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The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

Simple Summary: Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. There is a cancer cell subpopulation called cancer stem cells (CSCs), capable of forming spheres in vitro and resisting anti-tumor treatments. Melatonin has shown antitumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by spheres derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. Melatonin reduced viability only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. Moreover, the hormone triggers these effects in a way that does not involve its specific MT1 receptor. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1—multidrug resistance molecules—was decreased in response to the hormone. These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the expression of MDR1 and ABCG2. Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44+/CD24−/low phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44+/CD24−/low phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2. [ABSTRACT FROM AUTHOR]

Simple Summary: Canine mammary cancer (CMC) is the most prevalent neoplasm in intact female dogs, with approximately 50% of these cases diagnosed as malignant. Various chemotherapeutic agents have been applied in CMC treatment, but these agents come with notable disadvantages and limited information on efficacy. The benzo[6,7]oxepino[3,2-b] pyridine (BZOP) derivative, 2-((2-methylpyridin-3-yl)oxy)benzaldehyde (MPOBA), exhibited potent anticancer activity against a human colorectal cancer cell line. However, the anticancer effects and molecular mechanisms of MPOBA on CMC have not been elucidated. This study investigated the effects of MPOBA on proliferation, migration, and apoptosis and investigated its potential mechanism in two CMC cell lines, REM134 canine mammary carcinoma and CMGT071020 canine mammary tubulopapillary carcinoma. Furthermore, an in vitro cytotoxicity test was also evaluated in the Madin–Darby canine kidney (MDCK) cell line. The results revealed that MPOBA significantly inhibited proliferation and migration and induced apoptosis in CMC cell lines. In addition, MPOBA showed lower cytotoxicity in MDCK cells. According to the gene expression analysis, the mRNA expression levels of TP53 tumor suppressor (TP53), Bcl-2 associated X (BAX), and B-cell lymphoma-2 (BCL-2) in both CMC cells were significantly altered in the treatment groups compared to the control. These results suggested that MPOBA may have induced apoptosis in both CMC cell lines. This effect may be mediated through the downregulation of BCL-2 and upregulation of BAX gene expressions. These findings indicated that MPOBA may serve as an emerging candidate agent for CMC treatment. However, mRNA expression levels may not be directly proportional to protein expression levels. Therefore, additional studies, including apoptosis-related protein analysis and apoptosis marker assays, are required to confirm the major apoptosis signaling pathway regulated by MPOBA. CMC is the most frequently diagnosed cancer and one of the leading causes of death in non-spayed female dogs. Exploring novel therapeutic agents is necessary to increase the survival rate of dogs with CMC. MPOBA is a BZOP derivative that has a significant anticancer effect in a human cell line. The main goal of this study was to investigate the anticancer properties of MPOBA against two CMC cell lines (REM134 and CMGT071020) using a 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, a wound healing assay, a transwell migration assay, an Annexin V-FITC apoptosis assay with a flow cytometry analysis, a mRNA expression analysis using quantitative real-time PCR (qRT-PCR), and an immunohistochemistry (IHC). According to the accumulated studies, MPOBA caused significant concentration- and time-dependent reductions in cell proliferation and cell migration and induced apoptosis in both CMC cell lines. In gene expression analysis, nine canine genes, including TP53, BCL-2, BAX, epidermal growth factor receptor (EGFR), snail transcription factor (SNAIL), snail-related zinc-finger transcription factor (SLUG), TWIST, E-cadherin, and N-cadherin, were investigated. The mRNA expression results revealed that MPOBA induced upregulation of TP53 and overexpression of the pro-apoptotic gene BAX, together with an inhibition of BCL-2. Moreover, MPOBA also suppressed the mRNA expression levels of SNAIL, EGFR, and N-cadherin and induced upregulation of E-cadherin, crucial genes related to the epithelial-to-mesenchymal transition (EMT). However, there was no significant difference in the IHC results of the expression patterns of vimentin (VT) and cytokeratin (CK) between MPOBA-treated and control CMC cells. In conclusion, the results of the present study suggested that MPOBA exhibited significant anticancer activity by inducing apoptosis in both CMCs via upregulation of TP53 and BAX and downregulation of BCL-2 relative mRNA expression. MPOBA may prove to be a potential candidate drug to be further investigated as a therapeutic agent for CMC. [ABSTRACT FROM AUTHOR]

Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44+/CD24−/low phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44+/CD24−/low phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.

Canine mammary gland tumour (CMT) commonly affects the female dog. The objective of this study was to develop a doxorubicin-resistant CMT cell line and determine its in vitro and in vivo characteristics, including mRNA and microRNA (miRNA) expression profiles. Doxorubicin-resistant CMT-Star cells were developed from CMT-Stylo cells. The cells were characterized, including tumorigenicity in NOD/SCID mouse models. MiRNA and mRNA expression of the two cell lines were profiled and clustered. ATP binding cassette subfamily B member 1 (ABCB1) and subfamily G member 2 (ABCG2) expressions were significantly increased in the CMT-Star cell line. CMT-Star cells also had altered expression of 785 genes and 14 miRNAs. Downregulating plasminogen (PLG) and plasminogen activator urokinase (PLAU) while upregulating transforming growth factor beta receptor 3 (TGFBR3), epidermal growth factor receptor 1 (EGFR1) and ABCB1 rendered CMT-Star cells less proliferative, less invasive and more resistant to chemotherapeutic drugs. The upregulated miRNAs in CMT-Star cells include miRNA-191, -29a, -107, -99b, -874, -93 and -210, while the downregulated miRNAs include miRNAs-106a, -92a, -92b, -155 and -15b. TGFβR, EGF receptor 1 and Wnt signalling are enriched in doxorubicin-resistant CMT-Star cells and could be potential therapeutic targets in dogs with doxorubicin-resistant CMT.

CMC is the most frequently diagnosed cancer and one of the leading causes of death in non-spayed female dogs. Exploring novel therapeutic agents is necessary to increase the survival rate of dogs with CMC. MPOBA is a BZOP derivative that has a significant anticancer effect in a human cell line. The main goal of this study was to investigate the anticancer properties of MPOBA against two CMC cell lines (REM134 and CMGT071020) using a 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, a wound healing assay, a transwell migration assay, an Annexin V-FITC apoptosis assay with a flow cytometry analysis, a mRNA expression analysis using quantitative real-time PCR (qRT-PCR), and an immunohistochemistry (IHC). According to the accumulated studies, MPOBA caused significant concentration- and time-dependent reductions in cell proliferation and cell migration and induced apoptosis in both CMC cell lines. In gene expression analysis, nine canine genes, including TP53, BCL-2, BAX, epidermal growth factor receptor (EGFR), snail transcription factor (SNAIL), snail-related zinc-finger transcription factor (SLUG), TWIST, E-cadherin, and N-cadherin, were investigated. The mRNA expression results revealed that MPOBA induced upregulation of TP53 and overexpression of the pro-apoptotic gene BAX, together with an inhibition of BCL-2. Moreover, MPOBA also suppressed the mRNA expression levels of SNAIL, EGFR, and N-cadherin and induced upregulation of E-cadherin, crucial genes related to the epithelial-to-mesenchymal transition (EMT). However, there was no significant difference in the IHC results of the expression patterns of vimentin (VT) and cytokeratin (CK) between MPOBA-treated and control CMC cells. In conclusion, the results of the present study suggested that MPOBA exhibited significant anticancer activity by inducing apoptosis in both CMCs via upregulation of TP53 and BAX and downregulation of BCL-2 relative mRNA expression. MPOBA may prove to be a potential candidate drug to be further investigated as a therapeutic agent for CMC.

