Expression Of SSEA-3 And SSEA-4 In Breast Cancer

Background:In recent years, along with the in-depth understanding of stem cells and the progress of the biological experiments technique, stem cell research has affected nearly all of the life scientific fields and the bio-medical ones. Embryonic stem cells have drawn much more attention from scientists, due to their broad application prospects. Stage-specific embryonic antigens (SSEA) also receive much attention, because of the wide range of applications in the separation and identification of embryonic stem cells. Stage-specific embryonic antigen gained the name, because that in their initial discovery, they were specifically expressed in a specific stage of the mammal embryonic development process. Now we know they are all identified by using corresponding monoclonal antibody as a probe. And according to the orders of their discovery, these antigens are named as SSEA-1, SSEA-3 and SSEA-4 one by one. Stage-specific embryonic antigen-1 which is also called Lex or CD 15, because of its characteristics of broad distribution in the animal tissues, it has drawn more attention from the scientific community. Currently we know that proliferation of embryonic cells and tumor cells is much faster than that of adult ones. And when cancer occurs in normal tissues, it is often accompanied by the appearance of some embryonic antigens. However, it is largely unknown about the clinicopathological correlations of these factors in tumors. Furthermore, SSEA-3 and SSEA-4 can be transformed to LeX or SLeX by transferring fucose glycosylation or sialic-acidication. But in our country excepting studies in embryonic tissues of the animals, there are very few studies on SSEA-3 and SSEA-4 in the differentiated tissues as well as in some tumor tissues. Until now, no study of these factors on breast tumors is available. Therefore, further studies of these antigens on tumor tissues may help us understand the relationships between these embryonic stem cells antigens and tumourigenesis and tumour development.It has been shown that expression of SSEA-1 in some malignant tumors can be detected,and it even associats with tumor metastasis and poor prognosis. In the process of embryonic development, SSEA-3 and SSEA-4 appear first and SSEA-1 follows up later. Then we speculate whether SSEA-3 and SSEA-4 participate in the process of the tumorigensis and development in some malignant tumors just as LeX or SLex does, and by which mean they participate. These still need to be studied. However, studies about SSEA-3 and SSEA-4 on the breast tissues, including breast tumors, are still lacking in reported. Mainly from two levels of breast tissues and the breast cancer cell line, we detect expression and distribution of SSEA-3 and SSEA-4 in benign and malignant breast tissues, breast normal tissues, as well as in breast cancer cell line MDA-MB468 by using immunohistochemical or immunocyto-chemical staining method, in order to explore their correlation of the expression of these two factors with breast cancer. So as to provide a reference for the basic research and clinical treatment of breast cancer.Materials and Methods:1. Materials:Tissue specimens and Cell lines 1.1 Tissue specimens:Surgical specimens of 50 cases of breast cancer,50 cases of benign breast lesions, and 20 cases of normal breast tissues were collected from August 2006 to April 2007 surgical specimens in the First Affiliated Hospital of Zhengzhou University China. And these specimens were all reviewed and confirmed by two senior pathologists.1.2 Cell lines:Human breast cancer cell line MDA-MB468 was maintained with 10% newborn calf serum-DMEM medium (GIBCO/BRL, high-sugar). For passaging, the cells were treated with 0.02% EDTA and 0.25% trypsin 1:1 mixture. And it was cultured under conditions of 37℃,5% CO2 and saturated humidity adherently. The cells for additional experiments were all selected on their logarithmic growth phase growth (80% covolume).2. Methods2.1 Immunohistochemistry SP method was used to detect expression of SSEA-3 and SSEA-4 in the tissue specimens2.2 Logarithmic phase cells were harvested and 4% paraformaldehyde was added to fix the cells for 30min in 4℃refrigerator. Expression of SSEA-3 and SSEA-4 was immunocytochemically detected with the SP method just as above described.2.3 Scoring method:The intensity of the positivity was divided into the following 4 classes:negative; weak positive (+)…1 point; moderate positive (++)…2 points; strong positive (+++)…3 points. The average positivity according to the number of positive cells was scored as the following classes:negative; weak positive (+) positive cells were 25% or less…1 point; moderate positive (++) positive cells were 25%-49%…2 points; strong positive (+++) positive cells were more than 50%…3 points. Integral formula for calculating an integrated measurement was to multiply the above two scores.Resulting in the followings:<3 negative;>4 positive;>6 strong positive. Five high-power microscope (HP.) fields of vision for each specimen were evaluated and the average value was used in this study.2.4 Statistical analysis:All datas were processed by statistical software SPSS 13.0, and chi-squared test was used. It was considered statistically significant, when P was <0.05.Results:1. Expression of both SSEA-3 and SSEA-4 in breast cancer cell line MDA-MB468:Expressions of both SSEA-3 and SSEA-4 was much high and their positive rates were all above 90% at average.2 Expression of SSEA-3 in tissue specimens2.1 SSEA-3 positive staining could be seen in both malignant and benign breast lesion tissues;2.2 The positive rates of SSEA-3 detected in malignant and benign breast lesion tissues were 34%(17/50) and 16%(8/50), respectively. And expression of SSEA-3 on breast cancer has positive correlation with ER;2.3 No positive staining was observed in normal breast tissues;3. Expression of SSEA-4 in tissue specimens3.1 SSEA-4 positive staining was seen in both malignant and benign breast lesion tissues;3.2 The positive rates of SSEA-4 detected in malignant and benign breast lesion tissues were 58%(29/50) and 36%(18/50), respectively. And its expression in breast cancer has not significant correlation with ER;3.3 No positive staining was observed in normal breast tissues; ConclusionThe results show that SSEA-3 and SSEA-4 antigens are expressed both in benign and malignant breast lesion tissues. Furthermore, there are significant differences for the expression of both SSEA-3 and SSEA-4 in benign and malignant breast tissues as well as in normal breast tissues. The positive rates of SSEA-3 and SSEA-4 in breast cancer are much higher than that in benign breast lesions (P<0.05), whereas in normal breast tissues there is nearly not any positive expression. In breast cancer these two antigens become much more. This indicates that these two antigens may have some relevance with breast cancer. Expression of SSEA-3 and SSEA-4 becomes more during the occurrence of breast cancer, this supplies us new direction for breast cancer research. And these also suggest us that it is valuable to further studies on the specific relevance between these antigens and the occurrence, development and prognosis of breast cancer.In addition, SSEA-3 and SSEA-4 can be detected in breast cancer with the positive rates of 34%(17/50) and 58%(29/50) respectively, and positive rates of these two antigens in breast cancer cells were much high, whereas, in normal breast tissues we found no positive expression of the both antigens. Taking its limited expression and distribution in breast cancer and normal breast tissues into account, these antigens may serve as a kind of potential carbohydrate-based breast cancer vaccine. And this may provide a new hope for us to improve the treatment and prognosis of breast cancer.Expression of SSEA-3 on breast cancer has positive correlation with ER, however, expression of SSEA-4 has not significant correlation with ER in our experiment.There have been studies, which have shown that during the process of embryonic development, expression of SSEA-3 and SSEA-4 is constantly down regulated, and the rate of SSEA-3 reduction in the cells is much higher than that of SSEA-4. Maybe this the reason for that that expression of SSEA-4 in both benign and malignant breast tissues is higher than that of SSEA-3 in our experiment. Furthermore, this may indicate that SSEA-4 may has a much closer relationship with breast cancer...