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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 421-426

A modified method by differential adhesion and serum-free culture medium for enrichment of cancer stem cells


1 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Nanfang Hospital; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, China
2 Department of Neurology, TCM-Integrated Cancer Center of Southern Medical University, Guangzhou, China
3 Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Wan-Long Tan
Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.174533

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 > Abstract 

Objective: In this study, we showed a modified method for the isolation of cancer stem cells (CSCs) using a combination of differential adhesion method and serum-free culture medium (SFM) method.
Materials and Methods: Trypsin-sensitive cells and trypsin-resistant cells were isolated from MB49, EJ, and SK-OV-3 cells using a combination of differential adhesion method and SFM method. The CSCs markers expression of trypsin-resistant cells was verified by the flow cytometry, the Western blotting, and the quantitative polymerase chain reaction. Functional comparisons were verified by the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay.
Results: Trypsin-resistant cells were isolated successfully. They were identified with high expression of CSCs markers and possessed higher resistance to chemotherapy, greater migration in vitro and stronger tumorigenic abilities in vivo.
Conclusion: Trypsin-resistant cells showed specific CSCs characterizations. They were able to be isolated successfully with a modified method by a combination of differential adhesion method and SFM method.

Keywords: Cancer stem cells, resistant, sensitive, serum-free culture, trypsin


How to cite this article:
Zhu YT, Wang CY, Pang SY, Lei CY, Luo Y, Tan WL. A modified method by differential adhesion and serum-free culture medium for enrichment of cancer stem cells. J Can Res Ther 2018;14, Suppl S2:421-6

How to cite this URL:
Zhu YT, Wang CY, Pang SY, Lei CY, Luo Y, Tan WL. A modified method by differential adhesion and serum-free culture medium for enrichment of cancer stem cells. J Can Res Ther [serial online] 2018 [cited 2019 Jul 16];14:421-6. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/421/174533


 > Introduction Top


The vaccine was effective against metastatic bladder cancer in our previous study.[1] Recurrence of solid tumors may be due to the cancer stem cells (CSCs) were not eliminated. CSCs could not be eliminated as the inability of traditional chemotherapy and radiotherapy.[2],[3] The previous study found that repeated cycles of differential adhesion were able to enrich breast CSCs by 20-fold.[4] The serum-free culture medium (SFM) method was limited to isolate CSCs due to the deficiency of purity.[5] As we known, the combination of the differential adhesion method and SFM method has not been used to enrich the CSCs, which could gain the purity of cell sorting. The enrichment of CSCs would promote the development of our vaccine research. Thus, we provide a modified method here using combining the differential adhesion method and SFM method to enrich CSCs. In this study, the murine bladder cancer cell, the human bladder cancer, and the human ovarian cancer were selected to represent the feasibility.


 > Materials and Methods Top


Cell lines

MB49 cell line, the murine bladder cancer cell, was a gift from (provided by I. C. Summerhayes, Lahey Clinic, Burlington, MA).[1] EJ cell line, the human bladder cancer cell, and SK-OV-3 cell line, the human ovarian cancer cell were provided and preserved in Pathology Lab, Southern Medical University. These cells were cultured at 37°C in a 5% CO2 humidified incubator using RPMI1640 which contained 10% fetal bovine serum (FBS, Thermo Scientific HyClone, Logan, Utah).

The differential adhesion method

Cells were cultured confluence in a 6-well plate, washed 3 times with phosphate-buffered saline (PBS), and digested using trypsin solution (eBioscience, San Diego, California) at 37°C. After several minutes, cells were separated and collected after washing with PBS. Trypsin solution was added to the attached cells, the process of digested and collected was repeated several times. Cells collected after different times respectively were cultured in a 6-well plate on 3 days. Then the trypsin-resistant cells and trypsin-sensitive cells were digested with trypsin again, separated and collected as former, respectively. These steps were repeated 3 times. At last, those trypsin-resistant cells were cultured using SFM and grown to spheres which were considered to be CSCs.

The components of SFM were RPMI1640, epidermal growth factor (20 ng/ml), fibroblast growth factor basic (20 ng/ml), leukemia inhibitory factor (20 ng/ml), B-27 serum-free supplement (20 μl/ml), and bovine serum albumin (4 μg/ml).

Flow cytometry

The MB49, EJ, and SK-OV-3 cells and their trypsin-resistant cells were harvested, respectively. They were dissociated in autoMACS running buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), labeled with PE anti-prominin-1 (Miltenyi Biotec) and FITC anti-CD44 (Miltenyi Biotec), incubated and washed with PBS 2 times. The PE rat IgG1 κ isotype control (eBioscience) and the FITC rat IgG2b κ isotype control (eBioscience) were used as a control. The portion of CD44+ CD133+ cells was calculated using a BD FACSAria cell sorter (Becton-Dickinson, San Jose, California).