Canine mammary gland tumour (CMT) commonly affects the female dog. The objective of this study was to develop a doxorubicin-resistant CMT cell line and determine its in vitro and in vivo characteristics, including mRNA and microRNA (miRNA) expression profiles. Doxorubicin-resistant CMT-Star cells were developed from CMT-Stylo cells. The cells were characterized, including tumorigenicity in NOD/SCID mouse models. MiRNA and mRNA expression of the two cell lines were profiled and clustered. ATP binding cassette subfamily B member 1 (ABCB1) and subfamily G member 2 (ABCG2) expressions were significantly increased in the CMT-Star cell line. CMT-Star cells also had altered expression of 785 genes and 14 miRNAs. Downregulating plasminogen (PLG) and plasminogen activator urokinase (PLAU) while upregulating transforming growth factor beta receptor 3 (TGFBR3), epidermal growth factor receptor 1 (EGFR1) and ABCB1 rendered CMT-Star cells less proliferative, less invasive and more resistant to chemotherapeutic drugs. The upregulated miRNAs in CMT-Star cells include miRNA-191, -29a, -107, -99b, -874, -93 and -210, while the downregulated miRNAs include miRNAs-106a, -92a, -92b, -155 and -15b. TGFβR, EGF receptor 1 and Wnt signalling are enriched in doxorubicin-resistant CMT-Star cells and could be potential therapeutic targets in dogs with doxorubicin-resistant CMT. [ABSTRACT FROM AUTHOR]

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

Abstract Background The anti‐cancer effects of Gynura procumbens leaves extract (GPE) have been reported in various human cancers. However, the anti‐cancer effects and molecular mechanisms of this extract on canine mammary cancer (CMC) have not yet been elucidated. Objectives The main goal of this study was to investigate the anti‐cancer properties of GPE against two CMC cell lines (CHMp‐13a and CHMp‐5b). Methods The GP leaves were extracted with 80% ethanol. Anti‐cancer potentials of GPE on CHMp‐13a and CHMp‐5b cancer cell lines using dimethyl‐2‐thiazolyl‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), wound healing, transwell migration, and caspase 3/7 activity assays were evaluated. The mRNA expression levels of two oncogenes: epidermal growth factor receptor (EGFR) and twist family bHLH transcription factor 1 (TWIST) and one tumour suppressor gene: phosphatase and tensin homolog (PTEN) in these cell lines were determined by quantitative real‐time PCR (qRT‐PCR). In addition, The EGFR and PTEN protein levels as well as protein kinase B (AKT) and extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation levels expression were also evaluated by western blot analysis. Results The results showed that GPE caused a significant concentration‐ and time‐dependent reduction in cell proliferation of both CHMp‐13a and CHMp‐5b cells, detected by MTT assays. This extract also significantly suppressed cancer cell migration in both cell lines, tested by wound healing and transwell migration assays. Additionally, the increase in caspase 3/7 activity observed in both CMC cell treated with GPE suggests that GPE induced caspase 3/7 dependent apoptosis. Moreover, GPE significantly decreased EGFR mRNA and protein expression levels compared to control in both cell lines in a dose‐dependent manner. Conclusion These findings emphasized that GPE has an in vitro anti‐cancer activity against CMC by inhibiting EGFR signalling pathway. Thus, GPE may serve as an alternative therapy in CMC with high EGFR expression.

Simple Summary: There is worldwide interest in understanding the cancerous diseases that are causing increasing deaths in humans. In recent years, interest has grown in finding suitable models of different types of cancer in animals to lead the scientific community to a better understanding of the disease, in order to win the battle against cancer. The aim of this investigation was to compare breast cancer samples and canine mammary tumors from a hormonal point of view to validate the canine species as a model to study human breast cancer. There was a close similarity between premenopausal human breast cancer and canine mammary cancer in terms of hormonal receptors. In both species, all hormones assayed were increased in tumors compared to normal mammary gland samples. This research not only further supports canine mammary cancer as a spontaneous model for the study of human breast cancer but is also important in providing a deeper understanding of the hormonal pathogenesis of breast/mammary cancer in each independent species. The validity of spontaneous canine mammary cancer (CMC) as a natural model for the study of human breast cancer (HBC) from a hormonal point of view has never been thoroughly investigated. In this study, we analyzed the immunohistochemical expression of aromatase (Arom) and steroid receptors [estrogen receptor α (ER α), estrogen receptor β (ER β), progesterone receptor (PR) and androgen receptor (AR)] and intratumor steroid hormone levels of 17β-estradiol (E2), estrone sulfate (SO4E1), progesterone (P4), androstenedione (A4), dehydroepiandrosterone (DHEA), and testosterone (T) in 78 samples of mammary cancer—51 human breast cancer (HBC) and 27 canine mammary cancer (CMC)—and corresponding controls. Frequency of tumors expressing Arom, ERβ, PR, and AR was similar in both species, whereas ERα+ tumors were less frequent in the canine species. There was a closer similarity between premenopausal HBC and CMC. In HBC and CMC, all hormones assayed were increased in tumors compared to control samples. Intratumor androgen levels were similar in the two species, although levels of progesterone and estrogens were higher in the HBC samples than the CMC samples. Statistical associations among Arom, receptors, and hormones analyzed suggest that the major hormonal influence in both species is estrogenic through the ER, being the α isoform predominant in the human samples. Our findings further support CMC as a spontaneous model for the study of HBC, especially premenopausal HBC, although several differences, such as the more prevalent ERα immunoexpression and higher intratumor levels of estrogens and P4 in HBC, should be taken into account in comparative hormonal studies. [ABSTRACT FROM AUTHOR]

Masitinib mesylate, a selective tyrosine kinase inhibitor of the c-KIT receptor, is used for the treatment of mast cell tumours in dogs. Masitinib has previously been investigated in various cancers; however, its potential anticancer effect in canine mammary tumours (CMTs) is unknown. In the present paper, we investigated the antiproliferative effect of masitinib in CMT cells and its possible mechanisms of action.

