title: Spheroid-Formation (Colonosphere) Assay for in Vitro Assessment and Expansion of Stem Cells in Colon Cancer.
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.
authors.affiliation: Cancer Genetics and Stem Cell Group, Cancer Biology Unit, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, NG7 2UH, UK.
authors.forename: Sameerah
authors.initials: S
authors.lastname: Shaheen
authors.affiliation: Cancer Genetics and Stem Cell Group, Cancer Biology Unit, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, NG7 2UH, UK.
authors.forename: Mehreen
authors.initials: M
authors.lastname: Ahmed
authors.affiliation: Cancer Genetics and Stem Cell Group, Cancer Biology Unit, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, NG7 2UH, UK.
authors.forename: Federica
authors.initials: F
authors.lastname: Lorenzi
authors.affiliation: Cancer Genetics and Stem Cell Group, Cancer Biology Unit, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, NG7 2UH, UK. a.nateri@nottingham.ac.uk.
authors.forename: Abdolrahman S
authors.initials: AS
authors.lastname: Nateri
mesh_terms: D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation; D000066673:Cell Self Renewal; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D008856:Microscopy, Fluorescence; D055262:Mucin-2 / Q000378:metabolism; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D018874:Spheroids, Cellular / Q000378:metabolism* / Q000473:pathology
chemical_list: D018960:Hyaluronan Receptors; C524625:MUC2 protein, human; D055262:Mucin-2; D000071317:Nanog Homeobox Protein; D049452:Green Fluorescent Proteins
keywords: Cancer stem cell; Colonosphere; Colorectal cancer; Differentiation; Immunofluorescence; In vitro assay; Self-renewal