|Table of Contents|

Application of serum-free suspension spheroid culture method in enrichment and purification of human-derived prostate cancer stem cells

Journal Of Modern Oncology[ISSN:1672-4992/CN:61-1415/R]

Issue:
2023 16
Page:
2949-2956
Research Field:
Publishing date:

Info

Title:
Application of serum-free suspension spheroid culture method in enrichment and purification of human-derived prostate cancer stem cells
Author(s):
MAO Qiyuan1; LI Daorui1; ZHENG Qi1; CHEN Maokuan2; LIN Hongsheng1; WANG Xueqian1
1.Department of Oncology,Guang' anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053,China;2.Department of Urology,Surgery Department 1,Yongning District Hospital of Traditional Chinese Medicine,Guangxi Nanning 530000,China.
Keywords:
serum-free suspension spheroid culture; prostate cancer; tumor stem cells; DU145 cells; tumor stem cell microspheres（tumor spheres); experimental model
PACS:
R737.25
DOI:
10.3969/j.issn.1672-4992.2023.16.001
Abstract:
Objective：To enrich and purify human-derived prostate cancer stem cells by a serum-free suspension spheroid culture method and to validate it in vivo and vitro.Methods：The prostate cancer DU145 cell line was used to isolate,enrich and purify tumor stem cell microspheres（tumor spheres) by serum-free suspension spheroid culture.Stem cell phenotypes were identified by flow cytometry,and animal tumorigenesis assays were used to inoculate different quantitative levels of DU145 and tumor spheres into the bilateral axils of NOD/SCID mice to observe the tumorigenic rate and tumorigenic speed.The isolated enriched and cultured purified tumor spheres were assayed for induced differentiation,proliferation level,reactive oxygen species clusters,and cell cycle.The tumor stem cell characteristics of tumor spheres were further clarified.Specific proteins were detected by Western blot and relevant mRNA expression was detected by RT-PCR.Results：Serum-free suspension spheroid culture was used to enable the isolation and enrichment of logarithmic growth stage DU145 cells to form tumor spheres,which could be cultured for 3 generations to achieve purification,and the flow cytometric identification of their expression of prostate cancer stem cell phenotype CD44（+)CD24（-/low) ratio was （63±1)%,which was significantly higher than that of DU145 cells［（33±4)%］（P＜0.05).Animal tumorigenesis experiments showed that the tumorigenesis rate and tumorigenicity were higher on the tumor spheres side than on the DU145 cells side.Tumor spheres had a stronger self-renewal capacity and differentiation potential,and cell cycle assays revealed that （81.67±0.68)% of tumor spheres were in the G0/G1 phase of the cell cycle（P＜0.05),while tumor spheres had lower levels of reactive oxygen species cluster ROS levels（P＜0.05).Tumor spheres highly expressed tumor stem cell HES1 protein（P＜0.05) and tumor stem cell-associated mRNA of Notch1,HES1,HIF-1α,Sox2,and Jagged1（P＜0.05).Conclusion：The method of isolation and purification of DU145 tumor stem cells by serum-free suspension sphere culture is simple and easy,with high acquisition volume and high tumorigenic rate.Tumor spheres have clear tumor stem cell characteristics and can be used as a good experimental model for prostate cancer tumor stem cell research.

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Memo

Memo:
National Natural Science Foundation of China（No.82004179,82104656）;国家自然科学基金青年项目（编号：82004179,82104656）；“优秀青年科技人才（创新类）”培养专项（编号：ZZ14-YQ-013）；北京市科学技术协会-青年人才托举工程（编号：BYESS2023357）
Last Update: 1900-01-01