Background: The anti‐cancer effects of Gynura procumbens leaves extract (GPE) have been reported in various human cancers. However, the anti‐cancer effects and molecular mechanisms of this extract on canine mammary cancer (CMC) have not yet been elucidated. Objectives: The main goal of this study was to investigate the anti‐cancer properties of GPE against two CMC cell lines (CHMp‐13a and CHMp‐5b). Methods: The GP leaves were extracted with 80% ethanol. Anti‐cancer potentials of GPE on CHMp‐13a and CHMp‐5b cancer cell lines using dimethyl‐2‐thiazolyl‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), wound healing, transwell migration, and caspase 3/7 activity assays were evaluated. The mRNA expression levels of two oncogenes: epidermal growth factor receptor (EGFR) and twist family bHLH transcription factor 1 (TWIST) and one tumour suppressor gene: phosphatase and tensin homolog (PTEN) in these cell lines were determined by quantitative real‐time PCR (qRT‐PCR). In addition, The EGFR and PTEN protein levels as well as protein kinase B (AKT) and extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation levels expression were also evaluated by western blot analysis. Results: The results showed that GPE caused a significant concentration‐ and time‐dependent reduction in cell proliferation of both CHMp‐13a and CHMp‐5b cells, detected by MTT assays. This extract also significantly suppressed cancer cell migration in both cell lines, tested by wound healing and transwell migration assays. Additionally, the increase in caspase 3/7 activity observed in both CMC cell treated with GPE suggests that GPE induced caspase 3/7 dependent apoptosis. Moreover, GPE significantly decreased EGFR mRNA and protein expression levels compared to control in both cell lines in a dose‐dependent manner. Conclusion: These findings emphasized that GPE has an in vitro anti‐cancer activity against CMC by inhibiting EGFR signalling pathway. Thus, GPE may serve as an alternative therapy in CMC with high EGFR expression. [ABSTRACT FROM AUTHOR]

Obesity is a major health condition owing to its effects on chronic diseases and cancers in humans, but little information is available regarding the role of obesity in canine mammary cancer (CMC). In the present study, we performed immunohistochemistry to investigate the effect of obesity on CMC by analyzing the number of tumor-associated macrophages, intratumoral microvessel density (iMVD), and the expression of prognostic factors including epidermal growth factor receptor (EGFR), cyclooxygenase 2 (COX-2), and Ki67 in CMC specimens. These data were compared in CMC specimens from lean or ideal body weight (Group 1) versus overweight or obese (Group 2) female dogs (n = 60 for each group). Associations between obesity status and histologic characteristics, such as histologic subtype, grading, and lymphatic invasion, were also investigated. Compared with lean or ideal body weight dogs, TAM (tumor-associated macrophage) counts (P <.005) and iMVD (P <.001) were significantly higher in overweight or obese dogs. CMC specimens of dogs in the overweight or obese group also showed higher histologic grade (P <.001). In addition, although no association was found between obesity status and either COX-2 or EGFR expression, Ki67 expression was greater in CMC specimens of overweight or obese dogs (P <.005). The results of this study suggest that obesity may influence CMC development and progression, being associated with higher histologic grade, greater infiltration of TAMs, and increased tumor angiogenesis. [ABSTRACT FROM AUTHOR]

Abstract Dogs develop cancer spontaneously with age, with breed-specific risk underlying differences in genetics. Mammary tumors are reported as the most frequent neoplasia in intact female dogs. Their high prevalence in certain breeds suggests a genetic component, as it is the case in human familial breast cancer, distinctly in BRCA2-associated cancers. However, the molecular genetics of BRCA2 in the pathogenesis of canine cancer are still under investigation. Genetic variations of canine BRCA2 comprised single nucleotide polymorphisms, insertions and deletions. The BRCA2 level has been shown to be reduced in tumor gland samples, suggesting that low expression of BRCA2 is contributing to mammary tumor development in dogs. Additionally, specific variations of the BRCA2 gene affect RAD51 binding strength, critically damage the BRCA2-RAD51 binding and further provoke a defective repair. In humans, preclinical and clinical data revealed a synthetic lethality interaction between BRCA2 mutations and PARP inhibition. PARP inhibitors are successfully used to increase chemo- and radiotherapy sensitivity, although they are also associated with numerous side effects and acquired resistance. Cancer treatment of canine patients could benefit from increased chemo- and radiosensitivity, as their cancer therapy protocols usually include only low doses of drugs or radiation. Early investigations show tolerability of iniparib in dogs. PARP inhibitors also imply higher therapy costs and consequently are less likely to be accepted by pet owners. We summarized the current evidence of canine BRCA2 gene alterations and their association with mammary tumors. Mutations in the canine BRCA2 gene have the potential to be exploited in clinical therapy through the usage of PARP inhibitors. However, further investigations are needed before introducing PARP inhibitors in veterinary clinical practice.

The validity of spontaneous canine mammary cancer (CMC) as a natural model for the study of human breast cancer (HBC) from a hormonal point of view has never been thoroughly investigated. In this study, we analyzed the immunohistochemical expression of aromatase (Arom) and steroid receptors [estrogen receptor α (ER α), estrogen receptor β (ER β), progesterone receptor (PR) and androgen receptor (AR)] and intratumor steroid hormone levels of 17β-estradiol (E2), estrone sulfate (SO4E1), progesterone (P4), androstenedione (A4), dehydroepiandrosterone (DHEA), and testosterone (T) in 78 samples of mammary cancer—51 human breast cancer (HBC) and 27 canine mammary cancer (CMC)—and corresponding controls. Frequency of tumors expressing Arom, ERβ, PR, and AR was similar in both species, whereas ERα+ tumors were less frequent in the canine species. There was a closer similarity between premenopausal HBC and CMC. In HBC and CMC, all hormones assayed were increased in tumors compared to control samples. Intratumor androgen levels were similar in the two species, although levels of progesterone and estrogens were higher in the HBC samples than the CMC samples. Statistical associations among Arom, receptors, and hormones analyzed suggest that the major hormonal influence in both species is estrogenic through the ER, being the α isoform predominant in the human samples. Our findings further support CMC as a spontaneous model for the study of HBC, especially premenopausal HBC, although several differences, such as the more prevalent ERα immunoexpression and higher intratumor levels of estrogens and P4 in HBC, should be taken into account in comparative hormonal studies.

