Cargando…
Suspension culture strategies to enrich colon cancer stem cells
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 c...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
D.A. Spandidos
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9950343/ https://www.ncbi.nlm.nih.gov/pubmed/36844615 http://dx.doi.org/10.3892/ol.2023.13702 |
_version_ | 1784893142077538304 |
---|---|
author | Zhou, Guojun Lv, Xiaojiang Zhong, Xiaorong Ying, Wei Li, Wenbo Feng, Yanchao Xia, Qinghua Li, Jianshui Jian, Shunhai Leng, Zhengwei |
author_facet | Zhou, Guojun Lv, Xiaojiang Zhong, Xiaorong Ying, Wei Li, Wenbo Feng, Yanchao Xia, Qinghua Li, Jianshui Jian, Shunhai Leng, Zhengwei |
author_sort | Zhou, Guojun |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9950343 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | D.A. Spandidos |
record_format | MEDLINE/PubMed |
spelling | pubmed-99503432023-02-25 Suspension culture strategies to enrich colon cancer stem cells Zhou, Guojun Lv, Xiaojiang Zhong, Xiaorong Ying, Wei Li, Wenbo Feng, Yanchao Xia, Qinghua Li, Jianshui Jian, Shunhai Leng, Zhengwei Oncol Lett Articles 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. D.A. Spandidos 2023-02-06 /pmc/articles/PMC9950343/ /pubmed/36844615 http://dx.doi.org/10.3892/ol.2023.13702 Text en Copyright: © Zhou et al. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. |
spellingShingle | Articles Zhou, Guojun Lv, Xiaojiang Zhong, Xiaorong Ying, Wei Li, Wenbo Feng, Yanchao Xia, Qinghua Li, Jianshui Jian, Shunhai Leng, Zhengwei Suspension culture strategies to enrich colon cancer stem cells |
title | Suspension culture strategies to enrich colon cancer stem cells |
title_full | Suspension culture strategies to enrich colon cancer stem cells |
title_fullStr | Suspension culture strategies to enrich colon cancer stem cells |
title_full_unstemmed | Suspension culture strategies to enrich colon cancer stem cells |
title_short | Suspension culture strategies to enrich colon cancer stem cells |
title_sort | suspension culture strategies to enrich colon cancer stem cells |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9950343/ https://www.ncbi.nlm.nih.gov/pubmed/36844615 http://dx.doi.org/10.3892/ol.2023.13702 |
work_keys_str_mv | AT zhouguojun suspensionculturestrategiestoenrichcoloncancerstemcells AT lvxiaojiang suspensionculturestrategiestoenrichcoloncancerstemcells AT zhongxiaorong suspensionculturestrategiestoenrichcoloncancerstemcells AT yingwei suspensionculturestrategiestoenrichcoloncancerstemcells AT liwenbo suspensionculturestrategiestoenrichcoloncancerstemcells AT fengyanchao suspensionculturestrategiestoenrichcoloncancerstemcells AT xiaqinghua suspensionculturestrategiestoenrichcoloncancerstemcells AT lijianshui suspensionculturestrategiestoenrichcoloncancerstemcells AT jianshunhai suspensionculturestrategiestoenrichcoloncancerstemcells AT lengzhengwei suspensionculturestrategiestoenrichcoloncancerstemcells |