Milk fat miRNome changes in response to LPS challenge in Holstein cows.

Mastitis is an inflammatory disease in dairy cows, causing economic losses and reducing animal welfare. In order to contribute for the discovery of early and noninvasive indicators, our objective was to determine the effects of a lipopolysaccharide (LPS) challenge on the microRNA profile (miRNome) of milk fat, using microarray analyses in cows. Cows were fed a lactation diet at ad libitum intake (n = 6). At 27 ± 3 days in milk, cows were injected with 50 µg of LPS Escherichia coli in one healthy rear mammary quarter. Milk samples were collected just before LPS challenge (LPS-) and 6.5 h after LPS challenge (LPS +) from the same cows. Microarray analysis was performed using customized 8 × 60 K ruminant miRNA microarrays to compare LPS- to LPS + miRNome. In silico functional analyses were performed using OmicsNet and Mienturnet software. MiRNome comparison between LPS- and LPS + identified 37 differentially abundant miRNAs (q-value ≤ 0.05). The predicted target genes of the 37 differentially abundant miRNAs are mostly involved in cell life including apoptosis, cell cycle, proliferation and differentiation and in gene expression processes. MiRNome analyses suggest that miRNAs profile is related to the inflammation response of the mammary gland. In conclusion, we demonstrated that milk fat might be an easy and rapid source of miRNAs that are potential indicators of early mastitis in cows.

Usefulness of BRCA and ctDNA as Prostate Cancer Biomarkers: A Meta-Analysis.

Prostate cancer represents the most common male urologic neoplasia. Tissue biopsies are the gold standard in oncology for diagnosing prostate cancer. We conducted a study to find the most reliable and noninvasive diagnostic tool. We performed a systematic review and meta-analysis of two biomarkers which we believe are the most interesting: BRCA (BRCA1 and 2) and ctDNA. Our systematic research yielded 248 articles. Forty-five duplicates were first excluded and, upon further examination, a further 203 articles were excluded on the basis of the inclusion and exclusion criteria, leaving 25 articles. A statistical analysis of the obtained data has been performed. With a collective calculation, BRCA1 was expressed in 2.74% of all cases from 24,212 patients examined and BRCA2 in 1.96% of cases from 20,480 patients. In a total calculation using ctDNA, it was observed that 89% of cases from 1198 patients exhibited high expression of circulating tumor DNA. To date, no ideal PCa biomarker has been found. Although BRCA1 and BRCA2 work well for breast and ovarian cancers, they do not seem to be reliable for prostate cancer. ctDNA seems to be a much better biomarker; however, there are few studies in this area. Further studies need to be performed.

Silica-Calcite Sedimentary Rock (Opoka) Enhances the Immunological Status and Improves the Growth Rate in Broilers Exposed to Ochratoxin A in Feed.

Mycotoxins, such as Ochratoxin A (OTA), originating from fungi like Aspergillus and Penicillium, represent serious health hazards to poultry. The use of mycotoxin-adsorbing feed additives can reduce these risks. Opoka, a porous transitional rock, shows promise as one of these additives. This study is the first to examine the effect of Opoka administered with OTA on zootechnical parameters and the immune response of chickens. A 42-day investigation examined the impact of 1% of Opoka supplementation in feed on OTA-challenged broiler chickens. Seventy-two chickens were allocated into three groups of twenty-four individuals each: a control group, an OTA-exposed (2 mg/kg feed) group, and an OTA (2 mg/kg feed) plus 1% of Opoka group. Growth and blood parameters were monitored at predetermined intervals, and comprehensive biochemical, hematological, and cytometric analyses were conducted. The study showed that OTA exposure had a negative impact on chicken weight gain. However, adding Opoka to the diet improved weight gain, indicating its potential as a protective agent. Chickens fed with Opoka also had an increased white blood cell count, which suggests an improved immune response and elevated glucose and cholesterol concentrations. These findings indicate that Opoka may be useful in mitigating health complications caused by OTA exposure in broilers.

