothelial growth factor (VEGF) (P = 0.017) and CSF1R (P = 0.045). Among these biomarkers correlations were found, particularly between SNCG and tribbles 1 (r = 0.512, P = 0.001). The efficient metastasis of CIMC is intimately linked to components in the tumour microenvironment. This study suggests that upregulation and correlation of SNCG and tribbles 1 deserves to be further explored.     

VEGFR‐2 expression in malignant tumours of the canine mammary gland: a prospective survival study

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is the main receptor activated by vascular endothelial growth factor ‐A (VEGF‐A) to promote tumour angiogenesis. Its clinical prognostic value has not been studied in canine mammary tumours (CMTs). Dogs with mammary cancer were enrolled in a survival study and the immunohistochemical expressions of VEGFR‐2 and VEGF‐A were analysed and associated with clinicopathological features. VEGFR‐2 expression was associated with VEGF immunoreactivity in cancer cells, supporting the presence of an autocrine loop that may be involved in CMTs growth and survival. VEGFR‐2 was also expressed by endothelial cells from tumour vasculature and positively associated with stromal matrix metalloproteinase‐9 (MMP‐9), suggesting the existence of a link between endothelial cells activation and up‐regulation of matrix degrading proteins. Carcinosarcomas exhibited high VEGFR‐2 expression suggesting that it may be one of the activated molecular pathways in this aggressive histological type and that VEGFR‐2 inhibitors may constitute a potential treatment to improve the prognosis of these patients. Both VEGF and VEGFR‐2 immunoreactivities were independent of patients' overall survival (OS) and disease‐free survival (DFS).     

Effects of Wnt10b on dermal papilla cells via the canonical Wnt/β‐catenin signalling pathway in the Angora rabbit

Wnt10b is a member of Wnt family that plays a variety of roles in biological functions, including those in the development of hair follicles. To investigate the effect of Wnt10b on hair growth in the Angora rabbit and to determine the underlying molecular mechanism, we cultured dermal papilla (DP) cells with exogenous Wnt10b in vitro. We observed the expressions of downstream critical gene β‐catenin and lymphoid enhancer‐binding factor 1 (LEF1) in Wnt/β‐catenin pathway. The levels of β‐catenin mRNA and protein were higher in the Wnt10b group of DP cells than in the Control group, and the mRNA level of LEF1 in the Wnt10b group was higher than in the Control group. Moreover, translocation of β‐catenin from cytoplasm to nucleus was activated in the Wnt10b group. Furthermore, the mRNA levels of the hair follicle‐regulatory genes, insulin‐like growth factor‐1 (IGF‐1) and alkaline phosphatase (ALP), and the protein activity of ALP was also upregulated in the Wnt10b group compared to their corresponding levels in the Control group. These data suggest that Wnt10b could activate the canonical Wnt/β‐catenin signalling pathway to induce DP cells in the Angora rabbit. In addition, the proliferation of DP cells was significantly promoted when cultured with Wnt10b for 48 and 72 hr, suggesting that Wnt10b plays a pivotal role in the proliferation and maintenance of DP cells in vitro. In conclusion, this study demonstrates that Wnt10b may promote hair follicle growth in Angora rabbit through the canonical Wnt/β‐catenin signalling pathway that promotes the proliferation of DP cells.     

Serum and intratumoural GH and IGF-I concentrations: Prognostic factors in the outcome of canine mammary cancer

The biological implication of the growth hormone/insulin like growth factor-I (GH/IGF-I) axis in canine mammary tumours (CMT) has been recently demonstrated, however its clinical and prognostic implications are unknown. Our aim was to investigate its prognostic significance.Hormonal determinations were done by enzyme immunoassays techniques validated for canine species in serum and tumour tissue from 32 bitches with CMT and in serum and normal mammary tissue from 10 controls. Serum and tissular GH and IGF-I concentrations were significantly higher in the case of malignant tumour compared with benign and controls. GH and IGF-I elevated concentrations were significantly associated with tumour relapse and/or metastases during follow-up and in dogs with reduced survival times; however these parameters were not independent prognostic factors in multivariate analysis. This association demonstrates a link between high serum and intratumoural GH and IGF-I concentrations and a worse prognosis and opens the possibility to new anticancer endocrine therapies in dogs.     

