Klaudinexpressziós vizsgálatok kutyák szolid emlőrákjának tüdőáttéteiben

Abstract

The present study has evaluated the expression of claudin-1, -2, -3, -4, -5, and -7 in 20 cases of lung metastases of grade III simple infiltrating carcinomas of mammary gland in dogs {Figure 1, 2), and compared the results to normal mammary glands'and grade III simple infiltrating carcinomas expression on protein level. The results of the present study demonstrate strong expression of claudin-4, and -7; moderately expression of claudin-3; weak expression of claudin-5; and deletion of claudin-1 and -2 proteins in metastases of solid simple infiltrating carcinomas of mammary gland in canine. The tumour samples were fixed in 8% neutral buffered formalin solution for 24 hours at room temperature, dehydrated in a series of ethanol and xylene and embedded in paraffin. The 3-4 μm thick sections were routinely stained with hemalaun and eosin. For immunohistochemistry the sections (3-4 μm) were cut from paraffin blocks, and were deparaffinized in xylene and graded ethanol. After treatment with appropriate antigen retrieval (Target Retrieval Soluton, DAKO, Glostrup, Denmark, pH 6; microwave oven for 30 minutes), the sections were treated with the primary antibodies (Zymed Inc.) against claudin-1 (diluted 1:100, rabbit polyclonal), claudin-2 (diluted 1:80, mouse monoclonal), claudin-3, -7 (diluted 1:80, rabbit polyclonal), claudin-4, and -5 (diluted 1:100, mouse monoclonal) for 60 minutes at room temperature. Immunohistochemical staining was performed using the streptavidin-peroxidase procedure. Antigen-bound primary antibody was detected using standard avidin-biotin immunoperoxidase complex (DAKO LSAB2 Kit). The chromogen substrate was 3,3-diamino-benzidine tetrahydrochloride (DAB substratechromogen, DAKO, Denmark) in each case. Mayer's hemalaun was used for counter-staining. For each claudin a negative control with omission of the primary antibody was included. The authors used human external positive controls (13). The number of positive cells was calculated as follows: 10 randomly selected areas per slide were analyzed using 20x objective with 100 cells counted in each field. The scoring standardized for each group was as follows: 5 = 80 to 100%, 4 = 60 to 80 5, 3 = 40 to 60%, 2 = 20 to 40%, 1 = 5 to 20 %, and 0 = 0 to 5% of the cells showed positive reactions. The metastases were totally negative for claudin-1 (Figure 3) and -2 proteins. Claudin-3, -4, -5, and -7 were detected as intense membrane reaction in metastases: Claudin-3 scoring: 2.5 [range = 1.0 - 4.0]; 5-80% tumour cell positivity (Figure 4), Claudin-4 scoring: 5; 80-100% (!) tumour cell positivity ({Figure 5, 6), Claudin-S scoring: 1,4 [range = 0-2]; 0-40% tumour cell positivity (Figure 7) and Claudin-7 scoring: 3,9 [range=3-5]; 60-90% tumour cell positivity (Figure 8). The peritumoural intact epithelial cells of the alveoli, bronchioli and bronchi were positive for claudin-1, -3, -4 and -7 proteins and were negative for claudin-2 and -5 molecules. The mesothelial cells of the pleura were negative for all claudins. Recently claudin-3, and -4 were identified as receptors for Clostridium perfringens enterotoxin (20). In their study the malignant metastatic neoplastic epithelial cells of mammary glands highly expressed the claudin-4 protein, receptor of the CPE, which may sensitize these secunder tumours to CPE-mediated cytolysis. Further studies are needed to investigate a possibility of an antitumour therapeutic approach by CPE in cases of metastases of solid simple infiltrating carcinomas of mammary glands in dogs and careful studies need to be performed to accurately determine the presence, and nature of the immune response against CPE when administrated via intratumoural and systemic routes. In their recent investigation, angiogenesis {Figure 9) was evaluated and quantified by immunohistochemical evaluation of microvessel density (MVD) using claudin-5 as marker for vascular endothelium in 20 distant (lungs) metastases from grade III simple infiltrating carcinomas of mammary glands in dogs (Figure 10, 11). The endothelial cells of the intrapulmonal vessels and the intratumoural microvessels were positive for claudin-5 molecule. Computer image analysis was used to measure the intratumoural MVD (Table) (15). For claudin-5, mean MVD was in cases of metastases of solid simple carcinoma 6.22 pixel % per × 200 fields (range 4.79-7.49). This result is almost equivalent, moreover weakly higher than in their earlier study the mean MVD in case of grade III simple carcinoma: 5.33 pixel % per × 200 fields (range 4.03-6.33). The antivascular therapy of the metastases of grade III simple infiltrating carcinomas of mammary glands in dogs may be the either possibility of the anti-tumour therapies.