Enrichment of cancer stem cells by agarose multi-well dishes and 3D spheroid culture
As the theory of cancer stem cells (CSCs) is maturing, CSC-targeted therapy is emerging as an important therapeutic strategy and seeking the ideal method for rapid enrichment and purification of CSCs has become crucial. So far, based on the known CSC phenotypes and biological characteristics, the methods for enrichment CSCs mainly include low adhesion culture, low oxygen culture, chemotherapy drug stimulation and side population (SP) sorting but these methods cannot realize quick enrichment of the desired CSCs. Herein, we adopt a novel method that efficiently enriches a certain amount of CSCs through agarose multi-well dishes using rubber micro-molds to make cancer cells into cell spheroids (3D). These 3D cancer cell spheroids in the proportions of expression of CSC biomarkers (single stain of CD44, CD44v6 and CD133 or double stain of both CD44 and CD133) were significantly higher than those of the conventional adherent culture (2D) using flow cytometry analysis. In addition, the expression levels of stemness transcription factors such as OCT4, NANOG and SOX2 in 3D were also significantly higher than that in 2D through Western blot (WB) and quantitative polymerase chain reaction (qPCR) assays. In addition, the CSCs in 3D could form colonies with different sizes in soft agar. In conclusion, we developed a new method to enrich some kinds of CSCs, which might be a benefit for future CSC-targeted therapy studies and anti-CSC drug screening applications.
KeywordsCancer stem cell 2D 3D Spheroid Enrichment
This work was supported by the National Nature Science Foundation of China (81701426, 81730042 and 81771636), the Medical and Health Research Science and Technology Plan Project of Zhejiang Province (2018KY523, 2017KY473 and 2017ZB067) and the Public Welfare Science and Technology Plan Project of Wenzhou City (Y20170151, Y20160069 and ZS2017012).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Fujii H, Honoki K, Tsujiuchi T, Kido A, Yoshitani K, Takakura Y (2009) Sphere-forming stem-like cell populations with drug resistance in human sarcoma cell lines. Int J Oncol 34:1381–1386Google Scholar
- Heider KH, Kuthan H, Stehle G, Munzert G (2004) CD44v6: a target for antibody-based cancer therapy. Cancer Immunol, Immunother: CII 53:567–579Google Scholar
- Jin X, Jin X, Kim H (2017) Cancer stem cells and differentiation therapy. Tumour Biol: J Int Socr Oncodev Biol Med 39:1010428317729933Google Scholar
- Meirelles K, Benedict LA, Dombkowski D, Pepin D, Preffer FI, Teixeira J, Tanwar PS, Young RH, MacLaughlin DT, Donahoe PK, Wei X (2012) Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance. Proc Natl Acad Sci U S A 109:2358–2363CrossRefGoogle Scholar
- Rao Pattabhi S, Martinez JS, Keller TC 3rd (2014) Decellularized ECM effects on human mesenchymal stem cell stemness and differentiation. Differentiation 88:131–143Google Scholar
- Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115Google Scholar
- Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828Google Scholar
- Todaro M, Gaggianesi M, Catalano V, Benfante A, Iovino F, Biffoni M, Apuzzo T, Sperduti I, Volpe S, Cocorullo G, Gulotta G, Dieli F, De Maria R, Stassi G (2014) CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Cell Stem Cell 14:342–356CrossRefGoogle Scholar
- Zhang CL, Huang T, Wu BL, He WX, Liu D (2017) Stem cells in cancer therapy: opportunities and challenges. Oncotarget 8:75756–75766Google Scholar