Isolation of cancer stem cells and the establishment of a H<sub>2</sub>O<sub>2</sub>-resistant cancer stem cell model_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Qingxi ^1,2,3 , CHEN Hongran ^1,2 , LI Hui ^1,2 ,  ZHANG Tongcun ¹ ,  MA Wenjian ^1,2

1. College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, P.R.China;
2. Qilu Institute of Technology, Jinan Shandong, 250200, P.R.China;
3. IncoCell Tianjin Ltd., Tianjin, 300457, P.R.China;

Corresponding author：

ZHANG Tongcun, Email: tony@tust.edu.cn; MA Wenjian, Email: ma_wj@tust.edu.cn

Keywords：

Cancer stem cells; targeting; anti-cancer drugs; breast cancer cell

DOI：

10.7507/1002-1892.201809014

Video：

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Objective To isolate cancer stem cells (CST) from human breast cancer cell line (MCF-7) and study their sensitivity toward oxidative stress.Methods MCF-7 cells were cultured in serum-free suspension culture medium to identify cells forming the sphere phenotype. The morphological changes of MCF-7 cells were observed by inverted phase contrast microscope (compared with MCF-7 cells cultured in serum-free suspension culture medium). The expression of CST marker CD133 was detected by immunocytochemical staining in CST cell spheres (experimental group) with a diameter of 100 μm and MCF-7 cells (control group) with a fusion degree of 70%. The positive rate of CD133 was detected by flow cytometry in the third generation of tumor cells with diameter of 150 μm. The second generation of tumor globular cells (experimental group) with diameter of 150 μm and corresponding MCF-7 cells (control group) were taken to be damaged by 50 mol/L H₂O₂ for 120 minutes. The expression of DNA damage marker histone H2AX phosphorylation (γH2AX) was detected by immunocytochemical staining.Results Inverted phase contrast microscopy showed that MCF-7 cells grew initially in a single-cell adherent state, then aggregated and grew in serum-free suspension culture medium, and finally formed CST cell spheres, while the control MCF-7 cells cultured in MCF-7 cell culture medium grew extensively and could not grow in suspension. Fluorescence microscopy showed that the expression of CD133 in MCF-7 cells of control group was negative, while that in experimental group was positive. Flow cytometry showed that CD133 was positive in CST cells, and the positive rate was 92%. Inverted fluorescence microscopy showed that the expression of γH2AX in CST tumor spheres of experimental group was significantly lower than that in MCF-7 cells of control group after 120 minutes of H₂O₂ injury.Conclusion Serum-free suspension culture medium can produce globular CST cells from MCF-7 tumor cell line, which have strong antioxidant damage.

Citation： LIU Qingxi, CHEN Hongran, LI Hui, ZHANG Tongcun, MA Wenjian. Isolation of cancer stem cells and the establishment of a H₂O₂-resistant cancer stem cell model. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(11): 1433-1438. doi: 10.7507/1002-1892.201809014 Copy

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14.	Bulnes S, Bermúdez G, Lafuente JV. Association of Notch-1, osteopontin and stem-like cells in ENU-glioma malignant process. Oncotarget, 2018, 9(59): 31330-31341.
15.	Yang Z, Ni W, Cui C, et al. Identification of LETM1 as a marker of cancer stem-like cells and predictor of poor prognosis in esophageal squamous cell carcinoma. Hum Pathol, 2018, 81: 148-156.
16.	Liu YQ, Wang SK, Xu QQ, et al. Acetyl-11-keto-β-boswellic acid suppresses docetaxel-resistant prostate cancer cells in vitro and in vivo by blocking Akt and Stat3 signaling, thus suppressing chemoresistant stem cell-like properties. Acta Pharmacol Sin, 2019, 40(5): 689-698.
17.	Gordeeva O. Cancer-testis antigens: Unique cancer stem cell biomarkers and targets for cancer therapy. Semin Cancer Biol, 2018, 53: 75-89.
18.	刘庆喜, 吕丽慧, 孙贺, 等. 血清对神经细胞分化的影响. 中国修复重建外科杂志, 2018, 32(2): 223-227.
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20.	Velasco-Velázquez MA, Velázquez-Quesada I, Vásquez-Bochm LX, et al. Targeting breast cancer stem cells: a methodological perspective. Curr Stem Cell Res Ther, 2019, 14(5): 389-397.
21.	王瑞海. 肿瘤干细胞的分离培养及生物学特性. 中国组织工程研究与临床康复, 2011, 15(27): 5087-5090.
22.	Kaya C, Ashraf M. The mechanism of hydrogen sulfide mitigation of iron deficiency-induced chlorosis in strawberry (Fragaria × ananassa) plants. Protoplasma, 2019, 256(2): 371-382.
23.	Choi YH. Schisandrin A prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells. Biomed Pharmacother, 2018, 106: 902-909.