Different miRNA contents between mammary epithelial cells and milk fat globules: a random or a targeted process?

MicroRNAs (miRNAs) are small noncoding RNAs present in milk-derived extracellular vesicles and milk fat globules (MFG). Nucleic acid content between the lactating mammary tissue (MT) and MFG are quite similar but discrepancies exist in their miRNA content. Our objective was to identify the origin of these discrepancies, and to evaluate the existence of a possible mechanism sorting miRNAs that will or will not be exported from the mammary epithelial cells (MECs) in bovine MFG. miR-125b-5p, miR-126-3p, miR-141-3p, and miR-204-5p, chosen on the basis of their abundance in the MT, were quantified using RT-qPCR in lactating cow MT, MFG, and laser capture-microdissected MECs. Two miRNAs (miR-125b-5p and miR-141-3p) were detected in the MT as well as in MFG and MECs. miR-204-5p was detected only in the MT, suggesting that it is very likely expressed in a cell type other than MECs. miR-126-3p was detected both in the MT and in MECs but not in MFG, suggesting a targeting mechanism for miRNAs in MECs. These results highlights differences in miRNA content between MECs and MFG may be due to a possibly not random mechanism for loading MFG with miRNA cargos that could involve a variable distribution in MECs or a sorting mechanism.

Comparative Effects of Using New Multi-Strain Synbiotics on Chicken Growth Performance, Hematology, Serum Biochemistry and Immunity.

In this study, the influence of new multi-strain synbiotics on chicken growth performance, hematology, serum biochemistry and immunity was explored. Each synbiotic preparation (A, B and C) comprised three, four or five strains of Lactobacillus sp., respectively, as well as S. cerevisiae and inulin. All strains used in the synbiotics originated from wild-type strains from animal farms in Poland. Six groups of chickens, ROSS 308 line, were fed with three different synbiotics at a dose of 0.5 g/1 kg of feed. Body weight, as well as the biochemical and hematological parameters of the animals in each study group, were determined on the 7th, 14th, 28th and 42nd day of life. Body weight on day 42 differed between groups and was the highest in control group. This group also had the highestfeed conversion ratio (FCR) value. All measured biochemical parameters were in the normal ranges for poultry; however, we observed a lower alkaline phosphatase (AP) concentration on day 7 in the groups fed with synbiotics, which correlated with a lower level of triglycerides in those groups. The aspartate transaminase (AST) concentration was significantly lower in all groups on day 42 in comparison with the control. On day 7, the control group showed the highest concentration of Ca, K and P. Other parameters did not differ significantly throughout the experiment. All groups showed a similar tendency of increase in the red blood cells (RBC) count according to the age of the birds. Every white blood cells (WBC) population showed differences in the proportions between T and B lymphocytes. The T cell and monocyte counts increased until day 28 in all groups. The results showed that our newly developed synbiotic formulas do not have any unfavorable influence on chicken health and may modulate immune response and biochemical parameters. However, this hypothesis needs to be evaluated in future experiments.

Probiotic microorganisms detoxify ochratoxin A in both a chicken liver cell line and chickens.

BACKGROUND: The administration of probiotics and prebiotics (synbiotics) is a promising method for detoxification of ochratoxin A (OTA) in animals. The aim of this study was to investigate the ability of five probiotic strains of lactic acid bacteria (LAB) and one Saccharomyces cerevisiae yeast strain, from three different synbiotics for poultry, to detoxify OTA. In addition, we also investigated the genotoxicity of faecal water (FW) of chickens after administering OTA and/or synbiotics for 42 days. RESULTS: All tested LAB and yeast strains had the ability to detoxify OTA by significant (P < 0.05) reducing its concentration (by 31.3-47.7% and 31.9%, respectively, after 24 h incubation) and genotoxicity (by 22.6-51.8% and 52.7%, respectively). Synbiotics composed of four and five probiotic strains significantly (P < 0.05) decreased FW genotoxicity of chicks, after exposure to OTA, to the level seen in the control group (21.8% ± 1.7%) and were more effective than synbiotics composed of three probiotic strains (31.5%). CONCLUSION: These results showed that there was a beneficial effect of the synbiotics on the gastrointestinal tract of animals. Furthermore, synbiotic preparations containing four or five of tested strains can be considered as preventive agents in the contamination of poultry with OTA. © 2019 Society of Chemical Industry.

