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Abstract

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24– cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.

Keywords

Breast cancer stem cell mammosphere CD44+/CD24–MDA-MB-231 SKBR3 MCF7

Introduction

Breast cancer is one of the most common cancers among women. The prevalence of breast cancer in the world has been estimated to reach 2.3 million by 2030.¹ There are two types of breast cancer, ductal and lobular, which are divided into invasive and in situ (non-invasive) types, with several subtypes of breast cancer based on histology properties. Also, there are several subtypes of breast cancer according to molecular characteristics (estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 expression), luminal A and B (ER+/PR+/HER2– or ER+/PR–/HER2–), basal-like triple negative breast cancer (ER–/PR–/HER2–), and HER2+ and claudin low.² These receptors can be used as prognosis markers as well as therapeutic targets.¹ Breast cancer includes a variety of cancer cells such as cancer stem cells (CSCs) and differentiated cancer cells that can complicate breast cancer treatment. In comparison with differentiated cancer cells, breast cancer stem cells (BCSCs) have some specific properties including self-renewal, differentiation, metastasis, migration, and treatment resistance, making tumors more aggressive and progressive.³ These minor cell populations (almost 2% of tumor tissue) express a variety specific markers in different types of breast cancers such as CD44+/CD24–, aldehyde dehydrogenase (ALDH) activity, and epithelial specific antigen (ESA) or CD326 (EpCAM).³ CD24 is an expressed glycoprotein on the surface of differentiated cancer cells.⁴ Although CD44 is a trans-membrane glycoprotein which regulates growth signals in stem cells along with the tyrosine kinase receptor,⁴ it cannot be considered as a strong predictive marker to identify BCSCs in all types of breast cancers.^5,6 A number of studies have identified other markers such as CD133, CD166, Lgr5, CD47, and ABCG2 for the detection of BCSCs in all types of breast cancers.⁷ There are several main methodologies which identify and isolate CSCs from cancer cell lines or primary tumors; these include isolation of CSCs by cell sorting with flow cytometry according to CSCs markers, detection of CSCs by side population phenotypes through hoechst 33342 exclusion, enrichment of the putative CSCs by sphere formation in the serum-free medium, and/or isolation of CSCs through measurement with the ALDH activity. Isolated CSCs should show a reasonable expression of stemness markers and be capable of forming spheres and forming new tumors in vivo.⁸ Forming spheres in the serum-free culture is an efficient way to enrich CSCs from cancer cell lines. However, forming spheres from primary tumors is still a problem for researchers.⁹ Recently, given the therapy-resistant properties of CSCs, it has become important to detect and characterize CSCs to find new approaches for cancer therapy that can directly target CSCs without any effect on the normal epithelial cells through the desired doses of drugs.^10,11 In this study, two luminal subtype cell lines, namely, MCF7 (ER+/PR+/HER2–) and SKBR3 (ER–/PR–/HER2+), and a basal subtype cell line, MDA-MB-231 (ER–/PR–/HER2–), with stemness markers and different molecular properties were chosen. Then, the mammosphere formation efficiency (MFE), CD44+/CD24– cell ratio, stemness genes expression, proliferation rate, migration, drug resistance, and tumorigenicity of mammospheres generated from the cell lines were compared. This study then compared the stemness properties of mammospheres generated from three breast cancer cell lines with different molecular receptors. Isolating BCSCs with high stemness characteristics can be regarded as the first step in this approach.

Material and methods

Culture of cell lines

Three human breast cancer cell lines including MCF7, MDA-MB-231, and SKBR3 with different molecular receptors were purchased from the Pasteur Institute (Tehran, Iran). All lines were tested to confirm that they were mycoplasma free. Cells were maintained in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin, and 1% non-essential amino acids (all purchased from Thermo Fisher Scientific, UK) under the humidified atmosphere, at 37°C, with 5% CO₂.

Mammospheres culture from cell lines

For mammospheres formation, MCF7, MDA-MB-231, and SKBR3 cells were suspended at 25,000 cells/mL and seeded into an agar-coated flask in serum-free DMEM/F12 (1:1) supplemented with 10 ng/mL basic fibroblast growth factor (b-FGF), 20 ng/mL epidermal growth factor (EGF), and 2% B27 (all purchased from Sigma, USA). Growth factors were added every 2 days to the medium. In this condition, cells were grown as non-adherent spheres (called mammosphere) with a size of >60 µm. Primary mammospheres were collected every week by centrifugation and dissociated to single cells by trypsin/ethylenediaminetetraacetic acid (EDTA) 0.05% (v/w). For secondary mammospheres formation, Lombardo et al.’s protocol was used in this study.¹² Mammospheres were passaged during 3 weeks on the days 7, 14, and 21.^13–15

MFE

A number of mammospheres with a size range of 60–200 µm were acquired from MCF7, MDA-MB-231, and SKBR3 in each flask and counted under microscope on days 7, 14, and 21. For the detection of the long-term culture and regeneration of mammospheres, mammospheres forming efficiency (%) was calculated based on the following ratio

MFE (%) = \frac{(number of mammospheres per well)}{(number of cells seeded per well)} \times 100

Flow cytometry

Mammospheres from MCF7, MDA-MB-231, and SKBR3 were collected and enzymatically dissociated into single cells via 0.05% trypsin/EDTA. Mono-colonal antibodies against human CD44-FITC and CD24-PE (BD Biosciences, USA) were added to the cell suspension with the recommended concentration and incubated at room temperature in dark for 30 min. Then, the cells were washed with phosphate-buffered saline (PBS) and analyzed by the FACSCalibur flow cytometer (Becton Dickinson, USA).

