Spheroid-Formation (Colonosphere) Assay for in Vitro Assessment and Expansion of Stem Cells in Colon Cancer

Shaheen, Sameerah; Ahmed, Mehreen; Lorenzi, Federica; Nateri, Abdolrahman S.

Abstract

Colorectal cancers (CRCs) form a disorganized hierarchy of heterogeneous cell populations on which current chemotherapy regimens fail to exert their distinctive cytotoxicity. A small sub-population of poorly differentiated cancer stem-like cells (CSCs), also known as cancer initiating cells, may exhibit embryonic and/or adult stem-cell gene expression signatures. Self-renewal and survival signals are also dominant over differentiation in CSCs. However, inducers of differentiation exclusive to CSC may affect cellular pathways required for the formation and progression of a tumor, which are not utilized in normal adult stem-cells. Nevertheless, assays for targeting CSCs have been hindered by expanding and maintaining rare CSCs in vitro. However, CRC-CSCs are able to form floating spheroids (known as colonospheres) 3-dimentinionally (3D) in a serum-free defined medium. Therefore, great efforts have been paid to improve colonosphere forming assay as a preclinical model to study tumor biology and to conduct drug screening in cancer research. The 3D-colonosphere culture model may also represent in vivo conditions for the spontaneous aggregation of cancer cells in spheroids. This protocol describes the development of an enrichment/culture assay using CRC-CSCs to facilitate colorectal cancer research through immunofluorescence staining of colonospheres. We have developed colonospheres from HCT116 CRC cell line to compare and link CRC-CSC markers to the NANOG expression level using an immunofluorescence assay. Our data also show that the immunostaining assay of colonosphere is a useful method to explore the role and dynamics of CRC-CSCs division between self-renewal and cell lineage differentiation of cancer cells. In principle, this method is applicable to a variety of primary cells and cell lines of epithelial origin. Furthermore, this protocol may also allow screening of libraries of compounds to identify bona fide CRC-CSC differentiation inducers.

Citation

Shaheen, S., Ahmed, M., Lorenzi, F., & Nateri, A. S. (2016). Spheroid-Formation (Colonosphere) Assay for in Vitro Assessment and Expansion of Stem Cells in Colon Cancer. Stem Cell Reviews and Reports, 12(4), 492-499. https://doi.org/10.1007/s12015-016-9664-6

• Journal Article Type: Article
• Acceptance Date: May 13, 2016
• Online Publication Date: May 20, 2016
• Publication Date: 2016-08
• Deposit Date: Aug 13, 2018
• Publicly Available Date: Jan 14, 2019
• Journal: Stem Cell Reviews and Reports
• Print ISSN: 1550-8943
• Electronic ISSN: 1558-6804
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 12
• Issue: 4
• Pages: 492-499
• DOI: https://doi.org/10.1007/s12015-016-9664-6
• Keywords: Cell Biology; Cancer Research
• Public URL: https://nottingham-repository.worktribe.com/output/1109626
• Publisher URL: https://link.springer.com/article/10.1007%2Fs12015-016-9664-6
• Contract Date: Jan 14, 2019