Canine mammary tumors (CMTs) are the most common neoplasm in intact female dogs. Canine mammary cancer (CMC) represents 50% of CMTs, and besides surgery, which is the elective treatment, additional targeted and non-targeted therapies could offer benefits in terms of survival to these patients. Also, CMC is considered a good spontaneous intermediate animal model for the research of human breast cancer (HBC), and therefore, the study of new treatments for CMC is a promising field in comparative oncology. Dogs with CMC have a comparable disease, an intact immune system, and a much shorter life span, which allows the achievement of results in a relatively short time. Besides conventional chemotherapy, innovative therapies have a large niche of opportunities. In this article, a comprehensive review of the current research in adjuvant therapies for CMC is conducted to gather available information and evaluate the perspectives. Firstly, updates are provided on the clinical–pathological approach and the use of conventional therapies, to delve later into precision therapies against therapeutic targets such as hormone receptors, tyrosine kinase receptors, p53 tumor suppressor gene, cyclooxygenases, the signaling pathways involved in epithelial–mesenchymal transition, and immunotherapy in different approaches. A comparison of the different investigations on targeted therapies in HBC is also carried out. In the last years, the increasing number of basic research studies of new promising therapeutic agents on CMC cell lines and CMC mouse xenografts is outstanding. As the main conclusion of this review, the lack of effort to bring the in vitro studies into the field of applied clinical research emerges. There is a great need for well-planned large prospective randomized clinical trials in dogs with CMC to obtain valid results for both species, humans and dogs, on the use of new therapies. Following the One Health concept, human and veterinary oncology will have to join forces to take advantage of both the economic and technological resources that are invested in HBC research, together with the innumerable advantages of dogs with CMC as a spontaneous animal model.

Despite extensive research over many decades, human breast cancer remains a major worldwide health concern. Advances in pre-clinical and clinical research has led to significant improvements in recent years in how we manage breast cancer patients. Although survival rates of patients suffering from localized disease has improved significantly, the prognosis for patients diagnosed with metastatic disease remains poor with 5-year survival rates at only 25%. In vitro studies using immortalized cell lines and in vivo mouse models, typically using xenografted cell lines or patient derived material, are commonly used to study breast cancer. Although these techniques have undoubtedly increased our molecular understanding of breast cancer, these research models have significant limitations and have contributed to the high attrition rates seen in cancer drug discovery. It is estimated that only 3–6% of drugs that show promise in these pre-clinical models will reach clinical use. Models that can reproduce human breast cancer more accurately are needed if significant advances are to be achieved in improving cancer drug research, treatment outcomes, and prognosis. Canine mammary tumors are a naturally-occurring heterogenous group of cancers that have several features in common with human breast cancer. These similarities include etiology, signaling pathway activation and histological classification. In this review article we discuss the use of naturally-occurring canine mammary tumors as a translational animal model for human breast cancer research.

Abstract Background Mammary cancer has a high incidence in canines and is an excellent model of spontaneous carcinogenesis. Molecular iodine (I2) exerts antineoplastic effects on different cancer cells activating re-differentiation pathways. In co-administration with anthracyclines, I2 impairs chemoresistance installation and prevents the severity of side effects generated by these antineoplastic drugs. This study is a random and double-blind protocol that analyzes the impact of I2 (10 mg/day) in two administration schemes of Doxorubicin (DOX; 30 mg/m2) in 27 canine patients with cancer of the mammary gland. The standard scheme (sDOX) includes four cycles of DOX administered intravenously for 20 min every 21 days, while the modified scheme (mDOX) consists of more frequent chemotherapy (four cycles every 15 days) with slow infusion (60 min). In both schemes, I2 or placebo (colored water) was supplemented daily throughout the treatment. Results mDOX attenuated the severity of adverse events (VCOG-CTCAE) in comparison with the sDOX group. The overall tumor response rate (RECIST criteria) for all dogs was 18% (interval of reduction 48–125%), and no significant difference was found between groups. I2 supplementation enhances the antineoplastic effect in mDOX, exhibiting a significant decrease in the tumor epithelial fraction, diminished expression of chemoresistance (MDR1 and Survivin) and invasion (uPA) markers and enhanced expression of the differentiation factor known as peroxisome proliferator-activated receptors type gamma (PPARγ). Significant tumor lymphocytic infiltration was also observed in both I2-supplemented groups. The ten-month survival analysis showed that the entire I2 supplementation (before and after surgery) induced 67–73% of disease-free survival, whereas supplementation in the last period (only after surgery) produced 50% in both schemes. Conclusions The mDOX+I2 scheme improves the therapeutic outcome, diminishes the invasive capacity, attenuates the adverse events and increases disease-free survival. These data led us to propose mDOX+I2 as an effective treatment for canine mammary cancer.

Despite extensive research over many decades, human breast cancer remains a major worldwide health concern. Advances in pre-clinical and clinical research has led to significant improvements in recent years in how we manage breast cancer patients. Although survival rates of patients suffering from localized disease has improved significantly, the prognosis for patients diagnosed with metastatic disease remains poor with 5-year survival rates at only 25%. In vitro studies using immortalized cell lines and in vivo mouse models, typically using xenografted cell lines or patient derived material, are commonly used to study breast cancer. Although these techniques have undoubtedly increased our molecular understanding of breast cancer, these research models have significant limitations and have contributed to the high attrition rates seen in cancer drug discovery. It is estimated that only 3–6% of drugs that show promise in these pre-clinical models will reach clinical use. Models that can reproduce human breast cancer more accurately are needed if significant advances are to be achieved in improving cancer drug research, treatment outcomes, and prognosis. Canine mammary tumors are a naturally-occurring heterogenous group of cancers that have several features in common with human breast cancer. These similarities include etiology, signaling pathway activation and histological classification. In this review article we discuss the use of naturally-occurring canine mammary tumors as a translational animal model for human breast cancer research. [ABSTRACT FROM AUTHOR]

The identification of biomarkers that distinguish diseased from healthy individuals is of great interest in human and veterinary fields. In this research area, a metabolomic approach and its related statistical analyses can be useful for biomarker determination and allow noninvasive discrimination of healthy volunteers from breast cancer patients. In this study, we focused on the most common canine neoplasm, mammary gland tumor, and herein, we describe a simple method using ultra-high-performance liquid chromatography to determine the levels of tyrosine and its metabolites (epinephrine, 3,4-dihydroxy-L-phenylalanine, 3,4-dihydroxyphenylacetic acid, and vanillylmandelic acid), tryptophan and its metabolites (5-hydroxyindolacetic acid, indoxyl sulfate, serotonin, and kynurenic acid) in canine mammary cancer urine samples. Our results indicated significantly increased concentrations of three tryptophan metabolites, 5-hydroxyindolacetic acid (p < 0.001), serotonin, indoxyl sulfate (p < 0.01), and kynurenic acid (p < 0.05), and 2 tyrosine metabolites, 3,4-dihydroxy-L-phenylalanine (p < 0.001), and epinephrine (p < 0.05) in urine samples from the mammary gland tumor group compared to concentrations in urine samples from the healthy group. The results indicate that select urinary tyrosine and tryptophan metabolites may be useful as non-invasive diagnostic markers as well as in developing a therapeutic strategy for canine mammary gland tumors. [ABSTRACT FROM AUTHOR]

