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Keywords:

  • CD90;
  • Thy-1;
  • breast cancer;
  • cancer stem cell;
  • CD14;
  • breast cancer stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. © 2012 International Society for Advancement of Cytometry


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Breast cancer is the leading cause of cancer-related death in women worldwide (1, 2). In most patients, death is caused by the metastatic disease, which may evolve from the primary tumor. Emerging data suggest that one of the mechanisms accounting for progression of this tumor is the existence of subpopulations of cells present within human cancers, which display stem cell properties. These cells are defined to possess the capacity of self-renewal and differentiation into all of the heterogeneous cancer cells lineages, which comprise the tumor. In particular, cancer cells with stem cell–like properties have been proposed to play a critical role in metastatic progression and tumor resistance to commonly used cancer treatment (3, 4).

The current methods for determining whether cell populations isolated from solid tumors are in fact cancer stem cells (CSCs) involve purification of these cells, by cytometric technologies (5), from tumor samples, based on the properties of normal stem cells, namely: the expression of specific cell surface markers. Using this methodology, Al-Hajj et al. (6) identified and isolated breast CSCs using the combined expression of CD44+/CD24−/low/lin. This subpopulation of cells is highly enriched for cells, which are able to initiate tumors, can serially propagate tumors in mice (illustrating their capacity of self-renewal) and are resistant to conventional chemotherapy (7).

Although evidence suggests that the CD44+/CD24−/low/lin5 population may represent the breast CSC, recently, some studies have demonstrated that the clinical value and prognostic significance of these markers in breast cancer are uncertain. One report has found that the CD44+/CD24 phenotype exerts no significant impact on patients' prognosis (8). Conversely, Ahmed et al. (9) demonstrated that patients with tumors exhibiting the CD44/CD24+ phenotype had the worst prognosis, compared to tumors rich in CD44+/CD24 cells. Furthermore, Abraham et al. (10) suggested that CD44+/CD24 tumor cells in breast cancer may not be associated with the clinical outcome or the patients' survival, emphasizing the fact that the putative tumorigenic ability may not be limited to cells displaying this phenotype and, also, that other breast CSC markers remain to be identified.

Seeking for new breast CSC markers, which are truly associated with the degree of breast cancer malignancy, we have analyzed 10 stem cell markers, characterized according to the International Society for Cellular Therapy (11), in human breast cancer cell lines displaying different degrees of malignancy. Our results indicate that CD90 and CD14, two commonly used stem cell markers, are differentially expressed in these cell lines and might be related to their malignancy grade, constituting interesting markers that should be further studied to assess their actual role in human breast cancer.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Cell Culture Material

The following items were used to cultivate the cells: DMEM/F12, RPMI, or DMEM medium (Gibco, Rockville, MD), fetal calf serum (FCS; Atená Biotecnologia, Campinas, SP, Brazil), streptomycin/ampicillin (Sigma-Aldrich, St. Louis, MO), epidermal growth factor (Gibco), cholera enterotoxin (Sigma-Aldrich), insulin (Sigma-Aldrich), and cortisol (Sigma-Aldrich).

Cell Culture

The nontumorigenic human MCF-10A breast cell line was grown as described by Soule et al. (12). The breast cancer cell lines MCF-7, MDA-MB-213, and MDA-MB-435 were cultured in RPMI supplemented with 10% FCS and 1% streptomycin/ampicillin. The Hs578-T human breast cancer cell line was cultured in DMEM, supplemented with 10% FCS and 1% streptomycin/ampicillin. All cultures were maintained at 37°C under a water-saturated atmosphere containing 2% CO2. In the Supporting Information, the features of breast cancer cell lines used in this study are shown.

RNA Isolation and Gene Expression Analysis by Quantitative Reverse Transcriptase Polymerase Chain Reaction