Western blotting

The protein extracts were divided by electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, MA, USA). Membranes were blocked and incubated using the primary antibody anti-CD44 (Abcam, MA, USA), anti-CD133 (Abcam) and anti-β-actin antibody (Abcam). Then membranes were cultivated with anti-mouse secondary antibodies (Abcam).[6] At last, protein bands were measured by Fluor Chem FC2 (Alpha Innotech, CA, USA).

Quantitative polymerase chain reaction

Total RNA was isolated by Arcturus PicoPure RNA isolation kit (Arcturus, Life Technologies, CA, USA). The RNA quality was verified by Bioanalyzer RNA Pico Chip (Agilent Technologies, CA, USA). cDNAs were synthesized by superscript III reverse transcriptase (Invitrogen, CA, USA), amplified by SYBR green polymerase chain reaction (PCR) master mix (Bio-Rad, California) using 7500 real-time PCR system (AB Applied Biosystems, Singapore). The primers sequences were listed in [Table 1]. Normalization and fold changes were calculated by the △△Ct method.[7]
Table 1: Primers of selected genes

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Chemotherapy-resistance ability

Cells were seeded at a density of 1 × 104 per well using a 96-well plate. The agent, paclitaxel (Sigma, MO, USA), was added at different concentrations. CCK-8 was added after 4 days, and the absorbance value was detected. Cell viability was measured using the percentage of the absorbance values in treated wells with respect to untreated control wells.[7]

Migration abilities in vitro

Cells were seeded onto the upper well in pure RPMI1640 (at a density of 1 × 104 cells/0.25 ml/well), and a 6.5 mm pore size polycarbonate membrane chamber was bedded into the transwell apparatus (Costar, MA, USA). RPMI1640 having 10% FBS was added into the lower well. Cells were cultivated and migrated to the bottom surface after 1 day, fixed, stained, rinsed, and detected by inverted microscopy.

Tumorigenic abilities in vivo

All animal experiments were approved by the Ethics Committee of Southern Medical University. The MB49, EJ, and SK-OV-3 cells and their trypsin-resistant cells were injected into nude mice (Guangzhou, China) subcutaneously. The volume of tumors was calculated using the formula d2 × D/2, where D and d were the longest and the shortest diameters respectively.[8]

Statistical analysis

Data were expressed as the mean ± standard deviation and analyzed using one-way ANOVA. Statistical analyses were performed by the SPSS version 19.0 software (SPSS, Chicago, IL, USA), setting significance at P < 0.05.


 > Results Top


Cancer stem cells separable by differential adhesion and serum-free culture medium method

The average digested time of MB49, EJ, and SK-OV-3 cells, using differential adhesion method, was 2, 3 and 5 min, respectively [Figure 1]. Cells detached by more time of trypsinization exhibited round shaped morphologies. On the other hand, cells detached by fewer time retained original shaped morphologies, and cells detached by middle time had mixed morphologies [Figure 2]a. The SFM method generated CSC spheres within 3 weeks [Figure 2]b.
Figure 1: Diagram illustration to the proposed model for isolation of the cancer stem cells by differential adhesion method and serum-free culture medium method

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Figure 2: Morphology of the cancer stem cells by differential adhesion method and serum-free culture medium method. (a) Morphology of trypsin-resistant cells and trypsin-sensitive cells in MB49, EJ, and SK-OV-3 cells. (b) The representative image of cancer stem cells sphere formation in serum-free culture medium

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Expression of cancer stem cells markers

The flow cytometry (FCM) analysis showed that the fraction of CD133+ CD44+ cells in trypsin-resistant cells was much more than those in trypsin-sensitive cells of MB49, EJ and SK-OV-3 cells [Figure 3]a. The Western blotting (WB) analysis showed that the CD133 and CD44 proteins in trypsin-resistant cells were expressed higher than those in trypsin-sensitive cells [Figure 3]b. The quantitative-PCR analysis showed that the relative levels of CD133 and CD44 mRNAs were abundantly expressed in trypsin-resistant cells, but much less in trypsin-sensitive cells [Figure 3]c.
Figure 3: Comparison of specific markers in trypsin-resistant cells and trypsin-sensitive cells of MB49, EJ, and SK-OV-3 cells. (a) The flow cytometry analysis showed that the fraction of CD133+ CD44+ cells in trypsin-resistant cells was much more than those in trypsin-sensitive cells. (b) The Western blotting analysis showed that the CD133 and CD44 proteins in trypsin-resistant cells were expressed higher than those in trypsin-sensitive cells. (c) The quantitative polymerase chain reaction analysis showed that the relative levels of CD133 and CD44 mRNAs were abundantly expressed in trypsin-resistant cells, but much less in trypsin-sensitive cells

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Functional comparison

The CCK-8 analysis showed that trypsin-resistant cells displayed higher cell viabilities than those in trypsin-sensitive cells after being exposed to different concentrations of paclitaxel, which suggested that trypsin-resistant cells of MB49, EJ, and SK-OV-3 cells had a lower susceptibility to traditional anticancer agents [Figure 4]a.
Figure 4: Functional characteristics comparison in trypsin-resistant cells and trypsin-sensitive cells of MB49, EJ, and SK-OV-3 cells. (a) The CCK-8 analysis showed that trypsin-resistant cells displayed higher cell viabilities than those in trypsin-sensitive cells after being exposed to different concentrations of paclitaxel. (b) The transwell migration analysis showed that the number invaded the bottom chamber in trypsin-resistant cells was more than those in trypsin-sensitive cells. (c) The xenograft formation analysis showed that tumor volumes produced by trypsin-resistant cells was larger than those in trypsin-sensitive cells

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The transwell migration analysis showed that the number invaded the bottom chamber in trypsin-resistant cells was more than those in trypsin-sensitive cells, which suggested that trypsin-resistant cells of MB49, EJ, and SK-OV-3 cells had higher invasion ability than trypsin-sensitive cells [Figure 4]b.