Real-time polymerase chain reaction (PCR)

Real-time PCR was used for the detection of stemness gene expression (Nanog and Oct4) in the mammospheres. RNA extraction and cDNA synthesis were performed by trizol (Sigma) and the TaKaRa cDNA synthesis kit (TaKARA, Japan), respectively. SYBR Green PCR Master Mix (TaKaRa) was used for the amplification of genes with specific primers for human Nanog (forward: 5′-CAGCTACAAACAGGTGAAGAC-3′ and reverse: 5′-TGGTGGTAGGAAGAGTAAAGG-3′) and Oct4 (forward: 5′-TCTATTTGGGAAGGTATTCAGC-3′ and reverse: 5′-ATTGTTGTCAGCTTCCTCCA-3′). Meanwhile, GAPDH was used as a reference gene (forward: 5′-CCACTCCTCCACCTTTGACG-3′ and reverse: 5′-CCACCACCCTGTTGCTGTAG-3′).

Western blotting

Immunoblotting was performed for detection of stemness protein expression (Nanog and Oct4) in the mammospheres. Briefly, proteins were extracted by lysis buffer. Protein concentration was detected by Bradford assay. Equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PDVF) membrane (Bio-Rad, USA). Membranes were incubated with primary antibodies (Rabbit anti-Oct4 (Lifespan Biosciences, USA), Rabbit anti-Nanog (Abcam, UK), and human anti-GAPDH antibody) according to antibody data sheet for 2 h at room temperature. Then the membranes were incubated with an appropriate secondary antibody (horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, USA) and HRP-conjugated goat anti-mouse IgG (Dako, Denmark)) for 1 h at room temperature. Protein bands were visualized by an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, USA).

Brdu proliferation rate

For the measurement of 5-bromo-2-deoxyuridine (BrdU) incorporation into DNA, cell lines and mammospheres-derived cells were plated at a seeding concentration of 4 × 10⁵ in the T25 flask. Twenty-four hours after cell seeding, Brdu was added to the culture medium at the final concentration of 10 µM. After 24 h (approximately 60% confluency of the cell), cells were collected, washed, and prepared for antibody staining according to the recommended procedure.¹⁶ Anti-Brdu and goat anti-mouse IgG (both obtained from Sigma) were used as the primary and secondary antibodies, respectively. Then, cells were analyzed by flow cytometry.

Migration assay (scratch assay)

Scratch assay was used for the detection of mammospheres-derived cells migration. Cell lines and mammospheres-derived cells were plated in 6-well dishes at a seeding concentration of 2 × 10⁵ cells/well. In order to arrest proliferation, 20 µg/mL mytomycin (Sigma) was added to the culture dishes with 80%–90% confluency for 2 h. A thin scratch/wound was created through scratching with a 100 µL pipette tip. Images were taken by an Olympus camera (Japan) attached to an inverted microscope at 40× magnifications. Migration rate was detected with the modulation of distance between the two lines of scratch being 0, 6, 12, and 24 h after scratching. Wound closure (%) was evaluated based on the following ratio

% Wound closure = \frac{A_{Δ h}}{A_{0 h}} \times 100 %

where A stands for the size of gap (µm) and 0h is the start time, while Δh stands for the time difference of the second measurement of scratch with the start time.

MTS assay

Doxorubin (Dox) is one of the most effective and active chemotherapy drugs used for the patients with the invasive and non-invasive breast cancer.¹⁷ The cell viability of MCF7, MDA-MB-231, SKBR3, and their derived mammospheres in the normal and treatment conditions was measured by the MTS assay. In brief, cells were transferred into a single-cell suspension and plated into 96-well plates with a density of 5 × 10³–10⁴ cells/100 μL per well. Cells were cultured in the normal condition and treated with different doses of Dox (0–100 µg/mL) at 37ºC for 24 h. The twenty μL MTS solution (Sigma) was added per well and incubated at 37ºC for 3 h. Finally, optical density (OD) was measured under the 492-nm wavelength, and the survival rates were calculated.¹⁸

Chick chorioallantoic membrane (CAM) tumor formation assay

CAM in chicken embryo has been identified as an immune-deficient host model for tumor formation in vivo.^19–22 In order to inoculate cells onto the CAM, on day 3 (starting from the day of hatched chicken egg incubation), 2-mL albumin was withdrawn and a window was opened in the shell with an electric drill equipped with a 0.75-inch wheel tip (Dremel Moto-Tool; Emerson Electric Co., USA). After 9 days of incubation, cells were inoculated carefully onto the CAM, the top of the ectoderm layer at a concentration of 10⁶ cells/100 µL of the extracellular matrix (ECM) without touching CAM. Window was closed with parafilm, and the embryo was incubated for up to 5 more days. This experiment was repeated 6 times. On day 14, the embryos were opened and the CAMs were dissected out to determine the size and volume of the formed tumors on the top of CAM. A dissecting microscope determined the size of tumors.^21,23 The volume of tumors was calculated according to the following formula

V = \frac{(W^{2} \times L)}{2}

where W is the width and L is the length of the tumor.

Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)

Tumors were fixed in 10% formalin, sectioned and prepared for H&E staining and IHC, according to the Lokman²² protocol. Cytokeratin antibody (Abcam) was used for detecting invasion and migration into the mesoderm and endoderm layers. Images from each tumor were assessed by a pathologist. Detection and quantification of IHC was performed by Image J.

Statistical analysis

The results are presented as mean ± SEM for at least three independent experiments for each analysis. Student’s t-test with the p values of 0.05 or less was used to indicate statistically significant differences.

Results

MFE during three passages in MCF7, MDA-MB-231, and SKBR3

Mammospheres with a size over 60 µm were obtained from three cell lines including MCF7, MDA-MB-231, and SKBR3, which had been formed after 7 days in the CSCs specific culture medium (Figure 1(a)). Secondary and tertiary mammospheres, derived from primary and secondary mammospheres, respectively, were formed and MFE was calculated through counting mammospheres during passages 1–3 (P1–P3, respectively). MFE for MCF7 was 0.08, 0.15, and 0.33, while for MDA-MB-231, it was 0.05, 0.11, and 0.22; also, for SKBR3, it was 0.03, 0.08, and 0.19 in passages 1–3, respectively. Data showed that MFE was significantly increased during three passages in all three types of breast cancer cell lines (p < 0.05). Among cell lines, MCF7 had the highest MFE in passages 1–3 (Figure 1(b)).

Figure 1.

The protocol used for mammospheres formation and characterization of them. (a) Enrichment of breast cancer stem cells (BCSCs) for mammospheres formation in CSCs specific culture medium during 3 weeks (3 passages, P stands for passage). As shown, the distinct morphology of mammospheres emerged after passage 3. Bar was 200 µm. (b) Mammospheres with a size over 60 µm were evaluated. Bar was 100 µm. (c) Quantitative mammospheres forming efficiency (MFE) of MCF7, MDA-MB-231, and SKBR3. As detailed in the “Material and methods” section, MFE was during 3 passages of mammospheres. MCF7 showed the highest efficiency of mammospheres formation. (d) Flow cytometry detection of CD44 and CD24 markers on the surface of MCF7, MDA-MB-231, and SKBR3 cell lines (2D) and cell lines–derived mammospheres (3D) during 3 passages. (e) Percentages of CD24 positive cells in MCF7, MDA-MB-231, and SKBR3 are shown after passage 3 and compared with their originated cell lines. Please note that the percentage of mammosphere CD24 positive cells was significantly lower than their level counterpart in CD24 in MCF7 and SKBR3 cell lines (p < 0.05).

CD24 and CD44 expression analysis in MCF7, MDA-MB-231, and SKBR3 (2D), and cell lines–derived mammospheres (3D)

The first step to confirm the emerging mammospheres (3D) involved the use of BCSCs for the detection of the CD44 marker on the surface of mammospheres-derived cells. Flow cytometry results for CD44 and CD24 markers in three consecutive passages of mammospheres (3D) of MDA-MB-231 and the originated breast cancer cell line (2D: MDA-MB-231) showed the high expression rate of CD44 in three consecutive passages (P1, P2, and P3). However, it was not considerable in MCF7, SKBR3, and their derived mammospheres. Furthermore, the expression of the CD24 marker in the MCF7 cell line was 67.99%; while in MCF7-derived mammospheres during passages 1–3, it was shifted to 58.09%, 52.43%, and 44.63%, respectively. A similar trend was observed for the CD24 expression in SKBR3, which was 97.2% and then changed to 84.31%, 68.31%, and 59.09% in SKBR3-derived mammospheres within passages 1–3, respectively. Despite the steady-state level of the CD44 marker in MCF7 and SKBR3 mammospheres, the level of CD24 (differentiation marker) was decreased during three passages in MCF7 and SKBR3 mammospheres–derived cells (Figure 1(c)). CD24 expression was significantly decreased during passage 3 in both MCF7- and SKBR3-derived mammospheres (p < 0.001; Figure 1(d)). CD24 decreasing rate in MCF7-derived mammospheres in passage 3 was 23.36%, while it was 38.11% in SKBR3 ones. It has been shown that CD24 decreasing rate in SKBR3-derived mammospheres is more prominent in comparison to MCF7 ones.

mRNA expression analysis of Nanog and Oct4 in MCF7, MDA-MB-231, SKBR3 (2D), and cell lines–derived mammospheres (3D)