Total RNA was isolated using the RNAEasy kit. The RNA quality control was evaluated through the 280/260-nm and 230/260-nm absorbance ratios and by observing the 18S and 28S band integrity in denaturing gels. cDNA was obtained using Super Script III as recommended by the manufacturer. Amplification of the resulting cDNAs was performed using the SYBR GREEN Dye (Life Technologies), under the following conditions: 50°C for 2 min, 95°C for 10 min, 40 rounds of 95°C for 15 s and 60°C for 1 min. A dissociation cycle was performed after each run to check for nonspecific amplification or contamination. The mRNA expression of GAPDH, HPRT, and H-MBS genes was subjected to the GeNorm computational program analysis (13). The GAPDH and HPRT transcriptional expression levels, classified as the two most stable genes according to the GeNorm analysis, were used to calculate the GeNorm normalization factor utilized as the endogenous control for the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The amplification efficiency analyzed was calculated for each gene from the given slope in a linear regression curve of Ct values versus log of cDNA concentration. The corresponding PCR efficiency (E) of one cycle in the exponential phase was calculated according to the equation: E = 10[−1/slope]. Relative expression levels were calculated according to the previously described model by Pfaffl (14). Supporting Information Table 1 shows the primers used with their optimal concentrations (in the range of 10–600 nM).

Flow Cytometry Materials

All experiments performed comply with the MIFlowCyt standard (15) (Supporting Information). In brief, for the analysis of surface markers, we used the antibodies CD44 (Cat. No. 559942: mouse monoclonal [G44-26] to CD44 APC, abs/em = 650/660 nm), CD90 (Cat. No. ab11155: mouse monoclonal [F15-42-1] to CD90/Thy1 FITC, abs/em = 488/525 nm) (Abcam, Cambridge, United Kingdom), CD14 (Cat. No. ab25390: mouse monoclonal [61D3] to CD14 PE/Cy5.5®, abs/em = 488/694 nm) (Abcam) and with double tagging CD90+/CD14. The cells were analyzed on a BD FACSAria I flow cytometer (BD Bioscience, Franklin Lakes, NJ).

Flow Cytometry Analysis

For analysis of the surface markers, the cell culture plates were placed in crushed ice and detached to single cell suspensions using ice-cold 0.5M EDTA (Versene) solution. The cells were counted and then washed with phosphate-buffered saline (PBS). After blocking for 1 h in 5% bovine serum albumin, a total of 1 × 106 cells were incubated with specific antibodies for 60 min at room temperature. Unbound antibody was washed out through two cycles of washing with PBSA and cells were analyzed on a BD FACSAria flow cytometer. The data were analyzed using FlowJo 7.6 and at least 50,000 events per sample were collected.

Statistical Analysis

The results were analyzed for Gaussian distribution and passed the normality test. The statistical differences between group means were tested by the one-way ANOVA with Tukey post hoc test or Games-Howell for different variances if necessary using IBM SPSS Statistic software. A value of P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Stem Cell Markers Are Differentially Expressed in Breast Cancer Cell Lines

In order to compare the relative mRNA expression of stem cell markers between breast cancer cell lines and a nontumorigenic breast cell line we used quantitative RT-PCR. The results show that ABCG2, often used to isolate a subpopulation with stem cell–like characteristics, was expressed in the noninvasive and nonmetastatic MCF-7 breast cancer cell line (Fig. 1A; MCF10-A vs. MCF: 7 25 ± 2 fold [mean ± SEM]), corroborating the literature, which reports that expression of this gene is repressed during the epithelial-mesenchymal transition (16). As shown in Figure 1, the stem cell marker CD29 (Fig. 1B), CD31 (Fig. 1C), and CD105 (Fig. 1D), as well as the breast CSC markers, CD24 and CD44 (Figs. 1E and 1F, respectively), were expressed in MCF10-A cells and at in least one tumor cell line. While c-kit mRNA expression was not significantly different between the panel of cell lines tested (Fig. 1G), CD200 mRNA (Fig. 1H) was only detected in MDA-MB-435 cells. It is important to note that the origin of this cell line is controversial, since it is speculated to be a melanoma-derived cell line rather than a breast-cancer derived one (17). Interestingly, our results show that CD90 and CD14 were differentially expressed according to degree of aggressiveness-malignancy displayed by the cell lines isolated from primary breast tissues (MCF10-A and Hs578-T), as shown in the MIflowCyt Standard (Supporting Information). In fact, the highly invasive and metastatic Hs578-T cells presented the highest CD90 mRNA levels (Fig. 1I; MCF10-A vs. Hs578-T: 1,676.000 ± 96.795), while the level of CD14 mRNA expression was higher in the nontumorigenic MCF-10A cells (Fig. 1J; MCF10-A vs. MCF-7: 0.00030 ± 0.00002-fold; vs. MB-MDA-231: 0.032 ± 0.005; vs. MB-MDA-435: 0.00080 ± 0.00007; Hs578-T: 0.014 ± 0.004).