Dogs develop cancer spontaneously with age, with breed-specific risk underlying differences in genetics. Mammary tumors are reported as the most frequent neoplasia in intact female dogs. Their high prevalence in certain breeds suggests a genetic component, as it is the case in human familial breast cancer, distinctly in BRCA2-associated cancers. However, the molecular genetics of BRCA2 in the pathogenesis of canine cancer are still under investigation. Genetic variations of canine BRCA2 comprised single nucleotide polymorphisms, insertions and deletions. The BRCA2 level has been shown to be reduced in tumor gland samples, suggesting that low expression of BRCA2 is contributing to mammary tumor development in dogs. Additionally, specific variations of the BRCA2 gene affect RAD51 binding strength, critically damage the BRCA2-RAD51 binding and further provoke a defective repair. In humans, preclinical and clinical data revealed a synthetic lethality interaction between BRCA2 mutations and PARP inhibition. PARP inhibitors are successfully used to increase chemo- and radiotherapy sensitivity, although they are also associated with numerous side effects and acquired resistance. Cancer treatment of canine patients could benefit from increased chemo- and radiosensitivity, as their cancer therapy protocols usually include only low doses of drugs or radiation. Early investigations show tolerability of iniparib in dogs. PARP inhibitors also imply higher therapy costs and consequently are less likely to be accepted by pet owners. We summarized the current evidence of canine BRCA2 gene alterations and their association with mammary tumors. Mutations in the canine BRCA2 gene have the potential to be exploited in clinical therapy through the usage of PARP inhibitors. However, further investigations are needed before introducing PARP inhibitors in veterinary clinical practice. [ABSTRACT FROM AUTHOR]

Abstract Background MicroRNAs may act as oncogenes or tumour suppressor genes, which make these small molecules potential diagnostic/prognostic factors and targets for anticancer therapies. Several common oncogenic microRNAs have been found for canine mammary cancer and human breast cancer. On account of this, large-scale profiling of microRNA expression in canine mammary cancer seems to be important for both dogs and humans. Methods Expression profiles of 317 microRNAs in 146 canine mammary tumours of different histological type, malignancy grade and clinical history (presence/absence of metastases) and in 25 control samples were evaluated. The profiling was performed using microarrays. Significance Analysis of Microarrays test was applied in the analysis of microarray data (both unsupervised and supervised data analyses were performed). Validation of the obtained results was performed using real-time qPCR. Subsequently, predicted targets for the microRNAs were searched for in miRBase. Results Results of the unsupervised analysis indicate that the primary factor separating the samples is the metastasis status. Predicted targets for microRNAs differentially expressed in the metastatic vs. non-metastatic group are mostly engaged in cell cycle regulation, cell differentiation and DNA-damage repair. On the other hand, the supervised analysis reveals clusters of differentially expressed microRNAs unique for the tumour type, malignancy grade and metastasis factor. Conclusions The most significant difference in microRNA expression was observed between the metastatic and non-metastatic group, which suggests a more important role of microRNAs in the metastasis process than in the malignant transformation. Moreover, the differentially expressed microRNAs constitute potential metastasis markers. However, validation of cfa-miR-144, cfa-miR-32 and cfa-miR-374a levels in blood samples did not follow changes observed in the non-metastatic and metastatic tumours.

Mammary cancer is a frequent neoplasia in female dogs, in which most important risk factors are hormonal. Sexual hormones as estradiol play an important role in mammary carcinogenesis, being able to induce carcinogenic initiation, promotion and progression. However, the molecular mechanisms involved are incompletely understood. Estradiol is synthesized mainly in the ovaries, nevertheless, high concentrations of estradiol and some of its hormonal precursors have also been described in malignant mammary tumor tissue. The mechanisms of action of estradiol include the classic genomic effects that modulate gene transcription, and non-genomic effects, which trigger quick effects after estradiol binds to its specific receptors. These responses modulate various intracellular signaling pathways, triggering post-translational modification of several proteins. This review will discuss the well-known underlying mechanisms associated with the action of estradiol in the malignant progression of canine mammary tumors.

Mammary tumours are the most frequent in female dogs as in women and half are malignant. Tumorigenicity and invasiveness are important acquired characteristics for the development and progression of cancers and could be regulated by transcription factors associated with epithelial‐mesenchymal transition (EMT) as ZEB1, ZEB2, SNAI1, SLUG and STAT3. Thus, here, we evaluate the expression of EMT‐associated transcription factors in canine mammary cancer (CMC) cell lines characterized for invasiveness and tumorigenicity to determine if these could be considered good targets for future development of therapies. Five CMC cell lines were characterized regarding their morphology, doubling time and expression of intermediate and actin filaments. In addition, gene expression of SLUG, STAT3, ZEB1, ZEB2 and CDH1, tumorigenicity and invasiveness were assessed. Two of these cells presented an epithelial‐like morphology (E20 and E37) and three a mesenchymal‐like morphology (M5, M25 and CF41.Mg). M25 and CF41.Mg presented higher invasiveness. Furthermore, only mesenchymal‐like cells formed tumorspheres and CF41.Mg made more and larger tumorspheres. The mesenchymal‐like cells are more malignant than the epithelial‐like cells being the CF41.Mg the most malignant. This cell presented higher ZEB1 and ZEB2 and lower CDH1 gene expression. Finally, our results revealed that there is a positive correlation between ZEBs and the tumorsphere number and size. In conclusion, these findings support ZEB1 and ZEB2 as potential therapeutic targets for CMC cells, demonstrating a great potential of canine models for comparative and translational studies. [ABSTRACT FROM AUTHOR]

