Isolation and enrichment of liver cancer stem cells by magnetic cell sorting and serum-free suspension culture_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

CHEN Kefei , LIU Jun ,  LI Bo

Department of Hepatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;

Corresponding?author：

LI Bo, Email: cdhxlibo@126.com

Keywords：

liver cancer stem cell; magnetic cell sorting; flow cytometry; serum-free suspension culture

DOI：

10.7507/1007-9424.201611088

Video：

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Objective The aim is to sort CD90⁺ subpopulation cells in human liver cancer cell lines and investigate efficiency of magnetic cell sorting (MACS) on sorting the liver cancer stem cells. Methods ①Expressions of CD90. Immunohistochemical method was used to determine the expressions of CD90 in normal liver tissues in 8 cases, liver cancer and adjacent liver cancer tissues in 58 cases. ②Screened the cell lines. Huh-7, MHCC97-H, Bel-7402, and SMMC-7721 cell lines were divided into blank control group and experimental group (5.5×10⁵ cells per hole, 1 hole), cells of the experimental group were added with 5 μL CD90^–PE while cells of the blank control group were treated with 5 μL CD90^–PE non fluorescent antibody. Determined the proportion of CD90⁺ cells in the 2 groups by flow cytometry (FCM). ③MACS. Huh-7 and MHCC97-H cell lines were labeled with magnetic beads respectively and sorted by MACS, 1 mL cell suspensionsorted by magnetic sorting (MS) was collected as CD90^– group, and 1 mL PBS after MS wash was collected as CD90⁺ group, as well as blank control group and experimental group. Determined the proportion of CD90⁺ cells in 4 groups by FCM. Two times of MACS were performed in Huh-7 cells. ④Serum free culture and serum culture. Huh-7 cells were divided into serum-free culture group and serum culture group (1 hole), and proportions of CD90⁺ cells were determined by FCM at 1 week after culture. Results ①The positive rate of CD90 was 0 (0/8), 65.5% (38/58), and 20.7% (12/58) in normal liver tissues, liver cancer tissues, and adjacent liver cancer tissues respectively, and the positive rate of CD90 was higher in liver cancer tissues than those of normal liver tissues (χ²=6.78, P<0.05) and adjacent liver cancer tissues (χ²=20.83, P<0.05). ②For Huh-7, MHCC97-H, SMMC-7721, and Bel7402 cell lines, the proportions of CD90⁺ cells in the experimental group was 0.851%, 1.090%, 2.710%, and 4.050% respectively, the proportions of CD90⁺ cells in the blank control group was 0.241%, 0.688%, 1.890%, and 2.080% respectively, so we chose Huh-7 and MHCC97-H cell lines to perform MACS. ③Results of MACS for Huh-7 cell line. For the first MACS, the proportions of CD90⁺ cells in the blank control group, experimental group, CD90^– group, and CD90⁺ group was 0.241%, 0.851%, 0.574%, and 1.100% respectively. For the second MACS, the proportions of CD90⁺ cells in the blank control group, experimental group, CD90^– group, and CD90⁺ group was 0.032%, 0.961%, 0.426%, and 9.700% respectively. Conclusions The normal liver tissues do not express the CD90, but the liver cancer tissues express CD90 highly. There is a few CD90⁺ cells in Huh-7 and MHCC97-H liver cancer cell lines. The MACS has a certain effect on improving the proportion of CD90⁺ cells in the cell lines. The serum-free suspension culture has no effect on enriching CD90⁺ cells.

Citation： CHEN Kefei, LIU Jun, LI Bo. Isolation and enrichment of liver cancer stem cells by magnetic cell sorting and serum-free suspension culture. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2017, 24(8): 946-952. doi: 10.7507/1007-9424.201611088 Copy

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15.	Durnez A, Verslype C, Nevens F, et al. The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin. Histopathology, 2006, 49(2): 138-151.
16.	Münz M, Kieu C, Mack B, et al. The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene, 2004, 23(34): 5748-5758.
17.	Magee JA, Piskounova E, Morrison SJ. Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell, 2012, 21(3): 283-296.
18.	Yamashita T, Ji J, Budhu A, et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology, 2009, 136(3): 1012-1024.
19.	Lázaro CA, Chang M, Tang W, et al. Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes. Am J Pathol, 2007, 170(2): 478-489.
20.	Arzumanyan A, Friedman T, Ng IO, et al. Does the hepatitis B antigen HBx promote the appearance of liver cancer stem cells? Cancer Res, 2011, 71(10): 3701-3708.
21.	Ali N, Allam H, May R, et al. Hepatitis C virus-induced cancer stem cell-like signatures in cell culture and murine tumor xenografts. J Virol, 2011, 85(23): 12292-12303.
22.	Visvader JE. Cells of origin in cancer. Nature, 2011, 469(7330): 314-322.
23.	Chaffer CL, Brueckmann I, Scheel C, et al. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci U S A, 2011, 108(19): 7950-7955.
24.	Chen Y, Wong PP, Sjeklocha L, et al. Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture. Hepatology, 2012, 55(2): 563-574.
25.	Yang ZF, Ho DW, Ng MN, et al. Significance of CD90⁺ cancer stem cells in human liver cancer. Cancer Cell, 2008, 13(2): 153-166.
26.	Sell S. Cellular origin of hepatocellular carcinomas. Semin Cell Dev Biol, 2002, 13(6): 419-424.
27.	Zender L, Spector MS, Xue W, et al. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell, 2006, 125(7): 1253-1267.

