Interactions between various cell types in a tumor microenvironment (TME) are important for cancer progression and metastasis. Tumor-associated macrophages (TAMs) play a key role in this intratumoral dialogue regulating Wnt signaling pathway in cancer cells. Co-culture of canine and murine tumor cells with TAMs lead to inhibition of the canonical Wnt pathway and activation of the non-canonical Wnt pathway in tumor cells resulting in change in tumor cell phenotype and subsequent higher potential to invasion and metastasis. These molecular changes in cancer cells that are induced by TAMs resemble the quercetin activity. This natural product is a plant flavonoid from the group of polyphenols. Its biological function is inhibition of the canonical Wnt pathway. As quercetin supplementation is broadly discussed in cancer patients, findings of this study show that this compound should be used with caution, as it may enhance cancer spread.

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.

BACKGROUND:The angiogenesis process is regulated by many factors, such as Hypoxia-Inducible Factor-1 (HIF-1) and Vascular Endothelial Growth Factor (VEGF). Metformin has demonstrated its ability to inhibit cell growth and the LY294002 is the major inhibitor of PI3K/AKT/mTOR pathway that has antiangiogenic properties.METHODS:Canine mammary tumor cell lines CMT-U229 and CF41 were treated with metformin and LY294002. Cell viability, protein and gene expression of VEGF and HIF-1 were determined in vitro. For the in vivo study, CF41 cells were inoculated in female athymic nude mice treated with either metformin or LY294002. The microvessel density by immunohistochemistry for CD31 as well as the gene and protein expression of HIF-1 and VEGF were evaluated.RESULTS:The treatment with metformin and LY294002 was able to reduce the cellular viability after 24 hours. The protein and gene expression of HIF-1 and VEGF decreased after treatment with metformin and LY294002. In the in vivo study, there was a decrease in tumor size, protein and gene expression of HIF-1 and VEGFA, in addition to the decreasing of CD31 expression after all treatments.CONCLUSION:Our results demonstrate the effectiveness of metformin and LY294002 in controlling the angiogenesis process in mammary tumors by VEGF and HIF-1, the most important angiogenic markers.

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.

Differentially expressed microRNAs for the metastasis factor. Significance Analysis of Microarrays (SAM) test and one-way ANOVA test. Legend: avg. Hy3 − average array signal intensity of the probes, d(i) − observed relative difference, (No) − non-metastatic tumour, (Yes) − metastatic tumour, FDR − false discovery rate. (XLSX 69 kb)

Morphologically, canine soft-tissue sarcomas (STSs) resemble human STSs. In humans, proper classification of STSs is considered essential to improve insight in the biology of these tumors, and to optimize diagnosis and therapy. To date, there is a paucity of data published on the significance of detailed classification of STSs in the dog. We revised a cohort (n = 110) of proliferative lesions obtained from a study in Golden Retrievers that were considered "soft tissue sarcoma, not otherwise specified or of uncertain subtype" in order to optimize the diagnoses of these lesions. The criteria according to the veterinary WHO classification, recent veterinary literature, and the WHO classification for humans were applied. Revision was initially based on morphologic characteristics of hematoxylin and eosin-stained histologic sections of the neoplasms. If considered necessary (n = 76), additional immunohistochemistry was applied to aid characterization. The diagnosis of STS was confirmed in 75 neoplasms (68%). Of this group, diagnosis of a specific subtype of the STSs was possible in 58 neoplasms. Seven neoplasms had morphologic characteristics that were suggestive for sarcoma subtypes only described in the WHO classification for humans. Seventeen neoplasms remained "unclassified STSs." Thirty-one lesions (28%) were diagnosed "neoplasm, not being STS." Four lesions (4%) were considered nonneoplastic. Because incorrect classification of a tumor could lead to inappropriate therapeutic intervention and prognostication, the results of our study clearly illustrate the importance of revision and further diagnosis of "unclassified STSs" in dogs.

Morphologically, canine soft-tissue sarcomas (STSs) resemble human STSs. In humans, proper classification of STSs is considered essential to improve insight in the biology of these tumors, and to optimize diagnosis and therapy. To date, there is a paucity of data published on the significance of detailed classification of STSs in the dog. We revised a cohort ( n = 110) of proliferative lesions obtained from a study in Golden Retrievers that were considered “soft tissue sarcoma, not otherwise specified or of uncertain subtype” in order to optimize the diagnoses of these lesions. The criteria according to the veterinary WHO classification, recent veterinary literature, and the WHO classification for humans were applied. Revision was initially based on morphologic characteristics of hematoxylin and eosin–stained histologic sections of the neoplasms. If considered necessary ( n = 76), additional immunohistochemistry was applied to aid characterization. The diagnosis of STS was confirmed in 75 neoplasms (68%). Of this group, diagnosis of a specific subtype of the STSs was possible in 58 neoplasms. Seven neoplasms had morphologic characteristics that were suggestive for sarcoma subtypes only described in the WHO classification for humans. Seventeen neoplasms remained “unclassified STSs.” Thirty-one lesions (28%) were diagnosed “neoplasm, not being STS.” Four lesions (4%) were considered nonneoplastic. Because incorrect classification of a tumor could lead to inappropriate therapeutic intervention and prognostication, the results of our study clearly illustrate the importance of revision and further diagnosis of “unclassified STSs” in dogs.

