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

Abstract

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.

Figure 1

MUC1/CA 15–3 expression in canine mammary cancer cell lines and the human MCF7 cell line.A. Pictures of MCF7 human mammary carcinoma cells and canine mammary cancer cell lines: P114, CMT-U27, CMT-U309, CMT-W1, CMT-W1M, CMT-W2 and CMT-W2M obtained with an Olympus BX60 microscope (at the magnification of 200x). The MUC1 antigen is reflected as a brown colour. B. The graph of the mean MUC1 expression (and SE) in the examined cell lines. Ten pictures in each slide were analysed. The brown colour intensity was counted by a computer-assisted image analyser (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA). The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). The ANOVA and Dunett`s Multiple Comparison post-hoc test were applied to analyse the MUC1 expression in the examined cell lines (the MCF7 cell line was regarded as a control); p < 0.001 was regarded as highly significant and marked as ***, whereas p < 0.05 was regarded as significant and marked as *.

Figure 2

Invasion of canine mammary cancer cell lines.A. The graph of fluorescence intensity related to the invasion of canine mammary cancer cell lines: CMT-U27, CMT-U309, P114, CMT-W1, CMT-W1M, CMT-W2 and CMT-W2M. The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). The ANOVA + Tukey’s test were applied to analyse the results. p < 0.05 was regarded as significant and marked as *, whereas p < 0.001 was regarded as highly significant and marked as ***. B. Growth characteristics of the CMT-U27, CMT-U309, P114, CMT-W1, CMT-W1M, CMT-W2 and CMT-W2 cell lines (phase contrast micrographs) grown on Matrigel matrix for 24 hours.

Figure 3

Western blot analysis of the cellular extracts from human and canine mammary cell lines. Representative Western blot picture of MUC1 expression in the examined cancer cell lines: Lane 1: human breast cancer cell line MCF-7; Lane 2: canine spindle-cell tumour cell line CMT-U309; Lanes 3 and 4: canine mammary adenocarcinoma cell lines CMT-W1 and CMT-W2; Lane 5: canine mammary carcinoma cell line CMT-U27; Lane 6: canine mammary anaplastic cancer cell line P114; Lanes 7 and 8: canine mammary metastatic cancer cell lines CTM-W1 and CTM-W2.

Figure 4

MUC1 expression in the canine normal mammary gland and in benign and malignant mammary lesions. Sections of specimens stained immunohistochemically. MUC1 immunoreactivity was present in normal tissues samples, showing luminal and apical expression (A). Moderate and heterogeneous labelling of epithelial cells in complex adenoma (B). Higher level of immunoreactivity in complex carcinoma (C), in cancer cells invading the adipose tissue (D), in simple carcinoma (E) and in intra lymphatic metastatic cells (F). The images were digitalised using a video camera connected to a microscope (DMR Fluo HC, Leica, USA), and the semi-quantitative evaluation was estimated by counting 1.000 positive cells in randomly selected fields (magnification 200x) avoiding necrosis.