Open Access

Suspension culture strategies to enrich colon cancer stem cells

  • Authors:
    • Guojun Zhou
    • Xiaojiang Lv
    • Xiaorong Zhong
    • Wei Ying
    • Wenbo Li
    • Yanchao Feng
    • Qinghua Xia
    • Jianshui Li
    • Shunhai Jian
    • Zhengwei Leng
  • View Affiliations

  • Published online on: February 6, 2023     https://doi.org/10.3892/ol.2023.13702
  • Article Number: 116
  • Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

How to efficiently obtain high‑purity cancer stem cells (CSCs) has been the basis of CSC research, but the optimal conditions for serum‑free suspension culture of CSCs are still unclear. The present study aimed to define the optimal culture medium composition and culture time for the enrichment of colon CSCs via suspension culture. Suspension cell cultures of colon cancer DLD‑1 cells were prepared using serum‑free medium (SFM) containing variable concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to produce spheroids. Culture times were set at 10, 20 and 30 days. A total of nine different concentrations of EGF and bFGF were added to SFM to generate nine experimental groups. The proportions of CD44+, CD133+, and CD44+CD133+ double‑positive spheroid cells were detected via flow cytometry. mRNA expression of stemness‑, epithelial‑mesenchymal transition‑ and Wnt/β‑catenin pathway‑associated genes was determined via reverse transcription‑quantitative PCR. Self‑renewal ability was evaluated by a sphere‑forming assay. Tumorigenesis was studied in vitro using a colony formation assay and in vivo via subcutaneous cell injection in nude mice. It was found that the highest expression proportions of CD133+ and CD44+ spheroid cells were observed in group (G)9 (20 ng/ml EGF + 20 ng/ml bFGF) at 30 days (F=123.554 and 99.528, respectively, P<0.001), CD133+CD44+ cells were also observed in G9 at 30 days (and at 10 days in G3 and 20 days in G6; F=57.897, P<0.001). G9 at 30 days also displayed the highest expression of Krüppel‑like factor 4, leucine‑rich repeat‑containing G protein‑coupled receptor 5, CD44, CD133, Vimentin and Wnt‑3a (F=22.682, 25.401, 3.272, 7.852, 13.331 and 17.445, respectively, P<0.001) and the lowest expression of E‑cadherin (F=10.851, P<0.001). G9 at 30 days produced the highest yield of cell spheroids, as determined by a sphere forming assay (F=19.147, P<0.001); colony formation assays also exhibited the greatest number of colonies derived from G9 spheroids at 30 days (F=60.767, P<0.01), which also generated the largest mean tumor volume in the subcutaneous tumorigenesis xenograft model (F=12.539, P<0.01). In conclusion, 20 ng/ml EGF + 20 ng/ml bFGF effectively enriched colon CSCs when added to suspension culture for 30 days, and conferred the highest efficiency compared with other combinations.
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March-2023
Volume 25 Issue 3

Print ISSN: 1792-1074
Online ISSN:1792-1082

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Copy and paste a formatted citation
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Spandidos Publications style
Zhou G, Lv X, Zhong X, Ying W, Li W, Feng Y, Xia Q, Li J, Jian S, Leng Z, Leng Z, et al: Suspension culture strategies to enrich colon cancer stem cells. Oncol Lett 25: 116, 2023
APA
Zhou, G., Lv, X., Zhong, X., Ying, W., Li, W., Feng, Y. ... Leng, Z. (2023). Suspension culture strategies to enrich colon cancer stem cells. Oncology Letters, 25, 116. https://doi.org/10.3892/ol.2023.13702
MLA
Zhou, G., Lv, X., Zhong, X., Ying, W., Li, W., Feng, Y., Xia, Q., Li, J., Jian, S., Leng, Z."Suspension culture strategies to enrich colon cancer stem cells". Oncology Letters 25.3 (2023): 116.
Chicago
Zhou, G., Lv, X., Zhong, X., Ying, W., Li, W., Feng, Y., Xia, Q., Li, J., Jian, S., Leng, Z."Suspension culture strategies to enrich colon cancer stem cells". Oncology Letters 25, no. 3 (2023): 116. https://doi.org/10.3892/ol.2023.13702