Real-time PCR was used for the detection of stemness markers (Nanog and Oct4) expression levels in MCF7, MDA-MB-231, and SKBR3 (2D), as well as their derived mammospheres (3D). Expression levels of Oct4 in MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres indicated a 2.5, 4.5, and 4.9-fold increase, as compared to MCF7, MDA-MB-231, and SKBR3, respectively. Among three different types of mammospheres, SKBR3-derived mammospheres showed the highest difference rate in the Oct4 expression level, in comparison with its derived cell line. Also, the expression level of Nanog in MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres was found to shown an 8, 68.9, and 11.8-fold increase, as compared to MCF7, MDA-MB-231, and SKBR3, respectively. MDA-MB-231-derived mammospheres showed the highest difference rate in the Nanog expression, in comparison with other cell lines–derived mammospheres. The obtained data, therefore, showed that all three types of mammospheres had a significantly higher expression of Nanog and Oct4, in comparison with their derived cell lines (p < 0.05) (Figure 2(a) and (b)).

Figure 2.

Detection of stemness markers expression in MCF7, MDA-MB-231, SKBR3, and their derived mammospheres. (a) Detection of Oct4 gene expression in MCF7, MDA-MB-231, SKBR3 (2D), and cell lines–derived mammospheres (3D). Cell lines–derived mammospheres showed a more significant transcript level of Oct4 in comparison to their respective breast cancer cell lines. (b) Detection of Nanog gene expression in MCF7, MDA-MB-231, SKBR3 (2D), and cell lines–derived mammospheres (3D). Cell lines–derived mammospheres showed a more significant mRNA expression level of Nanog in comparison to the breast cancer cell lines. All relative expressions were quantified and normalized with the GAPDH transcript level. (c) Detection of Oct4 and Nanog protein expression in MCF7, MDA-MB-231, SKBR3 (2D), and cell lines–derived mammospheres (3D). (d) Cell lines–derived mammospheres showed a more significant protein level of Oct4 in comparison to their respective breast cancer cell lines. (e) Cell lines–derived mammospheres showed a more significant protein expression level of Nanog in comparison to the breast cancer cell lines. All relative expressions were quantified and normalized with the GAPDH protein level. Represented value bars are the mean of triplicate independent experiments ± SEM.

Protein expression analysis of Nanog and Oct4 in MCF7, MDA-MB-231, SKBR3 (2D), and cell lines–derived mammospheres (3D)

Western blotting was used for the detection of stemness markers (Nanog and Oct4) expression levels in MCF7, MDA-MB-231, and SKBR3 (2D), as well as their derived mammospheres (3D) (Figure 2(c)). Expression levels of Oct4 in MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres indicated a 1.8, 2, and 2-fold increase, as compared to MCF7, MDA-MB-231, and SKBR3, respectively. Also, the expression level of Nanog in MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres was found to shown a 1.5, 4.1, and 2.5-fold increase, as compared to MCF7, MDA-MB-231, and SKBR3, respectively. The obtained data, therefore, showed that all three types of mammospheres had a significantly higher expression of Nanog and Oct4, in comparison with their derived cell lines (p < 0.05) (Figure 2(d) and (e)).

Proliferation rate in MCF7, MDA-MB-231, and SKBR3 (2D), and cell lines–derived mammospheres (3D)

Brdu proliferation assay was used for the detection of the proliferation rate and Brdu incorporation in DNA through the treatment of cells with Brdu for 24 h. The results obtained from the flow cytometry showed the increment in the proliferation rate of MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres, which was a 1.43, 1.24, and 1.74-fold increase, as compared to MCF7, MDA-MB-231, and SKBR3 cell lines, respectively. Although this increase in the proliferation rate was significant in all three types of mammospheres (p < 0.05), the difference in the proliferation rate between SKBR3 and SKBR3-derived mammospheres was higher than that in the other groups (Figure 3).

Figure 3.

Detection of proliferation rate in MCF7, MDA-MB-231, and SKBR3 (2D), and the related mammospheres (3D). Brdu proliferation rate assay showed a more significant proliferation rate of cell lines–derived mammospheres in comparison to their originated cancer cell lines. Represented value bars are the mean of triplicate independent experiments ± SEM.

Migration rate in MCF7, MDA-MB-231, and SKBR3 (2D), and cell lines–derived mammospheres (3D)

