K. M. Pawłowski

Transcriptomic signature of cell lines isolated from canine mammary adenocarcinoma metastases to lungs

Metastasis is a final step in the progression of mammary gland cancer, usually leading to death. Potentially, a molecular signature of metastasis can be defined via comparison of primary tumors with their metastases. Currently, there is no data in the literature regarding the molecular portrait of metastases in dogs and only few reports regarding human cancer. This is the first report describing the transcriptomic signature of canine cancer metastatic cells. Two adenocarcinoma cell lines isolated from the canine mammary gland (CMT-W1 and CMT-W2) were compared with cell lines isolated from their lung metastases (CMT-W1M and CMT-W2M) with regards to the following cytometric parameters: cell cycle, ploidy, Bcl-2 expression, susceptibility to induced apoptosis, and transcriptomic profile. Cytometric analyses revealed significant differences in cell cycle and antiapoptotic potential between the examined cells. Using oligonucleotide microarrays, we found 104 up-regulated genes in the metastatic cell line CMT-W1M and 21 up-regulated genes in the primary CMT-W1 cell line. We also found 83 up-regulated genes in the CMT-W2M cell line and only 21 up-regulated genes in the CMT-W2 cell line. Among the up-regulated genes in both metastatic cell lines, we found 15 common genes. These differently expressed genes are involved mainly in signal transduction, cell structure and motility, nucleic acid metabolism, developmental processing, and apoptosis (GHSR, RASSF1, ARF1GAP, WDR74, SMOC2,SFRP4, DIAPH1, FSCN1, ALX4, SNX15, PLD2, WNT7B, POU6F2, NKG7, andPOLR2F). Seven ofthem are involved in a cellular pathway dependent on ghrelin via growth hormone secretagogue receptor (GHSR). Our results suggest that this pathway may be essential for mammary cancer cells to have a metastatic potential.

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

BackgroundIn 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.ResultsThe 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.ConclusionsOur results have indicated the treatment of cells involving use of the siRNA targeting efflux pumps could be a beneficial approach in the future.

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

BackgroundSolid 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.ResultsMicroarray 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.ConclusionsThe 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.

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.

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.

Growth hormone receptor (GHR) RNAi decreases proliferation and enhances apoptosis in CMT-U27 canine mammary carcinoma cell line.

Canine mammary gland has been identified as a major site of the extrapituitary growth hormone (GH) production. This finding is linked to its role in tumourigenesis of the mammary gland. Our previous studies indicated the role of GH and GH receptor (GHR) in regulation of proliferation and apoptosis. Thus, we have optimized the ghr RNA interference method in canine mammary carcinoma cell line CMT-U27. We have analysed the effect of GHR reduction on the intracellular signalling and the cell cycle and apoptosis. The results showed that GHR reduction decreased the p-ERK1/2 expression and caused increase of apoptosis and decrease in number of cells at S and G2M phases. This study indicates that GHR besides proliferative effect promotes growth by increasing cell survival. It can tilt the balance between proliferation and death in cancer cells.

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

BackgroundIt 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.ResultsA 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.ConclusionThe 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.

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.

Derivation of feline vaccine-associated fibrosarcoma cell line and its growth on chick embryo chorioallantoic membrane – a new in vivo model for veterinary oncological studies

Feline vaccine associated fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated fibrosarcoma cell line and its growth on CAM is described. The cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.

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

BackgroundMammary 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.ResultsImmunocytochemical 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.ConclusionsThe 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.