In recent years, significant emphasis has been placed on combination chemotherapy in cancer using cytotoxic agents and plant derived-bioactive substances that are capable of selectively arresting cell growth and inducing apoptosis in tumour cells. The present study was undertaken to evaluate the possibility that the combination of curcumin and cyclophosphamide could show synergistic anti-proliferative effects towards CMT-U27 and CMT-U309 canine mammary cancer cells and, if so, to clarify the mechanism involved. The anti-proliferative activities of curcumin, cyclophosphamide and a combined treatment on CMT cells were determined using the MTT and LDH assays. The concentration of drug required for 50% inhibition of cell viability (IC50) and combination index (CI) values were calculated from log dose-response curves of fixed-combinations of curcumin and cyclophosphamide generated from MTT assays. Apoptosis was detected using a DNA fragmentation assay and Annexin-V/propidium iodide staining followed by flow cytometry. Cell cycle analyses were also performed using flow cytometry. The expression of the apoptosis-related proteins Bax and Bcl-2 was determined by immunocytochemical staining. MTT and LDH assays showed that curcumin and cyclophosphamide induced a dose- and a time-dependent decrease in cell viability. Isobole analysis revealed that the substances exhibited a synergistic interaction when IC50 and 1/2 IC50 concentrations of curcumin and cyclophosphamide were added concurrently to the cultures. This synergy was characterised by a significant increase in the percentage of early and late apoptotic CMT-U27 and CMT-U309 cells. However, internucleosomal fragmentation of DNA was not observed in the DNA fragmentation assay. Cells treated with curcumin and cyclophosphamide arrested at the G2/M and S phases of the cell cycle, respectively. In combined treatments cells were arrested in both phases of the cell cycle. Furthermore, immunocytochemical stainings demonstrated that the curcumin induced apoptosis in CMT cells by the modulation of Bcl-2/Bax protein expression, as the expression of Bcl-2 was decreased and that of Bax increased. This effect was more pronounced in combination treatments. In conclusion, our study shows that a combination of curcumin and cyclophosphamide shows synergistic growth inhibitory activity on CMT cells via induction of apoptosis and cell cycle arrest accompanied by modulation of Bcl-2/Bax protein expression. This finding provides a molecular basis for the development of natural compounds as novel anticancer agents and will allow lowering the dose of cytotoxic agents, which will in turn lead to more specific and less toxic therapies for mammary cancer in dogs.

Graphical abstract Highlights • A novel triple negative canine mammary gland cancer cell line, CMT-7364, was established. • The CMT-7364 cell line shows character of epithelial-mesenchymal transition. • The CMT-7364 cells are capable of tumorigenesis and metastasis in immune-complete mice. Abstract Canine mammary tumor (CMT) has always been an ideal animal model for human breast cancer (HBC) research, however, there is a lack of various established CMT cell lines corresponding to HBC cell lines. This study was designed to establish a new type of CMT cell line. The primary tumor, CMT-7364, was identified as the intraductal papillary carcinoma, and showed negative immunoreactivity to estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2) by immunohistochemistry (IHC) analysis. The CMT-7364 cell line from this primary tumor also shows a negative immunoreactivity to ER, PR, and HER-2, and was negative to epithelial cell markers and positive to mesenchymal cell markers by immunocytochemistry (ICC) analysis. This cell line, which has been stably cultured for more than 115 passages, and was characterized by epithelial origin with the expression of the epithelial antigen by ICC analysis and invasion ability by transwell analysis. In vivo , tumor mass and metastases in the lung were found after inoculating the CMT-7364 cells in the nude mice model, and the immune-complete mice model respectively. The tissues from the xenograft tumor were also negative to ER, PR, and HER-2 by IHC analysis. Thus, a novel triple negative canine mammary cancer cell line, CMT-7364, was successfully established, which could be used as a promising model for the research of immunotherapy and Epithelial-Mesenchymal Transition (EMT) mechanism of the triple-negative breast cancer both in canine and human. [ABSTRACT FROM AUTHOR]

p16 is an important tumor suppressor gene encoded by the INK4A/ARF/INK4B gene locus that is conserved in humans, rodents, and canids. p16 regulates cell cycle in early G1 phase inhibiting transition out of cell cycle from G1/S phase by regulating a multi-protein control complex. p16-associated proteins, cyclin D, CDK4, and CDK6, experience expression level decreases or do not change during cell differentiation and quiescence in contrast to constant p16 expression in post-proliferative cell phases. We hypothesized that p16 has alternate binding partners, other than classical proliferation-associated proteins such as CDKs, in these post-proliferative cell phases. Using co-immunoprecipitation, we have identified 14-3-3σ as a potential alternate binding partner for p16 in quiescent post-proliferative canine mammary cancer cells. Additionally, expression of 14-3-3σ was maintained as fibroblasts exit cell cycle and differentiate to adipocytes simultaneously with continued expression of p16. Based on these results, we suggest that 14-3-3σ protein may be an alternative binding partner for p16 active during cell quiescence and may associate with p16 during cell differentiation. [ABSTRACT FROM AUTHOR]

Introduction and aim. Canine mammary cancer (CMC) is one of the most common malignancy in female dogs. Although there are many cell markers useful in the diagnosis of cancer in veterinary medicine, the expression of SOX 10 has not yet been well characterized. In human oncology, SOX10 expression is positively correlated with negative prognostic factors for breast cancer. Studies indicate similar mechanisms in the process of oncogenesis in humans and dogs. The aim of our work is to confirm that SOX 10 can be a new cellular marker in the diagnosis of mammary carcinomas in bitches. Material and methods. The study was carried out on 53 samples of canine mammary carcinoma, 14 samples of adenoma and 10 samples of normal glandular tissue. The material was stained with hematoxylin and eosin (H&E) to confirm the histological type and histological grade (Grading) according to the Elston and Ellis classification Immunohistochemistry of SOX10 expression was performed. The percentage of cells showing positive expression was assessed according to the following scale: 0% of cells 0 points, 1-10% of cells 1 point, 11- 50% 2 points, 51-80% 3 points, 81-100% 4 points. Results. Statistical analysis showed significantly higher expression in simple carcinoma compared to normal tissue (p=0.008) and in cancer compared to normal tissue (p=0.008) and significantly lower protein expression in benign tumors compared to G2 tumors (p=0.004). Conclusion. The obtained results may suggest a significant role of SOX10 in the progression of carcinogenesis in mammary tumors in bitches, similarly to the case of triple-negative breast cancer in females. [ABSTRACT FROM AUTHOR]

During the past four decades, an increased number of similarities between canine mammary tumors and human breast cancer have been reported: molecular, histological, morphological, clinical and epidemiological, which lead to comparative oncological studies. One of the most important goals in human and veterinary oncology is to discover potential molecular biomarkers that could detect breast cancer in an early stage and to develop new effective therapies. Recently, cancer cell lines have successfully been used as an in vitro model to study the biology of cancer, to investigate molecular pathways and to test the efficiency of anticancer drugs. Moreover, establishment of an experimental animal model for the study of human breast cancer will improve testing potential anti-cancer therapies and the discovery of effective therapeutic schemes suitable for human clinical trials. In this review, we collected data from previous studies that strengthen the value of canine mammary cancer cell lines as an in vitro model for the study of human breast cancer. [ABSTRACT FROM AUTHOR]