A wound-healing assay and the calculation of gap filling after 0, 6, 12, and 24 h were used for the detection of the migration rate. At first we detected CD24 rate of cells, 24 h after attachment of cancer cells–derived mammospheres to dish supplemented with DMEM/F12 and 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin, and 1% non-essential amino acids. No significant difference was observed in CD24 rates of adherent mammospheres-derived cells in comparison to non-adherent MCF7- and SKBR3-derived mammospheres (Supplementary Figure 1). A series of images and calculation of the migration rate after 0, 6, 12, and 24 h showed that all types of cell lines (2D) and their derived mammospheres (3D) initiated to migrate after 6 h; however, the migration rate of monolayers from MDA-MB-231-derived mammospheres was significantly higher than that of monolayers from MDA-MB-231 (p < 0.05; Figure 4(b)). Following the 6-h post-scratch, there was not any significant increase of migration for MCF7- and SKBR3-derived mammospheres, in comparison with MCF7 and SKBR3 cell lines, while after 12 h, the migration rate was significantly higher for MDA-MB-231- and SKBR3-derived mammospheres, in comparison with their cell lines (p < 0.05) (Figure 4). Finally, 24 h later, the migration rate was significantly higher in all types of cell lines–derived mammospheres (p < 0.05; Figure 4(a)–(c)). MCF7-derived mammospheres showed significant increased migration only after 24 h, in comparison with MCF7 (p < 0.05). After 24 h, the migration rate of MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres showed a 1.78, 1.3, and 2-fold increase, as compared to MCF7, MDA-MB-231, and SKBR3, respectively (Figure 4(d)). However, while MDA-MB-231 and MDA-MB-231-derived mammospheres showed the highest migration after 24 h (77% and 100%, respectively), monolayer mammospheres of SKBR3 displayed the higher migration rate in comparison to the corresponding cell line. This difference in the migration rate of SKBR3-derived mammospheres was consistent with the other cell lines (MDA-MB-231 and MCF7).

Figure 4.

Comparative analysis of the migration rates of MCF7, MDA-MB-231, and SKBR3-derived mammospheres monolayers with their cell lines monolayers. (a) A series images of the scratch assay from cancer cell lines and the derived mammospheres after 0, 6, 12, and 24 h of post-scratch. Cell lines were (a) MCF7, (b) MDA-MB-231, and (c) SKBR3. Bar was 200 µm. (d) Quantification of the migration rate of cancer cell line and their derived mammosphere monolayers. As shown, following the 6-h post-scratch, MDA-MB-231-derived mammospheres migrated more than their respective cell lines (p < 0.05). Twelve hours after the scratch, the migration rate of MDA-MB-231- and SKBR3-derived mammospheres was significantly higher than their cell lines (p < 0.05). Finally, 1 day later, the migration rate was significantly higher in all types of mammospheres (p < 0.05). Represented value bars are the mean of triplicate independent experiments ± SEM.

Drug resistance in MCF7, MDA-MB-231, and SKBR3 (2D), and cell lines–derived mammospheres (3D)

MTS assay was used to detect the toxicity potential of Dox on MCF7, MDA-MB-231, SKBR3, and their derived mammospheres. Increased concentrations of Dox (0–100 µg/mL) were used for 24 h. The results demonstrated that Dox at the concentration of 5 µg/mL induced the cell death in MDA-MB-231 and SKBR3. Also, the results showed that the higher concentration of Dox affected cell death in MDA-MB-231 and SKBR3 more considerably. However, the treatment with the increased concentration of Dox showed a significant drug resistance in MDA-MB-231- and SKBR3-derived mammospheres (p < 0.05). LD50 for MDA-MB-231 and MDA-MB-231-derived mammospheres were 10 and 40 µg/mL and for SKBR3 and SKBR3-derived mammospheres were 5 and 100 µg/mL, respectively. Although MCF7 showed drug resistance to the increased concentration of Dox, MCF7-derived mammospheres showed more drug resistance (p < 0.05; Figure 5).

Figure 5.

Drug resistance of MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres in comparison with their corresponding cell lines. Cell viability of cancer cell lines and derived mammospheres through the increasing concentration of Dox after 24 h. Cell lines were (a) MCF7, (b) MDA-MB-231, and (c) SKBR3. Although MCF7 showed drug resistance to Dox in the higher concentration of 20 µg/mL, MCF7-derived mammospheres displayed more drug resistance (p < 0.05). Also, MDA-MB-231- and SKBR3-derived mammospheres displayed more drug resistance to Dox in comparison with their corresponding cell lines (p < 0.05).

Tumorigenicity of MCF7, MDA-MB-231, and SKBR3, and cell lines–derived mammospheres in the CAM of chicken embryo

The CAM model, which has been used as the in vivo tumorigenicity assay, is a low cost and in vivo efficient model for cancer research to detect proliferation, angiogenesis, invasion, and metastasis of cancers.^19,22,24 Also, this model has been used to confirm the anti-tumor activity of drugs on the breast and ovarian cancer.^22,25 Therefore, in this study, to evaluate the tumorigenicity differences between breast cancer cell lines–derived mammospheres and their cell lines in vivo, cancer cells were inoculated on the CAM of chicken embryo, on the ectoderm layer. After 5 days, visible tumor masses were produced in all testing groups. Average size and volume of tumors were measured (Figure 6(a)). The average size of tumors from MCF7, MDA-MB-231, and SKBR3 (2D) was calculated to be 1.5, 3, and 3.2 mm, respectively, whereas the average size of tumors from MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres (3D) was 3.3, 3, and 3.4 mm, respectively. The data, therefore, showed that the average size of MCF7-derived mammospheres (3D) was significantly more than that of tumors from MCF7 (2D) (p < 0.05; Figure 6(b)). The increased size of tumors from SKBR3-derived mammospheres, in comparison with the average size of tumors from SKBR3, showed no significant difference. Also, there was not any statistically important difference between the average size of MDA-MB-231 (2D) and MDA-MB-231-derived mammospheres (3D) tumor masses (Figure 6(b)). Average width of the tumors produced by MCF7, MDA-MB-231, and SKBR3 (2D) was detected to be 1.7, 1.6, and 1.9 mm, respectively, whereas the average width of tumors produced by MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres (3D) was 2.5, 2.5, and 2.6 mm, respectively. Calculating tumor volume by length and width of tumors showed average volume of the tumors produced by MCF7, MDA-MB-231, and SKBR3 (2D) was detected to be 4.5, 8, and 12 mm³, respectively, whereas the average volume of tumors produced by MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres (3D) was 20.5, 20, and 24 mm³, respectively (Figure 6(c)). Tumors produced by MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres (3D) showed more volume in comparison with the corresponding cell lines. The average volume of the tumors produced by MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres was more than that of tumors produced by MCF7, MDA-MB-231, and SKBR3, respectively; however, the difference was not statistically significant (Figure 6(c)). Comparison of the test groups showed the highest difference in the size and volume of tumors produced by MCF7-derived mammospheres in comparison with the MCF7 cell lines (Figure 6).