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44+/CD24low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44+/CD24low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.

Uncontrolled proliferation is one of the main causes of the transformation of a normal cell into a malignant one.The Ki-67 and the proliferating cell nuclear antigen (PCNA) are currently being widely studied using immunohistochemical method to evaluate proliferation activity in tumours.PCNA is acid non-histonic auxiliary protein of the DNA polymerase-δ taking part in DNA replication.Its weight is 36 kDa and it consists of 261 amino acids.In the nucleus, protein appears at the end of the G1-phase of the cell cycle, the maximum concentration is achieved in the Sphase, and then gradually decreases in the G2-phase [8].However, the Ki-67 antigen, similarly to PCNA, is an intranuclear, non-histonic protein, which during electrophoresis yields a double beam AbstractPrognosis of postsurgical behaviour of mammary carcinomas in dogs is difficult using a routine histopathological examination alone.The aim of the present study was an assessment of prognostic value of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen expression in canine mammary carcinomas.Expression was evaluated with computer assisted microscopic image analysis in surgically removed, formalin-fixed, paraffin-embedded tissue samples by means of immunohistochemistry.The growth fraction parameters were compared with previously described data of flow cytometric DNA analysis as well as with clinical outcome based on 24-month long postoperative observation and careful postmortem examinations.In the group of dogs which died or were euthanatized due to metastases the mean Ki-67 index value was significantly higher in comparison to the group of animals which survived the observation period without malignant process progression.Such a relation was not observed for PCNA.Both Ki-67 and PCNA index values were significantly higher in the group of dogs with neoplasms that had increased levels of cells in the S-phase.The presence of a significant correlation between the Ki-67 antigen index and the clinical course after the operation, calculated using the t-student test, with the lack of such correlation in variance analysis test suggests that it should be treated only as a prognostic marker helper.The heterogenity of staining of the cell nuclei and the lack of correlations with the clinical course in case of PCNA seems to disqualify it as a prognostic factor.

This article describes five representative cases of canine mammary osteosarcomas and induced tumours by a cloned canine mammary osteosarcoma cell line in nude mice.All five primary tumours were combined mammary osteosarcomas i.e. composed of both cartilage and bone tissues.In four of the five cases the metastases were also combined osteosarcomas.However, for the metastases in one dog and in the nude mice, only bone forming and spindle cell tumours were seen.The metastases were spread directly to the lungs by the blood in three dogs, and via the lymph nodes in two dogs.The metastases differed in morphology, both within and between different metastatic sites.Some of the metastases had an even lower grade than the corresponding primary tumour.

OBJECTIVE:According to the current hypothesis, tumor-associated macrophages (TAMs) are "corrupted" by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell-TAM interactions are complicated and controversial we aimed to better define this phenomenon. METHODS AND RESULTS:Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial-mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs. CONCLUSIONS:We demonstrated that TAMs mediate a "switch" between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis. Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being "corrupted" by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis. These data challenge the conventional understanding of TAM-cancer cell interactions.

Background Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients. Methods Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway. Results In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration. Conclusions We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.

Canine mammary tumours (CMTs) represent nearly half of all tumours in female dogs and some 50% have malignant behaviour. Simple epithelial carcinomas have shorter disease free periods after surgery and a higher reduction of the proliferation index reduction after antiprogestin aglepristone treatment in vivo related to the expression of progesterone receptors (PR). These findings make simple carcinomas good candidates for endocrine therapy. To further explore this possibility, the effects of the antiprogestins mifepristone (RU486) and onapristone (ZK299) on cell viability and PR expression of the canine mammary carcinoma cell line isolated from a simple epithelial carcinoma CMT-U27 were studied. Twenty five percent of CMT-U27 control cells expressed PR. RU486 (p<0.05) and ZK299 (p<0.05) reduced the number of viable cells (WST-8 test) at 24h but only the latter treatment reduced significantly PR expression in viable tumour cells at 24h of incubation. The results suggest that both RU486 and ZK299 induce a decrease in the number of viable CMT-U27 tumour cells with different effects on PR expression. The canine mammary carcinoma cell line CMT-U27 is sensitive to the effects of antiprogestins and may serve to further explore the role of these drugs in canine mammary carcinomas.

Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand-independent mechanisms.