Spatial/temporal controls of development are regulated by the homeotic ( HOX) gene complex and require integration with oncogenes and tumour suppressors regulating cell cycle exit. Spontaneously derived neoplastic canine mammary carcinoma cell models were investigated to determine if HOX expression profiles were associated with neoplasia as HOX genes promote neoplastic potential in human cancers. Comparative assessment of human and canine breast cancer expression profiles revealed remarkable similarity for all four paralogous HOX gene clusters and several unlinked HOX genes. Five canine HOX genes were overexpressed with expression profiles consistent with oncogene-like character ( HOXA1, HOXA13, HOXD4, HOXD9 and SIX1) and three HOX genes with underexpressed profiles ( HOXA11, HOXC8 and HOXC9) were also identified as was an apparent nonsense mutation in HOXC6. This data, as well as a comparative analysis of similar data from human breast cancers suggested expression of selected HOX genes in canine mammary carcinoma could be contributing to the neoplastic phenotype. [ABSTRACT FROM AUTHOR]

In recent years, significant emphasis has been placed on combination chemotherapy in cancer using cytotoxic agents and plant derived-bioactive substances that are capable of selectively arresting cell growth and inducing apoptosis in tumour cells. The present study was undertaken to evaluate the possibility that the combination of curcumin and cyclophosphamide could show synergistic anti-proliferative effects towards CMT-U27 and CMT-U309 canine mammary cancer cells and, if so, to clarify the mechanism involved. The anti-proliferative activities of curcumin, cyclophosphamide and a combined treatment on CMT cells were determined using the MTT and LDH assays. The concentration of drug required for 50% inhibition of cell viability (IC50) and combination index (CI) values were calculated from log dose-response curves of fixed-combinations of curcumin and cyclophosphamide generated from MTT assays. Apoptosis was detected using a DNA fragmentation assay and Annexin-V/propidium iodide staining followed by flow cytometry. Cell cycle analyses were also performed using flow cytometry. The expression of the apoptosis-related proteins Bax and Bcl-2 was determined by immunocytochemical staining. MTT and LDH assays showed that curcumin and cyclophosphamide induced a dose- and a time-dependent decrease in cell viability. Isobole analysis revealed that the substances exhibited a synergistic interaction when IC50 and 1/2 IC50 concentrations of curcumin and cyclophosphamide were added concurrently to the cultures. This synergy was characterised by a significant increase in the percentage of early and late apoptotic CMT-U27 and CMT-U309 cells. However, internucleosomal fragmentation of DNA was not observed in the DNA fragmentation assay. Cells treated with curcumin and cyclophosphamide arrested at the G2/M and S phases of the cell cycle, respectively. In combined treatments cells were arrested in both phases of the cell cycle. Furthermore, immunocytochemical stainings demonstrated that the curcumin induced apoptosis in CMT cells by the modulation of Bcl-2/Bax protein expression, as the expression of Bcl-2 was decreased and that of Bax increased. This effect was more pronounced in combination treatments. In conclusion, our study shows that a combination of curcumin and cyclophosphamide shows synergistic growth inhibitory activity on CMT cells via induction of apoptosis and cell cycle arrest accompanied by modulation of Bcl-2/Bax protein expression. This finding provides a molecular basis for the development of natural compounds as novel anticancer agents and will allow lowering the dose of cytotoxic agents, which will in turn lead to more specific and less toxic therapies for mammary cancer in dogs. [ABSTRACT FROM AUTHOR]

Surgical procedures can affect host immunity proportionally to the extent of surgical trauma. In cancer cases, surgery-induced immunosuppression can potentially promote tumour metastasis. The aim of this study was to investigate, in bitches with malignant mammary tumours, whether major surgery (total unilateral mastectomy or bilateral regional mastectomy) has a more negative effect than minor surgery (unilateral regional mastectomy) on components of host immunity. Twenty bitches with mammary cancer of clinical stage II or III were allocated to group A (minor surgery) or group B (major surgery) of 10 animals each receiving the same anaesthetic protocol for mastectomy. Immune cell measurements in blood [number of leukocytes, neutrophils, lymphocytes and platelets, and relative percentages of T-lymphocytes (CD3+) and their CD4+, CD8+ and CD5low+ subpopulations] were performed before anaesthesia (day 0) and on days 3 and 10 post-mastectomy. On day 3, leukocytes, neutrophils and platelets numbers were higher (p = 0.016, 0.032 and 0.017, respectively) in group B than in group A. For all 20 bitches, T-lymphocytes and the CD4+, CD5low+ T-cells were significantly decreased on day 3, but no significant differences were noted between groups. Minor mastectomy seemed to preserve innate immunity better than major mastectomy, but cellular immunity was rather equally affected. • Leukocytes and neutrophils are increased on day 3 post-mastectomy in canine cancer. • Blood neutrophils and platelets numbers are higher after major than minor mastectomy. • Minor mastectomy for canine cancer preserves innate immunity better than major one. • T-lymphocytes (CD3+) and CD4+, CD5low+ T-cells are decreased on day 3 post-mastectomy. • Canine cellular immunity is rather equally affected by minor/major cancer mastectomy. [ABSTRACT FROM AUTHOR]

Highlights: [•] Lipidomics, high resolution mass spectrometry, polarity switching, serum, canine mammary cancer. [ABSTRACT FROM AUTHOR]

Background: In both women and female dogs, the most prevalent type of malignant neoplasm is the spontaneous mammary tumor. In dogs, half of these are malignant. The treatment of choice for the canine patients is surgical mastectomy. Unfortunately, it often fails in high-risk, locally invasive mammary tumors as of during the time of the surgery the micro-metastases are present. Moreover, there are neither large studies conducting to prove of the benefit from the chemotherapy in dogs nor established chemotherapy treatment protocols available. Additionally, the effectiveness of each individual chemotherapeutic agent and drug resistance of canine mammary cancer have not yet been characterized. That has become the aim of our study, to assess the expression of PGP, BCRP, MRP1 and MRP3 in canine mammary cancer cell lines and to investigate their role in cancer resistance to vinblastine, cisplatin and cyclophosphamide with using RNAi approach. Results: The results suggested that in canine mammary cancer, the vinblastine efflux was mediated by PGP and MRP1 proteins, cisplatin efflux was mediated by all four examined efflux pumps (PGP, BCRP, MRP1 and MRP3), whereas cyclophosphamide resistance was related to BCRP activity. RNAi silencing of these efflux pumps significantly decreased IC50 doses of the examined drugs in canine mammary carcinoma cells. Conclusions: Our results have indicated the treatment of cells involving use of the siRNA targeting efflux pumps could be a beneficial approach in the future. [ABSTRACT FROM AUTHOR]