Mammary Gland Transcriptome and Proteome Modifications by Nutrient Restriction in Early Lactation Holstein Cows Challenged with Intra-Mammary Lipopolysaccharide.

: The objective is to study the effects of nutrient restrictions, which induce a metabolic imbalance on the inflammatory response of the mammary gland in early lactation cows. The aim is to decipher the molecular mechanisms involved, by comparing a control, with a restriction group, a transcriptome and proteome, after an intra-mammary lipopolysaccharide challenge. Multi-parous cows were either allowed ad libitum intake of a lactation diet (n = 8), or a ration containing low nutrient density (n = 8; 48% barley straw and dry matter basis) for four days starting at 24 ± 3 days in milk. Three days after the initiation of their treatments, one healthy rear mammary quarter of 12 lactating cows was challenged with 50 µg of lipopolysaccharide (LPS). Transcriptomic and proteomic analyses were performed on mammary biopsies obtained 24 h after the LPS challenge, using bovine 44K microarrays, and nano-LC-MS/MS, respectively. Restriction-induced deficits in energy, led to a marked negative energy balance (41 versus 97 ± 15% of Net Energy for Lactation (NEL) requirements) and metabolic imbalance. A microarray analyses identified 25 differentially expressed genes in response to restriction, suggesting that restriction had modified mammary metabolism, specifically ß-oxidation process. Proteomic analyses identified 53 differentially expressed proteins, which suggests that the modification of protein synthesis from mRNA splicing to folding. Under-nutrition influenced mammary gland expression of the genes involved in metabolism, thereby increasing ß-oxidation and altering protein synthesis, which may affect the response to inflammation.

Enhancing anti-tumor efficacy of Doxorubicin by non-covalent conjugation to gold nanoparticles - in vitro studies on feline fibrosarcoma cell lines.

BACKGROUND: Feline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity. METHODS: Stability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline fibrosarcoma cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA). RESULTS: A novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline fibrosarcoma cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance). CONCLUSIONS: The results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline fibrosarcoma cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.

Comparison of stem/progenitor cell number and transcriptomic profile in the mammary tissue of dairy and beef breed heifers.

Bovine mammary stem cells (MaSC) are a source of ductal and lobulo-alveolar tissue during the development of the mammary gland and its remodeling in repeating lactation cycles. We hypothesize that the number of MaSC, their molecular properties, and interactions with their niche may be essential in order to determine the mammogenic potential in heifers. To verify this hypothesis, we compared the number of MaSC and the transcriptomic profile in the mammary tissue of 20-month-old, non-pregnant dairy (Holstein-Friesian, HF) and beef (Limousin, LM) heifers. For the identification and quantification of putative stem/progenitor cells in mammary tissue sections, scanning cytometry was used with a combination of MaSC molecular markers: stem cell antigen-1 (Sca-1) and fibronectin type III domain containing 3B (FNDC3B) protein. Cytometric analysis revealed a significantly higher number of Sca-1(pos)FNDC3B(pos) cells in HF (2.94 ± 0.35%) than in LM (1.72 ± 0.20%) heifers. In HF heifers, a higher expression of intramammary hormones, growth factors, cytokines, chemokines, and transcription regulators was observed. The model of mammary microenvironment favorable for MaSC was associated with the regulation of genes involved in MaSC maintenance, self-renewal, proliferation, migration, differentiation, mammary tissue remodeling, angiogenesis, regulation of adipocyte differentiation, lipid metabolism, and steroid and insulin signaling. In conclusion, the mammogenic potential in postpubertal dairy heifers is facilitated by a higher number of MaSC and up-regulation of mammary auto- and paracrine factors representing the MaSC niche.