Insulin receptor is expressed in normal canine mammary gland and benign adenomas but decreased in metastatic canine mammary carcinomas similar to human breast cancer

Insulin receptor (INSR) or insulin-like growth factor (IGF) signalling is speculated to be involved in mammary tumour development. Expression levels of members of the insulin receptor family (INSR, IGF1R, IGF2R, GHR) and their ligands IGF1and IGF2 were quantified in macro- and microdissected tissue samples of normal canine mammary gland, adenomas, carcinomas and their lymph node metastases to evaluate their potential impact on the carcinogenesis of canine mammary tumours. Normal mammary gland and adenomas had strong INSR expression, while carcinomas and metastases had significantly decreased expression. No differences were observed for IGF1R expression. IGF1, IGF2 and GHR mRNA expressions were strongly decreased in adenomas, carcinomas and metastases. INSR and IGF1R are therefore expressed in normal gland and adenomas and an increased stimulus by their ligands may be a proliferative stimulus in those tissues. However, decreased INSR expression carcinomas and their metastases render questionable its impact at late stages of carcinogenesis.     

3-Phosphoinositide-dependent protein kinase-1/Akt signalling and inhibition in a canine prostate carcinoma cell line

Deregulation of the 3‐phosphoinositide‐dependent protein kinase‐1 (PDK‐1)/Akt signalling pathway is associated with prostate cancer development and progression. Inhibition of PDK‐1/Akt signalling can be achieved using structurally optimized celecoxib derivatives such as OSU‐03012. In this study, we treated the novel canine prostate cancer cell line, Ace‐1, with OSU‐03012 or dimethyl sulphoxide in vitro. We found that Akt was constitutively phosphorylated in the canine prostate cancer cell line Ace‐1 and that there was a dose‐dependent decrease in cell viability, and Akt and glycogen synthase kinase‐3β phosphorylation, in response to OSU‐03012 treatment. This was accompanied by a dose‐dependent increase in apoptosis. These data suggest that Akt signalling pathway inhibition is a potential strategy for the treatment of dogs with prostate cancer and that canine prostate cancer is a relevant large animal model for evaluating Akt pathway inhibitors such as OSU‐03012 for use in people.     

Loss of p27 expression in canine mammary tumors and their metastases

The p27 gene is a member of the cyclin-dependent kinase inhibitors, which arrest G1- to S-phase transition of the cell cycle. We have previously shown a significant reduction of p27 mRNA expression level in laser-microdissected mammary carcinomas and their lymph node metastases when compared to non-neoplastic mammary gland of the same dog. Here, p27 expression was analyzed on the protein level in non-neoplastic mammary gland, primary mammary carcinomas, their lymph node metastases and intravascular tumor cells of 49 dogs, adenomas of 49 dogs and non-neoplastic mammary gland of 98 dogs by immunohistochemistry. A significantly (p ? 0.05) decreased percentage of p27 positive tissue samples was found when normal gland was compared with adenomas, carcinomas and lymph node metastases. Specifically, 91% of normal gland epithelium displayed nuclear p27 expression. In contrast, only 22% of the adenomas, 20% of carcinomas, 12% of lymph node metastases and 32% of intravascular tumor cells had p27 reactivity. Cell cycle control by p27 is therefore lost in the majority of canine mammary tumors. The lack of significant differences between benign and malignant mammary tumors indicates that decreased p27 expression is an early step in carcinogenesis of canine mammary tumors and hinders the use of p27 as a marker of malignancy for this tumor type.     

In vitro study to assess the efficacy of CDK4/6 inhibitor Palbociclib (PD‐0332991) for treating canine mammary tumours

Therapy of canine mammary tumours (CMTs) with classical antitumour drugs is problematic, so better therapeutic options are needed. Palbociclib (PD‐0332991) is an innovative and effective anticancer drug for the treatment of breast cancer in women. Palbociclib is an inhibitor of cyclin‐dependent kinase 4 (CDK4) and CDK6, which are key regulators of the cell cycle machinery and thus cell proliferation. In the present in vitro study, we investigated whether Palbociclib also represents a candidate drug to combat CMT. For this purpose, the effect of Palbociclib was analysed in P114 and CF41 cells, two CMT cell lines with an endogenous CDK4/6 co‐expression. Incubation of P114 and CF41 cells with Palbociclib resulted in a dose‐ and time‐dependent loss of phosphorylated retinoblastoma protein (pRb), a classical CDK4/6 substrate within the cell cycle machinery. Moreover, treatment of CMT cells with Palbociclib‐induced cell cycle arrest affected cell viability, prevented colony formation and impaired cell migration activity. Palbociclib also inhibited the growth of P114 and CF41 cell spheroids. Immunohistochemical analysis of canine patient samples revealed a consistent expression of CDK6 in different canine mammary carcinoma types, but an individual and tumour‐specific expression pattern of phosphorylated pRb independent of the tumour grade. Together, our findings let us suggest that Palbociclib has antitumour effects on CMT cells and that canine patients may represent potential candidates for treatment with this CDK4/6 inhibitor.     