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most aggressive and fatal types of mammary cancer, and both have a very poor prognosis and low survival rate. Human IBC is characterised by exacerbated angiogenesis, lymphangiogenesis, and lymphangiotropism. Lymphangiotropism is also characteristic of IMC, but microvascular density (MVD) and lymphangiogenesis have not been previously studied in canine IMC. In this study immunohistochemical expression of several angiogenesis-related factors (cyclooxygenase [COX]-2, vascular endothelial growth factors A and D [VEGF-A, VEGF-D], and vascular endothelial growth factor receptor 3 [VEGFR-3]), MVD, lymphatic proliferation index (LPI), and Ki-67 tumour proliferation index (PI) were studied in 21 canine IMC samples, 20 canine high-grade malignant non-IMC mammary tumours (MMTs), and four normal mammary gland samples (NMGs). All mammary neoplasms were histologically categorised as grade III. COX-2 values were also analysed by RT-PCR in seven IMCs, six MMTs and four NMGs. The expressions of COX-2, VEGF-A, and VEGF-D were significantly higher in IMC, MVD and LPI tumours, but not PI. In MMTs, COX-2 immunoexpression was significantly associated with VEGF-A, while in IMCs COX-2 was associated with VEGF-D (lymphangiogenic factor), its receptor VEGFR-3, and LPI. These results suggested that lymphangiogenic pathway stimulation isa specific role of COX-2 in IMC angiogenesis, which justifies the use of COX-2-based targeted palliative therapies in dogs. The exacerbated angiogenesis and lymphangiogenesis and the increased expression of angiogenesis-related factors further support canine IMC as a natural model for the study of human IBC. [ABSTRACT FROM AUTHOR]

Because canine mammary tumours constitute a serious clinical problem and there are no good prognostic markers (only histopathological variables are used), the aim of the presented study was to find new malignancy markers as well as to identify intracellular pathways and biological processes characteristic for canine mammary malignancy. We compared gene expression of the most malignant mammary tumours (poorly differentiated cancers of the 3rd grade of malignancy) with less malignant tumours (well differentiated cancers of the 1st grade of malignancy). The results of our study indicated that in dogs the number of tumour-infiltrating myeloid cells or expression of myeloid-specific antigens by cancer cells is related to the cancer progression and may constitute a new marker of malignancy, however further studies in this field are required. [ABSTRACT FROM AUTHOR]

The very recent studies on human and mice models have indicated an important role of myeloid precursor cells (progenitors or not fully differentiated cells that express the Gr1 antigen also called Gr1-positive myeloid suppressor cells) in the tumor progression and metastasis. They are thought to suppress the immune system and promote angiogenesis via Signal transducer and activator of transcription 3 (STAT3) activation. As of now there is no data available on the correlation of Gr1-positive cell number, phosphorylated STAT3 (p-STAT3) expression and cancer ability to metastasis. Thus, we counted the myeloid precursor cell number and analyzed p-STAT3 expression in 50 canine mammary tumors that gave local/distant metastases and did not metastasize. We showed that the number of Gr1-positive cells and p-STAT3 expression are significantly higher ( p < 0.001) in the metastatic tumors than in the non-metastatic ones. We also observed higher expression of p-STAT3 in the canine mammary cancer cell lines with metastatic potential than in other cell lines ( p < 0.001). Moreover, the number of myeloid precursors and p-STAT3 expression in metastatic tumors correlate strongly. The tumor infiltrating myeloid precursor cells may invigorate the STAT3 activity (probably via vascular endothelial growth factor - VEGF) that contributes to the tumor angiogenesis and furthermore tumor`s ability to metastasize. The analysis of gene expression in canine mammary cancer cell lines with metastatic potential indicated that semaphorin 3B (SEMA3B) and neuropilin receptors (NRP) may also be important elements in this process. Thus, we discuss the possible interactions within the tumor that may be required for cancer metastatis. [ABSTRACT FROM AUTHOR]

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer. [ABSTRACT FROM AUTHOR]

Abstract: Mammary cancer is among the most prevalent of canine tumors frequently resulting in death due to metastatic disease. Most tumors fail to raise an effective immune reaction making improving immune recognition a priority. Hybrid-cell fusion strategies have been employed to load dendritic cell populations with tumor cell antigens to stimulate immune recognition; however, recovery, heterogeneity and quality of primary cells from patients present enormous challenges. We employed allogeneic cell lines to develop an improved hybrid-cell fusion strategy and evaluated immune reactions in normal laboratory beagles. Such a strategy relies on enhanced immune recognition of allogeneic tumor cell antigens by antigen presenting cells. Optimized PEG-promoted fusions between uniquely stained canine mammary tumor CMT12 or CMT28 cells and a dendritic cell-like DH82 cell fusion partner resulted in greater than 40% hybrid-cell fusion populations by flow cytometry and fluorescence microscopy. Hybrid-cell fusions were delivered by direct ultrasound guided injection into popliteal lymph nodes of laboratory beagles. Only hybrid-cell fusions provided statistically significant enhancement of cell-mediated immunity (51Cr-release assay) compared to innate reactions in naïve vehicle injected dogs while dogs vaccinated with either single cell component alone did not. Vaccination with hybrid-cell fusions enhanced IFN-γ expression in sorted CD8+ and CD4+ cells but not in CD4−/CD8− cells consistent with a CTL response. Cell-mediated immune assays revealed strong reactions against matched (vaccine component) CMT cells and unmatched CMT cells indicative of an immune response to mammary cancer antigens common to both cell lines. These results provide proof of principle for development of an allogeneic vaccination strategy against canine mammary cancer. [Copyright &y& Elsevier]

Simple Summary: Canine mammary cancer corresponds to a tumor disease that frequently affects female dogs, especially those that are reproductively intact. This disease can decrease the survival and quality of life of affected animals; therefore, it is important to know the underlying mechanisms. Hormonal factors are very important for the development of this pathology. Estradiol, a hormone produced primarily in the ovaries actively participates in the process of mammary tumor formation, but the principal mechanisms behind this carcinogenic role remain incompletely understood. This review will discuss how estradiol would induce its pro-tumor effect on the mammary tissue, thus contributing to the understanding of the mechanisms involved in the development of this disease. Mammary cancer is a frequent neoplasia in female dogs, in which most important risk factors are hormonal. Sexual hormones as estradiol play an important role in mammary carcinogenesis, being able to induce carcinogenic initiation, promotion and progression. However, the molecular mechanisms involved are incompletely understood. Estradiol is synthesized mainly in the ovaries, nevertheless, high concentrations of estradiol and some of its hormonal precursors have also been described in malignant mammary tumor tissue. The mechanisms of action of estradiol include the classic genomic effects that modulate gene transcription, and non-genomic effects, which trigger quick effects after estradiol binds to its specific receptors. These responses modulate various intracellular signaling pathways, triggering post-translational modification of several proteins. This review will discuss the well-known underlying mechanisms associated with the action of estradiol in the malignant progression of canine mammary tumors. [ABSTRACT FROM AUTHOR]