Isolation And Identification Of Triple Negative Breast Cancer Stem Cells And The Preliminary Research On The Biological Behaviours

Background and purpose:Breast cancer stem cells are considered as the important causes not only for the heterogeneity of breast cancer, but also for the source of breast cancer invasion and metastasis. With the capacity of high invasion and heterogeneity, triple negative breast cancer (TNBC) may be related to breast cancer stem cells. This study aimed to explore the correlations of different subtypes of breast cancers especially TNBC with Aldehyde dehydrogenase1(ALDH1)+and CD44+/CD24-phenotypes in the primary breast cancer tissues of Chinese population and cell lines, and then take TNBC cell line BT20for example, to observe the biological behaviors including proliferation, adhesion, invasion, migration, drug sensitivity and tumorigenicity as well as the expression of stem-cell-associated markers of CD44+/CD24-cells sorted from BT20cell line.Methods:To analyze the correlations between ALDH1+, CD44+/CD24-phenotypes and different breast cancer subtypes, immunohistochemistry and flow cytometry were performed in101breast cancer tissues from Chinese population and10breast cancer cell lines respectively. The expression of stem-cell-associated markers in10cell lines presented with different subtypes was detected by Western blot. Meanwhile, mammospheres were cultured from these10cell lines in serum-free stem cell medium. CD44+/CD24-cells were sorted from BT20cell line and then compared with non CD44+/CD24-cells and/or the parental cells about the capacity of proliferation, adhesion, invasion, migration, drug sensitivity and tumorigenicity. The expression of stem-cell-associated markers were detected by Western blot.Results:The expression rates of ALDH1+and CD44+/CD24-phenotypes differed significantly among different breast cancer subtypes (P=0.001), with the higher expression rate in TNBC compared with non-TNBC (44.0%vs18.4%, P=0.016;84.0%vs39.5%, P<0.001). The distribution of breast cacner cases expressed both ALDH1+and CD44+/CD24-phenotypes in TNBC was significantly more than that in non-TNBC (44.4%vs11.8%, P=0.013). Similarly, more ALDH1+and CD44+/CD24-cells were found in TNBC cell lines compared with those in non-TNBC cell lines (15.64%±2.45%vs4.44%±2.28%, P=0.01;74.24%±12.18%vs3.09%±2.91%, P<0.001). ALDH1 positive cells were more frequently found in ER negative than in ER positive breast cancers (36.2%vs9.3%; P=0.002), while CD44+/CD24-phenotype was correlated with HER-2negative (62.1%vs34.9%; P=0.007). Mammoshperes with the capacity to enrich CD44+/CD24-cells were successfully cultured from CAL-51and BT20cell lines which both belong to TNBC cell lines. In addition, compared with non CD44+/CD24-cells and/or the parental cells, CD44+/CD24-cells exhibited the lower adhesion rate and the higher capacity of proliferation, invasion, migration and tumorigenicity, as well as lower sensitivity to cisplatin but higher sensitivity to sunitinib and sorafenib. The results of western blot showed the higher expression of CXCR4, MMP3, ABCG2, β-catenin and OCT4as well as the lower expression of E-cadherin in CD44+/CD24-cells compared with those in non CD44+/CD24-cells.Conclusions:The expression rates of ALDH1+and CD44+/CD24-phenotypes as well as the formation efficiency of mammoshperes were higher in TNBC. The results suggested that the higher proportion of breast cancer stem cells existed in TNBC. This study was first discovered that TNBC stem cells had the higher capacity of proliferation, migration, invasion, tumorigenicity and resistance to chemotherapy. Hence, TNBC stem cells may be the causes of the recurrence, metastasis and poor prognoses of patients with TNBC.