Figure 6.

Size and volume of the developed tumors from MCF7, MDA-MB-231, and SKBR3 (2D), and their cell lines–derived mammospheres (3D) in the chicken embryo through the CAM assay. (a) Tumors generated from MCF7, MDA-MB-231, SKBR3, and cell lines–derived mammospheres. Bar was 1 mm. (b) Size of the developed tumors from MCF7, MDA-MB-231, SKBR3 (2D), and cell lines–derived mammospheres (3D). Among tumors, this from MCF7-derived mammospheres showed a significantly larger size in comparison to those from its breast cancer cell line (p < 0.05). (c) Volume of the developed tumors from MCF7, MDA-MB-231, SKBR3 (2D), and cell lines–derived mammospheres (3D). Tumors from three types of mammospheres showed a larger volume in comparison to their cell lines; however, it was not significant. Represented value bars are the mean of six independent experiments ± SEM. Star indicates the significant difference (p < 0.05) between mammosphere samples and the respective cell line at the same point.

H&E and IHC staining of tumors produced by MCF7, MDA-MB-231, and SKBR3 (2D), and cell lines–derived mammospheres (3D)

Finally, all the tumors resected from chicken embryo were fixed, sectioned, and processed for H&E staining and IHC with the cytokeratin antibody to confirm the trend of mammosphere-derived cells in the migration and invasion into mesoderm and endoderm layers. H&E staining of tumors indicated that MCF7 cells (2D) were discernible in the ectoderm layer (ET), whereas tumor development from MCF7-derived mammospheres (3D) was extended to mesoderm (M) and endoderm layers (ED) (Figure 7(a)). The tumors produced by MCF7-derived mammospheres (3D) had more migration and invasion affinity from ectoderm (ET) to mesoderm (M) and endoderm (ED) layers. Also, tumors generated from SKBR3 cells (2D) migrated from ectoderm to mesoderm layers, whereas those generated from SKBR3-derived mammospheres (3D) migrated from ectoderm to endoderm layers (Figure 7(a)). Despite the more migration tendency of the tumors generated from MCF7- and SKBR3-derived mammospheres, tumors generated from MDA-MB-231-derived mammospheres (3D) did not show more migration capability in comparison to those generated from MDA-MB-231 cells (2D) (Figure 7(a)). Also, IHC with the cytokeratin antibody was used to confirm the H&E staining findings. Data obtained from IHC, therefore, significantly confirmed more migration and invasion of the tumors produced by MCF7-derived mammospheres (3D) from ectoderm (ET) to mesoderm (M) and endoderm (ED) layers, in comparison with the migration of the tumors of MCF7, which mostly developed on the ectoderm layer (Figure 7(b) and (c)). Also, the tumors generated from SKBR3-derived mammospheres (3D) mostly significantly migrated from ectoderm to endoderm, whereas the migration of the tumors generated from SKBR3 cells (2D) was mostly observed from ectoderm to mesoderm (Figure 7(b) and (c)). Meanwhile, the IHC of the tumors generated from MDA-MB-231-derived mammospheres (3D) showed no significant difference, as compared to the tumors generated from MDA-MB-231 (2D) (Figure 7(b) and (c)). Surprisingly, the data related to MDA-MB-231 and MDA-MB-231-derived mammospheres was not consistent with the scratch assay (in vitro study). This could be attributed to the different attitude of the cells when cultured in different conditions (2D vs 3D). Overall, the results showed the more migration and invasion capability in the tumors generated from MCF7- and SKBR3-derived mammospheres (3D), in comparison with MCF7 and SKBR3 cells (2D), respectively (Figure 7).

Figure 7.