Macrophages mediate a switch between canonical and non-canonical Wnt pathways in canine mammary tumors.

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.

Migrastatin analogues inhibit canine mammary cancer cell migration and invasion.

BACKGROUND: Cancer spread to other organs is the main cause of death of oncological patients. Migration of cancer cells from a primary tumour is the crucial step in the complex process of metastasis, therefore blocking this process is currently the main treatment strategy. Metastasis inhibitors derived from natural products, such as, migrastatin, are very promising anticancer agents. Thus, the aim of our study was to investigate the effect of six migrastatin analogues (MGSTA-1 to 6) on migration and invasion of canine mammary adenocarcinoma cell lines isolated from primary tumours and their metastases to the lungs. Canine mammary tumours constitute a valuable tool for studying multiple aspect of human cancer. RESULTS: OUR RESULTS SHOWED THAT TWO OF SIX FULLY SYNTHETIC ANALOGUES OF MIGRASTATIN: MGSTA-5 and MGSTA-6 were potent inhibitors of canine mammary cancer cells migration and invasion. These data were obtained using the wound healing test, as well as trans-well migration and invasion assays. Furthermore, the treatment of cancer cells with the most effective compound (MGSTA-6) disturbed binding between filamentous F-actin and fascin1. Confocal microscopy analyses revealed that treatment with MGSTA-6 increased the presence of unbound fascin1 and reduced co-localization of F-actin and fascin1 in canine cancer cells. Most likely, actin filaments were not cross-linked by fascin1 and did not generate the typical filopodial architecture of actin filaments in response to the activity of MGSTA-6. Thus, administration of MGSTA-6 results in decreased formation of filopodia protrusions and stress fibres in canine mammary cancer cells, causing inhibition of cancer migration and invasion. CONCLUSION: Two synthetic migrastatin analogues (MGSTA-5 and MGSTA-6) were shown to be promising compounds for inhibition of cancer metastasis. They may have beneficial therapeutic effects in cancer therapy in dogs, especially in combination with other anticancer drugs. However, further in vivo studies are required to verify this hypothesis.

Five markers useful for the distinction of canine mammary malignancy.

BACKGROUND: Spontaneous canine mammary tumors constitute a serious clinical problem. There are significant differences in survival between cases with different tumor grades. Unfortunately, the distinction between various grades is not clear. A major problem in evaluating canine mammary cancer is identifying those, that are "truly" malignant. That is why the aim of our study was to find the new markers of canine malignancy, which could help to diagnose the most malignant tumors. RESULTS: Analysis of gene expression profiles of canine mammary carcinoma of various grade of malignancy followed by the boosted tree analysis distinguished a `gene set`. The expression of this gene set (sehrl, zfp37, mipep, relaxin, and magi3) differs significantly in the most malignant tumors at mRNA level as well as at protein level. Despite this `gene set` is very interesting as an additional tool to estimate canine mammary malignancy, it should be validated using higher number of samples. CONCLUSIONS: The proposed gene set can constitute a `malignancy marker` that could help to distinguish the most malignant canine mammary carcinomas. These genes are also interesting as targets for further investigations and therapy. So far, only two of them were linked with the cancer development.

Expression and role of PGP, BCRP, MRP1 and MRP3 in multidrug resistance of canine mammary cancer cells.

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.

CSF-1R as an inhibitor of apoptosis and promoter of proliferation, migration and invasion of canine mammary cancer cells.