The xenograft formation analysis showed that tumor volumes produced by trypsin-resistant cells was larger than those in trypsin-sensitive cells, which suggested that trypsin-resistant cells of MB49, EJ, and SK-OV-3 cells had stronger tumorigenicity than trypsin-sensitive cells [Figure 4]c.


 > Discussion Top


Three methods were mostly used to enrich CSCs from cancers: Side population cells, specific CSCs surface markers, and SFM. The deficiency of these methods was the purity was not enough for CSCs.[5] Repeated cycles of differential trypsinization had been used to gather CSCs by 20-fold in breast cancer cells, keratinocytes, and human mammary epithelial cells. Because the serum caused irreversible differentiation of stem cells, SFM method might be useful for CSCs expansion and be able to allow for maintenance of an undifferentiated stem cell status.[9] As we know, this is the first study about the enrichment of CSCs via a combination of differential adhesion method and SFM method, which is modified to gain the purity of CSCs.

The CD133 and CD44 markers were most used to ascertain CSCs in tumor tissues.[10],[11] CSCs used in experiments were enriched for CD133+ and CD44+ markers, but not seen 100% dual positive cells by FCM analysis. This study found elevated expression levels of CD133+ and CD44+ in trypsin-resistant cells. Targeting CD44+ and CD133+ cancer cells involving a CD44+ CD133+ cell subpopulation might be a way for cancer therapy.[12] Only cells both expressing CD133+ and CD44+ were viewed as CSCs by FCM analysis.[13] The expression of CD133 and CD44 was found elevated in trypsin-resistant cells at the mRNA expression level and the protein expression level. There were other markers that have been used to identify CSCs from tumors, such as ABC transporters, aldehyde dehydrogenase and so on. Detecting the status of these markers will help us to understand CSCs in further research.

We characterized the trypsin-resistant cells populations functionally by different techniques.[14],[15] Specifically, trypsin-resistant cells possessed a greater ability to penetrate wells. Moreover, although chemotherapy eliminated most tumor cancer cells, it could not eliminate CSCs. In additional, trypsin-resistant cells had a lower sensitivity to paclitaxel, which conform to the theory of resistance to chemotherapy.[16],[17] Tumorigenicity in nude mice was a standard way used to evaluate the tumorigenic ability of CSCs.[18] Trypsin-resistant cells possessed a greater ability to form subcutaneous tumors in nude mice. Hence, trypsin-resistant cells were considered to have specific CSC properties.


 > Conclusions Top


CSCs were enriched successfully with a modified method by a combination of differential adhesion method and SFM method. Trypsin-resistant cells had CSCs characteristics containing chemotherapy resistance and in vivo tumorigenic capacity.

Financial support and sponsorship

This research was supported by Scientific Initiative Research Foundation of Southern Medical University (No. PY2014N031).

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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Zhang X, Shi X, Li J, Hu Z, Guo F, Huang X, et al. Novel immunotherapy for metastatic bladder cancer using vaccine of human interleukin-2 surface-modified MB 49 cells. Urology 2011;78:721-2.  Back to cited text no. 1
    
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Han ME, Jeon TY, Hwang SH, Lee YS, Kim HJ, Shim HE, et al. Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research. Cell Mol Life Sci 2011;68:3589-605.  Back to cited text no. 11
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Zhu YT, Zhao Z, Fu XY, Luo Y, Lei CY, Chen W, et al. The granulocyte macrophage-colony stimulating factor surface modified MB49 bladder cancer stem cells vaccine against metastatic bladder cancer. Stem Cell Res 2014;13:111-22.  Back to cited text no. 13
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Dalerba P, Cho RW, Clarke MF. Cancer stem cells: Models and concepts. Annu Rev Med 2007;58:267-84.  Back to cited text no. 14
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Sung JM, Cho HJ, Yi H, Lee CH, Kim HS, Kim DK, et al. Characterization of a stem cell population in lung cancer A549 cells. Biochem Biophys Res Commun 2008;371:163-7.  Back to cited text no. 16
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Okamoto A, Chikamatsu K, Sakakura K, Hatsushika K, Takahashi G, Masuyama K. Expansion and characterization of cancer stem-like cells in squamous cell carcinoma of the head and neck. Oral Oncol 2009;45:633-9.  Back to cited text no. 17
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