H&E and IHC staining for the developed tumors from MCF7, MDA-MB-231, and SKBR3 (2D), and cell lines–derived mammospheres (3D). (a) H&E staining of tumors from MCF7, MDA-MB-231, SKBR3, and cell lines–derived mammospheres. It showed tumor migration and invasion of MCF7- and SKBR3-derived mammospheres from the ectoderm (ET) to mesoderm (M) and endoderm layers (ED). (b) Cytokeratin IHC staining for the developed tumors from MCF7, MDA-MB-231, and SKBR3 (2D), and cell lines–derived mammospheres (3D). IHC staining confirmed the results of H&E staining as it showed the higher migration and invasion of MCF7- and SKBR3-derived mammospheres (3D) from ectoderm to mesoderm and endoderm, in comparison with MCF7 and SKBR3 cell lines (2D). Generated tumors from MDA-MB-231-derived mammospheres (3D) showed no more migration and invasion, as compared to those from MDA-MB-231 (2D). (c) IHC quantification or cytokeratin intensity of the developed tumors. It significantly confirmed more migration and invasion of the tumors produced by MCF7-derived mammospheres (3D) from ectoderm (ET) to mesoderm (M) and endoderm (ED) layers. Also, the tumors generated from SKBR3-derived mammospheres (3D) mostly significantly migrated from ectoderm to endoderm. Generated tumors from MDA-MB-231-derived mammospheres (3D) showed no more significant migration and invasion, as compared to those from MDA-MB-231 (2D). Bar is 50 µM.

Discussion

CSCs make tumors more aggressive and progressive through self-renewal, differentiation, metastasis, migration, and therapy resistance. Targeting CSCs can prevent from tumor development and recurrence. Selecting the best mammospheres with the most stemness phenotype could help to discover the best therapeutic strategy through targeting BCSCs. In the current study, we used three breast cancer cell lines with different molecular phenotypes in order to enrich BCSCs. We introduced mammosphere as a stem cell enrichment system in the third passage of the mammosphere formed in the selective culture medium and the non-adherent condition. We compared three types of mammospheres from three breast cancer cell lines with different molecular markers through verification tests for stemness characterization, such as CD44/CD24 markers, Nanog and Oct4, proliferation rate, migration rate, and tumorigenicity in vivo (chick embryo model, an immune-deficient host). In 2008, Grimshaw et al.²⁶ used metastatic cells from pleural effusions collected from breast cancer patients in order to separate BCSCs in the free serum culture media and the non-adherent condition. Another study addressing the isolation of BCSCs through differential culture media and the formed mammosphere was performed on the mouse breast cancer tissue, TM40D. Anticancer drugs including paclitaxel and epirubicin were used for more purity, and CD44/CD24 antibody was applied for the detection of the purity.²⁷ In 2010, Van Phuc et al. isolated BCSCs from Vietnamese breast cancer patient tissues, confirming the presence of CSCs by colony formation, differentiation, and tumorigenicity assay in the animal model.²⁸ In addition to CSCs isolation via mammosphere-forming culture media, fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) could be used as an efficient method for the isolation of CSCs from cell lines and tissues through CSC markers such as CD44, CD133, ESA (or CD326), and ALDH activity.^29–32 Using FACS or MACS for the isolation of CSCs required a large number of cells, whereas CSCs are extremely low in a primary tumor, biopsy sample, or in the patient blood. Then, using free serum-specific culture media in the low attachment conditions is a more efficient technique for the enrichment of CSCs from a rare sample.³³ In addition to some technical difficulties, the isolation of CSCs from the primary tissues is a costly technique.¹⁵ Also, many primary tumors derived from mammospheres differentiate after a few passages, suggest that they do not contain long-term self-renewal cells.³⁴ In 2015, Shi et al. used mammospheres formation for the enrichment of CSCs from the gastric cancer cell line HGC-27.³⁵ They confirmed the presence of CSCs by proliferation, sphere differentiation, colony formation, chemotherapy resistance, and tumor formation assay in an animal model.³⁶ Many studies have shown that cell lines such as breast cancer, glioblastoma, and colorectal can serve as attractive sources of cells for the isolation of CSCs and CSCs research.^12,37–41 Our selected cell lines with different molecular properties could have different MFE and CSC properties. In the current study, MFE was shown to be increased during three passages in three cell lines, such that MCF7 had the highest MFE and MDA-MB-231 possessed the lowest one. These differences in MFE might be dependent on the E-cadherin expression. Those high expressed E-cadherin may contribute to form mammospheres with the longer term and the higher MFE.¹² MCF7 expresses E-cadherin (CDH1), whereas SKBR3 and MDA-MB-231 do not express CDH1.^41,42 Despite some other studies that have introduced CD44 as a CSC marker,^2,43 in the current study, there was no evidence of changes in the expression status of the CD44 marker on the surface of MCF7 and SKBR3 mammospheres; while there was a significant decrease in CD24, the differentiation marker in the passage 3 of mammospheres was detected. These results, therefore, agree with the studies demonstrating the increasing levels of CD44 during the serial passages, passages 2–10 (from 3.39% up to 72.42%) in TM40D murine BCSCs,²⁷ and in the human retinoblastoma stem cells.⁴⁴ Also, the study of MCF7, SKBR3, and T47D showed a significant decrement in the CD24 level of mammospheres within 10 days without any significant increase in the CD44 level.⁴⁰ The studies, therefore, demonstrated that CD44+ could not be considered as a common BCSC marker in all types of breast cancers due to the expression of a variety of markers on the surface of BCSCs and the specific markers which were probably not yet identified.^5,6,40 In the current study, stemness mRNA and protein expression, Oct4 and Nanog, were significantly higher in the mammospheres generated from MCF7, MDA-MB-231, and SKBR3.