BACKGROUND: Tumor-associated macrophages (TAMs) have high impact on the cancer development because they can facilitate matrix invasion, angiogenesis, and tumor cell motility. It gives cancer cells the capacity to invade normal tissues and metastasize. The signaling of colony-stimulating factor-1 receptor (CSF-1R) which is an important regulator of proliferation and differentiation of monocytes and macrophages regulates most of the tissue macrophages. However, CSF-1R is expressed also in breast epithelial tissue during some physiological stages i.g.: pregnancy and lactation. Its expression has been also detected in various cancers. Our previous study has showed the expression of CSF-1R in all examined canine mammary tumors. Moreover, it strongly correlated with grade of malignancy and ability to metastasis. This study was therefore designed to characterize the role of CSF-1R in canine mammary cancer cells proliferation, apoptosis, migration, and invasion. As far as we know, the study presented hereby is a pioneering experiment in this field of veterinary medicine. RESULTS: We showed that csf-1r silencing significantly increased apoptosis (Annexin V test), decreased proliferation (measured as Ki67 expression) and decreased migration ("wound healing" assay) of canine mammary cancer cells. Treatment of these cells with CSF-1 caused opposite effect. Moreover, csf-1r knock-down changed growth characteristics of highly invasive cell lines on Matrigel matrix, and significantly decreased the ability of these cells to invade matrix. CSF-1 treatment increased invasion of cancer cells. CONCLUSION: The evidence of the expression and functional role of the CSF-1R in canine mammary cancer cells indicate that CSF-1R targeting may be a good therapeutic approach.

Gene expression profiles in canine mammary carcinomas of various grades of malignancy.

BACKGROUND: The frequency of mammary malignancies in canine patients is even three times over than in human. In various types of cancer different intracellular signalling pathways are perturbed, thus the patients with pathologically the same type of cancer often have dissimilar genetic defects in their tumours and respond in a heterogeneous manner to anticancer treatment. That is why the objective of the hereby study was to assess the gene expression profiles in canine mammary carcinomas (in unsupervised manner) classified by pathologists as grade 1 (well differentiated), grade 2 (moderately differentiated) and grade 3 (poorly differentiated) and compare their molecular and pathological classifications. RESULTS: Our unsupervised analysis classified the examined tissues into three groups. The first one significantly differed from the others and consisted of four carcinomas of grade 3 and one carcinoma of grade 2. The second group consisted of four grade 1 carcinomas. The very heterogeneous (based on their pathological parameters) group was the last one which consisted of two grade 1 carcinomas, two grade 3 carcinomas and five grade 2 carcinomas. Hierarchical dendrogram showed that the most malignant tumour group had significantly distinct gene expression. CONCLUSIONS: Molecular classification of canine mammary tumours is not identical with pathological classification. In our opinion molecular and pathological characterization of canine mammary malignancy can complement one another. However, furthers studies in this field are required.

A role of ghrelin in canine mammary carcinoma cells proliferation, apoptosis and migration.

BACKGROUND: Ghrelin is a natural ligand of the growth hormone secretagogue receptor (GHS-R). They are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/GHS-R axis may have an important role in tumor growth and progression. However, a role of ghrelin in canine tumors remains unknown. Thus, the aim of our study was two-fold: (1) to assess expression of ghrelin and its receptor in canine mammary cancer and (2) to examine the effect of ghrelin on carcinoma cells proliferation, apoptosis, migration and invasion. The expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines (isolated from primary tumors and their metastases) was examined using Real-time qPCR and immunohistochemistry. For apoptosis analysis the Annexin V and propidium iodide dual staining was applied whereas cell proliferation was evaluated by MTT assay and BrdU incorporation test. The influence of ghrelin on cancer cells migration and invasion was assessed using Boyden chamber assays and wound healing assay. RESULTS: The highest expression of ghrelin was observed in metastatic cancers whereas the lowest expression of ghrelin receptor was detected in tumors of the 3rd grade of malignancy. Higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. In vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. Growth hormone secretagogue receptor inhibitor ([D-Lys3]-GHRP6) as well as RNA interference enhances early apoptosis. CONCLUSION: The presence of ghrelin and GHS-R in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. Our experiments conducted in vitro confirmed that ghrelin promotes cancer development and metastasis.

CA 15-3 cell lines and tissue expression in canine mammary cancer and the correlation between serum levels and tumour histological grade.

