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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.

Authors

Sameerah Shaheen

Mehreen Ahmed

Federica Lorenzi



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 Mar 29, 2024
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

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