The current study, therefore, confirmed the higher proliferation rates through the Brdu proliferation assay in three types of mammospheres, in comparison with their cell lines. Our study also showed the high migration of mammospheres generated from three breast cancer cell lines through the wound-healing assay as well as the high migration of CSCs from MDA-MB-231 in the 3D-aligned collagen matrices.⁴⁵ It was also shown that the highest migration rate was in MDA-MB-231 and MDA-MB-231-derived mammospheres, in comparison with other cell lines, MCF7, SKBR3, and their derived mammospheres. CD44 expression in MDA-MB-231 could promote migration in these cells.³⁶ Although MDA-MB-231 mammospheres showed the most migration rate, the migration rate difference between mammospheres and the corresponding cell lines was the least in MDA-MB-231-derived mammospheres. Drug resistance has also been identified as one of the most important properties of CSCs. In our study, MCF7-, MDA-MB-231-, and SKBR3-derived mammospheres showed more drug resistance than their corresponding cell lines. Although MCF7 showed drug resistance to Dox, the corresponding derived mammospheres showed more resistance. To our knowledge, drug resistance in MCF7 is due to the up-regulation of CYP1A1 and CYP1A2, drug efflux pumps (ABC family).^46,47 For the first time, we compared tumorigenicity of mammospheres from MCF7, MDA-MB-231, and SKBR3 in an immune-deficient host (chicken embryo) through the CAM assay. Our results showed that the size of tumors generated from MCF7 mammospheres–derived cells was significantly larger in comparison with the control group, whereas no significant difference was observed in the tumors generated from MDA-MB-231 and SKBR3 mammospheres. Although there was not any considerable difference between the size of the tumors produced by MDA-MB-231- and SKBR3-derived mammospheres (3D) and their corresponding cell lines, the volume of the tumors in mammospheres (3D) was more considerable than the corresponding cell line. H&E staining also showed more migration and the motility ability of mammospheres generated from MCF7 and SKBR3 from ectoderm into mesoderm and endoderm. Lack of migration and invasion in the tumors generated from MDA-MB-231 mammospheres–derived cells in vivo showed the different behavior of these cells in vivo, in comparison with their in vitro one. Although the stemness properties of MDA-MB-231-derived mammospheres, such as Nanog expression and migration rate, were the highest in vitro, these data were not consistent in vivo (CAM assay). Chicken embryo model assay may not be, therefore, a suitable model to study CSCs isolated from MDA-MB-231, a triple negative breast cancer. Nutritional conditions of chicken embryo contain a high dosage of estrogen.⁴⁸ Estrogen stimulates CSCs activity and expands BCSCs in the ER+ cells.^49,50 So, it seems that this model is more compatible with the hormone receptor positive breast cancer cell lines, which should be confirmed with an appropriate animal model and further studies in the future work.

The results obtained from our study, therefore, introduced efficient models for the study of CSCs, CSCs markers, metastasis, and therapeutic strategies in each type of breast cancer cell lines to establish an applicable approach in further usage.

Supplemental Material

Supplementary_figure_1_1 – Supplemental material for Characterization of the stemness potency of mammospheres isolated from the breast cancer cell lines

Supplemental material, Supplementary_figure_1_1 for Characterization of the stemness potency of mammospheres isolated from the breast cancer cell lines by Saghar Yousefnia, Kamran Ghaedi, Farzad Seyed Forootan and Mohammad Hossein Nasr Esfahani in Tumor Biology

Footnotes

Acknowledgements

We thank our great colleagues, Dr. Danial Naseri (PhD) and Mr. Abbas Kiani Esfahani (MSc), for their technical assistance on the chorioallantoic membrane (CAM) assay and flow cytometry, respectively.

Author contributions

S.Y. contributed in designing research studies, conducting experiments, acquiring data, analyzing data, providing reagents, and writing the manuscript. K.G. contributed to design of research studies, data interpretation, manuscript writing, and the final approval of the manuscript. F.S.F. contributed to design of research studies, data interpretation, manuscript writing, and the final approval of the manuscript. M.H.N.E. contributed to design of research studies, data interpretation, manuscript writing, and the final approval of the manuscript.

Availability of data and materials

Raw data and materials are available upon request.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

The permission to use cancer cell lines and chicken embryo based on the standard protocols was obtained from Ethics Committee of Royan Institute (ethics code: IR.ACECR.ROYAN.REC).

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was funded by a grant-in-aid of research from Iran National Science Foundation (Award no. 93021399) to K.G., as the principal investigator; this study was also partly supported by the University of Isfahan to help S.Y. to get her PhD.

ORCID iD

Kamran Ghaedi

Supplemental material

Supplemental material for this article is available online.

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