BACKGROUND: Mammary tumours are the most common malignancy diagnosed in female dogs and a significant cause of mortality and morbidity in this species. Carbohydrate antigen (CA) 15-3 is a mucinous glycoprotein aberrantly over-expressed in human mammary neoplasms and one of the most widely used serum tumour markers in women with breast cancer. The aim of this study was to investigate the antigenic analogies of human and canine CA 15-3 and to assess its expression in canine mammary cancer tissues and cell lines. Immunohistochemical expression of CA 15-3 was evaluated in 7 canine mammary cancer cell lines and 50 malignant mammary tumours. As a positive control, the human breast carcinoma cell line MCF7 and tissue were used. To assess CA 15-3 staining, a semi-quantitative method was applied. To confirm the specificity and cross-reactivity of an anti-human CA 15-3 antibody to canine tissues, an immunoblot analysis was performed. We also investigated serum CA 15-3 activity to establish whether its expression could be assigned to several tumour characteristics to evaluate its potential use as a serum tumour marker in the canine mammary oncology field. RESULTS: Immunocytochemical analysis revealed CA 15-3 expression in all examined canine mammary cancer cell lines, whereas its expression was confirmed by immunoblot only in the most invasive cells (CMT-W1, CMT-W1M, CMT-W2 and CMT-W2M). In the tissue, an immunohistochemical staining pattern was observed in 34 (68%) of the malignant tumours. A high statistical correlation (p = 0.0019) between serum CA 15-3 levels and the degree of tumour proliferation and differentiation was shown, which indicates that the values of this serum marker increase as the tumour stage progresses. CONCLUSIONS: The results of this study reveal that CA 15-3 is expressed in both canine mammary tumour cell lines and tissues and that serum levels significantly correlate with the histological grade of the malignancy.

The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro.

BACKGROUND: It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro. RESULTS: A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion. CONCLUSION: The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.

Global gene expression profiles of canine macrophages and canine mammary cancer cells grown as a co-culture in vitro.

BACKGROUND: Solid tumours comprise various cells, including cancer cells, resident stromal cells, migratory haemopoietic cells and other. These cells regulate tumour growth and metastasis. Macrophages constitute probably the most important element of all interactions within the tumour microenvironment. However, the molecular mechanism, that guides tumour environment, still remains unknown. Exploring the underlying molecular mechanisms that orchestrate these phenomena has been the aim of our study.A co-culture of canine mammary cancer cells and macrophages was established and maintained for 72 hrs. Having sorted the cells, gene expression in cancer cells and macrophages, using DNA microarrays, was examined. The results were confirmed using real-time qPCR and confocal microscopy. Moreover, their ability for migration and invasion has been assessed. RESULTS: Microarray analysis showed that the up-regulated genes in the cancer cell lines are involved in 15 highly over-manifested pathways. The pathways that drew our diligent attention included: the inflammation pathway mediated by chemokine and cytokine, the Toll receptor signalling pathway and the B cell activation. The up-regulated genes in the macrophages were involved in only 18 significantly over-manifested pathways: the angiogenesis, the p53 pathway feedback loops2 and the Wnt signalling pathway. The microarray analysis revealed that co-culturing of cancer cells with macrophages initiated the myeloid-specific antigen expression in cancer cells, as well as cytokine/chemokine genes expression. This finding was confirmed at mRNA and protein level. Moreover, we showed that macrophages increase cancer migration and invasion. CONCLUSIONS: The presence of macrophages in the cancer environment induces acquisition of the macrophage phenotype (specific antigens and chemokines/cytokines expression) in cancer cells. We presumed that cancer cells also acquire other myeloid features, such as: capabilities of cell rolling, spreading, migration and matrix invasion (what has also been confirmed by our results). It may, perhaps, be the result of myeloid-cancer cell hybrid formation, or cancer cells mimicking macrophages phenotype, owing to various proteins secreted by macrophages.