Isolation Set-up and Characterization of cancer stem cells from HT29 colon cancer cell line

Document Type : Research/Original Article

Authors

1 Autophagy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

2 Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran

3 Colorectal Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

Abstract
Background: Cancer stem cells (CSCs) have critical roles in tumor initiation, progression, metastasis, resistance to chemoradiotherapy, and recurrence in various cancers such as colon cancer. Therefore, extensive studies are required to understand the CSCs mechanism of action and design novel cancer therapies. Successful isolation and characterization of CSCs from tumor tissues or cancer cell lines provide the proper opportunity for these kinds of studies. This study aimed to isolate and characterize CSCs from HT29 colon cancer cell lines. Methods: Sphere formation assay was used to isolate CSCs from the HT29 cell line. The expression of four stemness genes, including LGR5, SOX2, c-Myc, and Oct4, was assessed by real-time PCR. The CSC markers, CD 44 and CD 24, were also evaluated and compared between the parental HT29 cell line and HT29-derived spheres by flow cytometry. Statistical analysis was performed using GraphPad Prism version 6.0 and the Mann-Whitney U test. Results:HT29-derived sphere cells were successfully formed in serum-free media. The proportion of CD 24 and CD 44-expressing cells and the expression of LGR5, SOX2, c-Myc, and Oct4 stemness genes were significantly higher in CSCs isolated from HT29 spheres compared with parental HT29 cells. Conclusion:Sphere formation assay is a proper method for the isolation of CSCs from the HT29 cell line. The stemness markers LGR5, SOX2, c-Myc, Oct4,CD 44,andCD 24are suitable for the characterization of these cells.

Keywords

References

1.            Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2018;68(6):394-424.
2.            Yang L, Shi P, Zhao G, Xu J, Peng W, Zhang J, et al. Targeting cancer stem cell pathways for cancer therapy. Signal transduction and targeted therapy. 2020;5(1):1-35.
3.            Atashzar MR, Baharlou R, Karami J, Abdollahi H, Rezaei R, Pourramezan F, et al. Cancer stem cells: A review from origin to therapeutic implications. Journal of cellular physiology. 2020;235(2):790-803.
4.            Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, et al. Identification of cells initiating human melanomas. Nature. 2008;451(7176):345-9.
5.            Batlle E, Clevers H. Cancer stem cells revisited. Nature medicine. 2017;23(10):1124-34.
6.            Zhou Y, Xia L, Wang H, Oyang L, Su M, Liu Q, et al. Cancer stem cells in progression of colorectal cancer. Oncotarget. 2018;9(70):33403.
7.            Das PK, Islam F, Lam AK. The roles of cancer stem cells and therapy resistance in colorectal carcinoma. Cells. 2020;9(6):1392.
8.            Wang T, Shigdar S, Gantier MP, Hou Y, Wang L, Li Y, et al. Cancer stem cell targeted therapy: progress amid controversies. Oncotarget. 2015;6(42):44191.
9.            Medema JP. Targeting the colorectal cancer stem cell. New England Journal of Medicine. 2017;377(9):888-90.
10.          Phi LTH, Sari IN, Yang Y-G, Lee S-H, Jun N, Kim KS, et al. Cancer stem cells (CSCs) in drug resistance and their therapeutic implications in cancer treatment. Stem cells international. 2018;2018.
11.          Cherciu I, Bărbălan A, Pirici D, Mărgăritescu C, Săftoiu A. Stem cells, colorectal cancer and cancer stem cell markers correlations. Current health sciences journal. 2014;40(3):153.
12.          Jariyal H, Gupta C, Bhat VS, Wagh JR, Srivastava A. Advancements in cancer stem cell isolation and characterization. Stem cell reviews and reports. 2019;15(6):755-73.
13.          Roy S, Majumdar AP. Cancer stem cells in colorectal cancer: genetic and epigenetic changes. Journal of stem cell research & therapy. 2012(6).
14.          Bahmad HF, Cheaito K, Chalhoub RM, Hadadeh O, Monzer A, Ballout F, et al. Sphere-formation assay: three-dimensional in vitro culturing of prostate cancer stem/progenitor sphere-forming cells. Frontiers in oncology. 2018;8:347.
15.          Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature medicine. 1997;3(7):730-7.
16.          Kazama S, Kishikawa J, Kiyomatsu T, Kawai K, Nozawa H, Ishihara S, et al. Expression of the stem cell marker CD133 is related to tumor development in colorectal carcinogenesis. Asian journal of surgery. 2018;41(3):274-8.
17.          Morath I, Hartmann T, Orian-Rousseau V. CD44: More than a mere stem cell marker. The international journal of biochemistry & cell biology. 2016;81:166-73.
18.          Weichert W, Knösel T, Bellach J, Dietel M, Kristiansen G. ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival. Journal of clinical pathology. 2004;57(11):1160-4.
19.          Elbadawy M, Usui T, Yamawaki H, Sasaki K. Emerging roles of C-Myc in cancer stem cell-related signaling and resistance to cancer chemotherapy: a potential therapeutic target against colorectal cancer. International journal of molecular sciences. 2019;20(9):2340.
20.          Roudi R, Barodabi M, Madjd Z, Roviello G, Corona SP, Panahi M. Expression patterns and clinical significance of the potential cancer stem cell markers OCT4 and NANOG in colorectal cancer patients. Molecular & Cellular Oncology. 2020;7(5):1788366.
21.          Takeda K, Mizushima T, Yokoyama Y, Hirose H, Wu X, Qian Y, et al. Sox2 is associated with cancer stem-like properties in colorectal cancer. Scientific reports. 2018;8(1):1-9.
22.          Shekarriz R, Montazer F, Alizadeh-Navaei R. Overexpression of cancer stem cell marker Lgr5 in colorectal cancer patients and association with clinicopathological findings. Caspian journal of internal medicine. 2019;10(4):412.
23.          Holah NS, Aiad HA-E-S, Asaad NY, Elkhouly EA, Lasheen AG. Evaluation of the role of ALDH1 as cancer stem cell marker in colorectal carcinoma: an immunohistochemical study. Journal of clinical and diagnostic research: JCDR. 2017;11(1):EC17.
24.          Munro MJ, Wickremesekera SK, Peng L, Tan ST, Itinteang T. Cancer stem cells in colorectal cancer: a review. Journal of clinical pathology. 2018;71(2):110-6.
 
 
 
Iranian Journal of Colorectal Research
Volume 9, Issue 3
September 2021
Pages 103-108

Files

Share

How to cite

Statistics