| Objective:The aim of this study is to make sure whether the subpopulation cells characterized by CD44+CD24-ESA+phenotype exist in primary breast cancer cells of primary cell culture. Mensurate and compare the proportion of CD44+CD24-ESA+subsets in primary cell culture and1028cell line culture. Based on the experimental method and technique founded by the former, make a comparion of the proportion of CD44+CD24"ESA+(CD44+CD24-/low) subsets between triple negative breast cancer and non-triple negative breast cancer, in order to clear the relationship of CD44+CD24-/lowphenotype cell subsets with lymph node metastasis, recurrence, systemic organ metastases, other clinicopathological features and prognosis in the triple negative breast cancer.Methods:1. Make a collection of fifteen fresh tumor tissue after surgery of primary breast cancer from May2011to October2011, and carry on primary cell culture with the collected postoperative tumor issue, continous cell culture with the1028cell line. The CD44+CD24-ESA+subpopulation was separated from primary cell culture and continous cell culture cells by flow cytometry.2. Fresh tumor specimens from eight triple negative breast cancer patients and eight non-triple negative breast cancer patients holding a corresponding clinical stage were collected from May2011to October2011to carry on primary cell culture. The CD44+CD24-ESA+subpopulation was separated from primary cell culture by flow cytometry,and make statistics analysis.3. Sixty postoperative paraffin specimens of triple negative breast cancer patients were collected from2005to2007. In order to make a randomized controlled trial, a number of sixty postoperative paraffin specimens from non-triple negative breast cancer patients holding a corresponding clinical stage were randomly selected among the patients during the same period. Identify the CD44+CD24-/lowsubpopulation by double-staining immunohistochemistry technique. Calculate the percentage of CD44+CD24-/low subpopulation in negative breast cancer patients and non-triple negative breast cancer patients respectively, and make statistics analysis.Results:1.Cultured human primary breast cancer cells successfully. Screened out the CD44+CD24-ESA+subpopulation from primary cultured breast cancer cells, and the proportion of CD44+CD24-ESA+subpopulation detected in primary cell and continous cell were (2.90±1.63)%and(6.32±0.25)%, repectively. Significant differences were observed.(t=5.034, P=0.000)。2. The CD44+CD24-ESA+subpopulation was separated from primary cell culture of triple negative breast cancer and non-triple negative breast cancer by flow cytometry, and the proportion of each was (4.0±0.91)%,(1.6±0.71)%, respectively. Significant differences were observed (t=6.931, P=0.000)3. Identify the CD44+CD24-/low subpopulation by double-staining immunohistochemistry technique. Calculate the percentage of CD44+CD24-/low subpopulation in triple negative breast cancer and non-triple negative breast cancer respectively. The positive proportion of each was60.0%,31.7%respectively. Significant differences were observed (χ2=9.701, P=0.002))4.Significant differences(P<0.05) were observed in lymph node metastasis(83.3%), recurrence(38.9%), systemic organ metastases(61.1%) in the CD44+CD24-/low subpopulation group than in the non-CD44+CD24-/low subpopulation, while the lymph node metastasis(45.8%), recurrence(4.2%), systemic organ metastases(33.3%),respectively. The5-year overall survival rates was50.0%and75.0%in the two group(χ2=4.882, P=0.027).Conclusions1.Cultured human primary breast cancer cells successfully. Separated the CD44+CD24-ESA+subpopulation using CD44+CD24-ESA+marks by flow cytometry from the primary breast cancer cells and the1028cell line successfully, and confirmed the presence of the CD44+CD24-ESA+subpopulation in the primary breast cancer cells.Discoveried that the percentage of CD44+CD24-ESA+cells in the1028cell line was significantly higher than in the primary breast cancer cells.2. The CD44+CD24-ESA+subpopulation was successfully separated from primary cell culture of triple negative breast cancer and non-triple negative breast cancer by flow cytometry. Discoveried that the percentage of CD44+CD24-ESA+cells in triple negative breast cancer patients was significantly higher than in non-triple negative breast cancer.A tentative judgement that the poor prognosis of the triple negative breast cancer related with the high proportion of CD44+CD24-ESA+subsets was made. 3.Identify the CD44+CD24-/low subpopulation by double-staining immunohistochemistry technique. Discoveried that the proportion of CD44+CD24-/low cells in the triple negative breast cancer patients group was significantly higher than the non-triple negative breast cancer patients.4.The final statistical results confirmed that CD44+CD24-/low cells subsets correlated with lymph node metastasis, recurrence, systemic organ metastasis of triple negative breast cancer. Furthermore, the poor prognosis of the triple negative breast cancer was confirmed relating with a higher proportion of CD44+CD24-/low cells subsets. |
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