1. Simard EP, Ward EM, Siegel R, et al. Cancers with increasing incidence trends in the United States: 1999 through 2008. CA Cancer J Clin, 2012, 62(2): 118-128.
2. Germano D, Daniele B. Systemic therapy of hepatocellular carcinoma: current status and future perspectives. World J Gastroenterol, 2014, 20(12): 3087-3099.
3. Koh KC, Lee H, Choi MS, et al. Clinicopathologic features and prognosis of combined hepatocellular cholangiocarcinoma. Am J Surg, 2005, 189(1): 120-125.
4. Willekens I, Hoorens A, Geers C, et al. Combined hepatocellular and cholangiocellular carcinoma presenting with radiological characteristics of focal nodular hyperplasia. World J Gastroenterol, 2009, 15(31): 3940-3943.
5. Yu XH, Xu LB, Zeng H, et al. Clinicopathological analysis of 14 patients with combined hepatocellular carcinoma and cholangiocarcinoma. Hepatobiliary Pancreat Dis Int, 2011, 10(6): 620-625.
6. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997, 3(7): 730-737.
7. Babaie Y, Herwig R, Greber B, et al. Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells, 2007, 25(2): 500-510.
8. Branda M, Wands JR. Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma. Hepatology, 2006, 43(5): 891-902.
9. Ishizaki Y, Ikeda S, Fujimori M, et al. Immunohistochemical analysis and mutational analyses of beta-catenin, Axin family and APC genes in hepatocellular carcinomas. Int J Oncol, 2004, 24(5): 1077-1083.
10. Zhang F, Chen XP, Zhang W, et al. Combined hepatocellular cholangiocarcinoma originating from hepatic progenitor cells: immunohistochemical and double-fluorescence immunostaining evidence. Histopathology, 2008, 52(2): 224-232.
11. Kühl M. Non-canonical Wnt signaling in Xenopus: regulation of axis formation and gastrulation. Semin Cell Dev Biol, 2002, 13(3): 243-249.
12. Sell S, Leffert HL. Liver cancer stem cells. J Clin Oncol, 2008, 26(17): 2800-2805.
13. Mishra L, Banker T, Murray J, et al. Liver stem cells and hepatocellular carcinoma. Hepatology, 2009, 49(1): 318-329.
14. Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology, 2006, 44(1): 240-251.
15. Durnez A, Verslype C, Nevens F, et al. The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin. Histopathology, 2006, 49(2): 138-151.
16. Münz M, Kieu C, Mack B, et al. The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene, 2004, 23(34): 5748-5758.
17. Magee JA, Piskounova E, Morrison SJ. Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell, 2012, 21(3): 283-296.
18. Yamashita T, Ji J, Budhu A, et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology, 2009, 136(3): 1012-1024.
19. Lázaro CA, Chang M, Tang W, et al. Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes. Am J Pathol, 2007, 170(2): 478-489.
20. Arzumanyan A, Friedman T, Ng IO, et al. Does the hepatitis B antigen HBx promote the appearance of liver cancer stem cells? Cancer Res, 2011, 71(10): 3701-3708.
21. Ali N, Allam H, May R, et al. Hepatitis C virus-induced cancer stem cell-like signatures in cell culture and murine tumor xenografts. J Virol, 2011, 85(23): 12292-12303.
22. Visvader JE. Cells of origin in cancer. Nature, 2011, 469(7330): 314-322.
23. Chaffer CL, Brueckmann I, Scheel C, et al. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci U S A, 2011, 108(19): 7950-7955.
24. Chen Y, Wong PP, Sjeklocha L, et al. Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture. Hepatology, 2012, 55(2): 563-574.
25. Yang ZF, Ho DW, Ng MN, et al. Significance of CD90⁺ cancer stem cells in human liver cancer. Cancer Cell, 2008, 13(2): 153-166.
26. Sell S. Cellular origin of hepatocellular carcinomas. Semin Cell Dev Biol, 2002, 13(6): 419-424.
27. Zender L, Spector MS, Xue W, et al. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell, 2006, 125(7): 1253-1267.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Isolation and enrichment of liver cancer stem cells by magnetic cell sorting and serum-free suspension culture

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