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Figure 1. Analysis of stem cells markers in normal and cancer mammary cell lines by qRT-PCR. The nontumorigenic MCF-10A breast cell line and cancer MCF-7, MDA-MB-213, MDA-MB-435, and Hs578-T cell lines were maintained as described in the Methods section. Upon reaching 80% confluence, total RNA was extracted and the expression levels of different stem cell markers were analyzed by qRT-PCR: (A) ABCG2, (B) C-KIT, (C) CD14, (D) CD24, (E) CD29, (F) CD31, (G) CD44, (H) CD90, (I) CD105, and (J) CD200. The normalization genes used in Genorm were HPRT and GAPDH. The asterisk shows statistically different expression of mRNA (P < 0.05). Quantitative RT-PCR was carried out in triplicate (n = 3 independent experiments).

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Identification of Stem Cell Markers in Breast Cancer Cell Lines by Flow Cytometry

In order to further investigate the protein expression levels of the set of genes displaying a higher differential profile, we estimated the presence of protein stem cell markers on the cell surface of the same panel of human breast cancer cell lines that was used for the qRT-PCR studies. In agreement with the observed mRNA expression levels, Hs578-T cells displayed the highest proportion of CD90-positive cells (more than 90%), indicating a large amount of cells expressing this membrane marker (Fig. 2A). The opposite was observed for CD14, for which an enriched (greater than 60%) subpopulation is found in MCF10-A cells (Fig. 2B), being almost absent in the malignant Hs578-T (Fig. 2A). The CD90+CD14 set of markers could be useful to establish a complete separation between the cells presenting the greatest difference in malignant potential such as MCF10-A and Hs578-T cells. Interestingly, the other cell lines, namely: MCF-7, MDA-MB-231, and MDA-MB-435, displayed intermediary levels of expression for these two markers (Fig. 2C). Indeed, the individual histogram distribution, shown in Supporting Information Figure 1, supports this statement. When we analyzed the CD44/CD90 double tagging, we observed that both the MCF10-A and Hs578-T cell lines displayed a subpopulation of CD44-positive cells, whereas CD90 expression was only detected in the most malignant cell line, namely, Hs578-T (Figs. 2A and 2B). The percentages of cells in each quadrant are indicated in Supporting Information.

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Figure 2. Flow cytometric analysis of surface markers in normal and breast cancer cell lines. The MCF10-A and Hs578-T cell lines were maintained as described in the Methods section; 106 single-cell suspensions of Hs578-T (A) and MCF10-A (B) was stained with anti-human CD90, CD14, CD44, double staining CD90/CD44, or CD90/CD14. The percentage of the CD90+/CD14 subpopulation in the MCF-10A, MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T cell lines is represented in (C). Cells were analyzed in a BD FACSAria flow cytometer. The percentages of cells in each quadrant are indicated in the Supporting Information. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Here, we have analyzed 10 different stem cell markers in human breast cancer cell lines, seeking for a set of markers which could, in fact, be associated with the degree of breast cancer malignancy. Among all markers analyzed, CD90 and CD14 were the most interesting ones. Differential expression of these markers was not detected in the malignant cell lines isolated from pleural effusion (MCF-7, MDA-MB-231, and MDA-MB-435), but only in malignant and nonmalignant cell lines isolated from primary breast tissue (Hs578-T and MCF10-A, respectively) (18), suggesting that the phenotype of breast cancer cell lines depends upon their origin.

The present study reveals that CD90 could be a potential marker of breast CSCs, since CD90+ cells represented more than 90% of the Hs578-T cell line, directly correlating with their high tumorigenicity and metastatic potential.