1. Kageyama Y, Yamaguchi R, Watanabe S, et al. Intraductal papillary neoplasm of the bile duct with rapidly progressive multicentric recurrence: A case report. Int J Surg Case Rep, 2018, 51: 102-106.
2. Givalos N, Manolidis G, Kochylas N, et al. Unusual case of intraluminal cecal recurrence of a low grade appendiceal mucinous neoplasm (LAMN)-Case report and brief literature review. Int J Surg Case Rep, 2018, 51: 112-116.
3. Mair MD, Nair S, Nikam S, et al. Longitudinal and cross-sectional assessment of quality of life in surgically treated advanced (T4) cancer of the buccal mucosa. Oral Surg Oral Med Oral Pathol Oral Radiol, 2017, 124(6): 529-536.
4. Chang KJ, Yang MH, Zheng JC, et al. Arsenic trioxide inhibits cancer stem-like cells via down-regulation of Gli1 in lung cancer. Am J Transl Res, 2016, 8(2): 1133-1143.
5. Lopez-Bertoni H, Li Y, Laterra J. Cancer stem cells: dynamic entities in an ever-evolving paradigm. Biol Med (Aligarh), 2015, 7(Suppl 2): pii: 001.
6. De Rose F, Fogliata A, Franceschini D, et al. Hypofractionation with simultaneous boost in breast cancer patients receiving adjuvant chemotherapy: A prospective evaluation of a case series and review of the literature. Breast, 2018, 42: 31-37.
7. Weiner CM, Mathewos A, Addissie A, et al. Characteristics and follow-up of metastatic breast cancer in Ethiopia: A cohort study of 573 women. Breast, 2018, 42: 23-30.
8. Li J, Chen JN, Zeng TT, et al. CD133+ liver cancer stem cells resist interferon-gamma-induced autophagy. BMC Cancer, 2016, 16: 15.
9. Takahashi K, Ehata S, Koinuma D, et al. Pancreatic tumor microenvironment confers highly malignant properties on pancreatic cancer cells. Oncogene, 2018, 37(21): 2757-2772.
10. Mansoori M, Madjd Z, Janani L, et al. Circulating cancer stem cell markers in breast carcinomas: a systematic review protocol. Syst Rev, 2017, 6(1): 262.
11. Türker Şener L, Güven C, Şener A, et al. Nicotine reduces effectiveness of doxorubicin chemotherapy and promotes CD44. Exp Ther Med, 2018, 16(1): 21-28.
12. Kuai Y, Gong X, Ding L, et al. Wilms' tumor 1-associating protein plays an aggressive role in diffuse large B-cell lymphoma and forms a complex with BCL6 via Hsp90. Cell Commun Signal, 2018, 16(1): 50.
13. Ren X, Ji Y, Jiang X, et al. Downregulation of CYP2A6 and CYP2C8 in tumor tissues is linked to worse overall survival and recurrence-free survival from hepatocellular carcinoma. Biomed Res Int, 2018, 2018: 5859415.
14. Bulnes S, Bermúdez G, Lafuente JV. Association of Notch-1, osteopontin and stem-like cells in ENU-glioma malignant process. Oncotarget, 2018, 9(59): 31330-31341.
15. Yang Z, Ni W, Cui C, et al. Identification of LETM1 as a marker of cancer stem-like cells and predictor of poor prognosis in esophageal squamous cell carcinoma. Hum Pathol, 2018, 81: 148-156.
16. Liu YQ, Wang SK, Xu QQ, et al. Acetyl-11-keto-β-boswellic acid suppresses docetaxel-resistant prostate cancer cells in vitro and in vivo by blocking Akt and Stat3 signaling, thus suppressing chemoresistant stem cell-like properties. Acta Pharmacol Sin, 2019, 40(5): 689-698.
17. Gordeeva O. Cancer-testis antigens: Unique cancer stem cell biomarkers and targets for cancer therapy. Semin Cancer Biol, 2018, 53: 75-89.
18. 刘庆喜, 吕丽慧, 孙贺, 等. 血清对神经细胞分化的影响. 中国修复重建外科杂志, 2018, 32(2): 223-227.
19. Parte SC, Batra SK, Kakar SS. Characterization of stem cell and cancer stem cell populations in ovary and ovarian tumors. J Ovarian Res, 2018, 11(1): 69.
20. Velasco-Velázquez MA, Velázquez-Quesada I, Vásquez-Bochm LX, et al. Targeting breast cancer stem cells: a methodological perspective. Curr Stem Cell Res Ther, 2019, 14(5): 389-397.
21. 王瑞海. 肿瘤干细胞的分离培养及生物学特性. 中国组织工程研究与临床康复, 2011, 15(27): 5087-5090.
22. Kaya C, Ashraf M. The mechanism of hydrogen sulfide mitigation of iron deficiency-induced chlorosis in strawberry (Fragaria × ananassa) plants. Protoplasma, 2019, 256(2): 371-382.
23. Choi YH. Schisandrin A prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells. Biomed Pharmacother, 2018, 106: 902-909.

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Isolation of cancer stem cells and the establishment of a H₂O₂-resistant cancer stem cell model