Expression of tissue factor in canine mammary tumours and correlation with grade,stage and markers of haemostasis and inflammation

Tissue factor (TF) expression in human cancers has been associated with a procoagulant state and facilitation of metastasis. This study was conducted in order to evaluate if TF was expressed in canine mammary tumours. Forty epithelial mammary tumours from 28 dogs were included. TF expression of the tumours was evaluated by immunohistochemistry using a polyclonal antibody against recombinant canine TF. In addition, thromboelastography, haemostatic and inflammatory parameters were evaluated in the patients. TF was recognized in 44% of benign and 58% of malignant tumours. TF localized to the cytoplasmic membrane of neoplastic luminal epithelial cells and/or diffusely in the cytoplasm. No association was found between TF expression and stage or grade of disease. A significant association between TF expression and antithrombin and plasminogen was found, and extensive TF expression was seen in a lymph node metastasis classified as anaplastic mammary carcinoma from a dog with concomitant disseminated intravascular coagulation (DIC).     

Effect of recombinant feline interferon-ω alone and in combination with chemotherapeutic agents on putative tumour-initiating cells and daughter cells derived from canine and feline mammary tumours

Interferons (IFNs) are naturally produced cytokines with multiple important biological functions. The activity of recombinant feline IFN-ω (rFeIFN-ω) alone and in combination with chemotherapeutic drugs was tested on canine and feline mammary carcinoma cell lines (REM134 and CAT-MT) and putative tumour-initiating cells (TIC) derived from these cell lines by sphere assay. Viability was measured by chemoluminescence and a one-way analysis of variance and Student's t -tests were used for statistical analysis. rFeIFN-ω showed in vitro antitumour activity on feline and canine mammary carcinoma cells and putative TICs with a dose-dependent and target cell-specific action. Putative TICs were more resistant to the action of rFeIFN-ω compared with daughter REM134 and CAT-MT cells. REM134 cells and TICs were more sensitive to rFeIFN-ω compared with the feline counterparts. An additive effect was noticed between rFeIFN-ω and conventional anticancer drugs, in particular following co-culture of cells with anthracycline drugs. The results suggest that rFeIFN-ω warrants further investigation as a therapeutic adjunct in feline and canine mammary tumours.     

Molecular phenotyping of malignant canine mammary tumours: Detection of high-risk group and its relationship with clinicomolecular characteristics

Canine mammary gland tumours (CMTs) constitute the most common cancer in female dogs and comprise approximately 50% of all canine cancers. With the advent of high-throughput technologies such as microarray and next-generation sequencing, the molecular phenotyping (classification) of various cancers has been extensively developed. The present study used a canine RNA-sequencing dataset, namely GSE119810, to classify 113 malignant CMTs and 64 matched normal samples via an unsupervised hierarchical algorithm with a view to evaluating the association between the resulting subtypes (clusters) (n = 4) and clinical and molecular characteristics. Finally, a molecular classifier was developed, and it detected 1 high-risk molecular subtype in the training dataset (GSE119810) and 2 independent validation datasets (GSE20718 and GSE22516). Our results revealed four molecular subtypes (C2–C5) in malignant CMTs. Furthermore, the normal samples constituted a distinct group in the clustering analysis. Marked significant associations were observed between the molecular subtypes (especially C5) and clinical/molecular features, including positive lymphatic invasion, high tumour grades, histopathology diagnoses, short survival and high TP53 mutation rates (ps <.05). The high-risk subtype (C5) was further characterized through the development of a cell cycle-based gene signature, which comprised 37 proliferation-related genes according to the support vector machine algorithm. This signature identified the high-risk group in both training and validation datasets (ps <.001). In the validation analysis, our potential classifier robustly predicted patients with positive lymphatic invasion, metastases and short survival.     

Tarantula cubensis extract alters the degree of apoptosis and mitosis in canine mammary adenocarcinomas

In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TCE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TCE. Subcutaneous injections of TCE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl-2) was found to be higher in pre-treatment compared to post-treatment tissues (p < 0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TCE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.