CD90 or THY-1 was originally discovered as a thymocyte antigen (19, 20) but further reports have shown expression of this protein in human fibroblasts, neurons, endothelial cells, as well as in murine T-cells (21, 22). This protein is involved in T-cell activation (23) and has widespread nonimmunologic functions, including inhibition of neurite outgrowth (24), apoptotic signaling (25), leukocyte and melanoma cell adhesion and migration (26, 27), tumor suppression (28, 29), and fibroblast proliferation and migration (30–32). It is an important marker of mesenchymal stem cells being associated with development, cancer, and CSC. According to the International Society for Cellular Therapy, CD90 is one of three markers (CD90, CD105, and CD73) commonly used to characterize multipotent mesenchymal stromal cells (11). Recent studies demonstrated that the CD90 marker was expressed on hepatic stem/progenitor cells during liver development (33) and in a stem cell population from human adult liver (34, 35). In cancer, Yang et al. (36) found that CD90 expression correlated with the tumorigenic potential of hepatocellular carcinoma cell lines and suggested CD90 as a putative marker for liver CSCs. Furthermore, Liu et al. (37) demonstrated that CD133+ glioblastoma CSCs from primary cultures presented high levels of CD90 mRNA and were resistant to several chemotherapeutic agents. According to True et al. (38), CD90 might serve as a molecular target for therapy and a cancer biomarker in prostate cancer.

In breast cancer, Cho et al. (39) showed that cells displaying the CD90+/CD24+/CD49f+/CD45 immunophenotype, identified in MMTV-wnt-1 murine breast tumors, were highly enriched for cells, which were able to form tumors when transplanted into syngeneic recipients. In humans, Donnenberg et al. (40) demonstrated that breast tumors contain a small population of CD44+/CD90+ localized at the tumor periphery, adjacent to the CD90+ stroma. Both of these reports showed the importance of CD90 in breast cancer. In addition to corroborating these findings, our data indicate the relationship between CD90 expression and the degree of malignancy in human breast cancer cell lines isolated from primary breast tissue.

Interestingly, in contrast with the CD90 expression profile, the CD14 marker apparently is mostly expressed in the nontumorigenic cell line. CD14 is a surface protein, which is expressed in monocytes/macrophages (41, 42), being often used, in the literature, as a stem cell marker (11). This protein is one of the useful markers used to identify mouse mammary progenitor cells (43). Our results show that the proportion of CD14+ cells is higher than 60% in the nontumorigenic MCF10-A cell line and less than 15% in the Hs578-T tumorigenic and metastatic cell line. This may be related to the epithelial features of the MCF10-A cell line, since, according to Kendrick et al. (44), this receptor can be found in the luminal estrogen receptor negative population, being likely that the CD14+ cells play a distinct role within the mammary epithelium as nonprofessional immune cells.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

In the present study, we analyzed the expression of 10 different stem cell markers in different breast cancer cell lines seeking for those which might be related with the malignancy grade of these lines. We demonstrate that two stem cell markers (CD90 and CD14) are differentially expressed in breast tumor cell lines, when compared with nontumorigenic lines of the same tissue origin. These two markers should be further analyzed in human breast cancer samples to confirm the results we obtained using human cell lines. It is important to further investigate the actual role of these protein markers during breast cancer progression, using a functional genomics approach. The study of the CD90 and CD14 markers could open new avenues for breast cancer diagnostics and therapeutics.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

The excellent technical support of Zizi de Mendonça, Sandra Regina de Souza, Debora Cristina da Costa, and Ricardo Krett de Oliveira is deeply appreciated. The authors are also grateful to Dr. Leticia Labriola for fruitful discussions and to Dr. Robert Schumacher for help with the FlowJo compensation analysis. The authors declare no potential conflicts of interest.

Literature Cited

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22220_sm_SuppFig1A.tiff470KSupporting Information Figure 1A. Flow cytometric analysis of markers breast cancer cell lines. The MCF-7, MB-MDA-231 and MB-MDA-435 cell lines were maintained as described in the Methods section. 106 single-cells suspension of these cells lines was stained with anti-human CD90 and anti-human CD14. Histograms display the percentage of cells stained with these markers: (A) MCF-7; (B) MB-MDA-231; (B) MB-MDA-435.
CYTO_22220_sm_SuppFig1B.tiff314KSupporting Information Figure 1B. Flow cytometric analysis of markers breast cancer cell lines. The MCF-7, MB-MDA-231 and MB-MDA-435 cell lines were maintained as described in the Methods section. 106 single-cells suspension of these cells lines was stained with anti-human CD90 and anti-human CD14. Histograms display the percentage of cells stained with these markers: (A) MCF-7; (B) MB-MDA-231; (B) MB-MDA-435.
Supplementary_Table-1.doc73KSupporting Information Table
MIFlowCyt_Item_Location.doc1243KSupporting Information MIFlowCyt

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