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

Stem Cell Rev Rep. 2016 Aug;12(4):492-9. doi: 10.1007/s12015-016-9664-6.


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.

Keywords: Cancer stem cell; Colonosphere; Colorectal cancer; Differentiation; Immunofluorescence; In vitro assay; Self-renewal.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Differentiation*
  • Cell Proliferation*
  • Cell Self Renewal
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / metabolism
  • Colorectal Neoplasms / pathology
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HCT116 Cells
  • Humans
  • Hyaluronan Receptors / metabolism
  • Microscopy, Fluorescence
  • Mucin-2 / metabolism
  • Nanog Homeobox Protein / genetics
  • Nanog Homeobox Protein / metabolism
  • Neoplastic Stem Cells / metabolism*
  • Neoplastic Stem Cells / pathology
  • Spheroids, Cellular / metabolism*
  • Spheroids, Cellular / pathology


  • Hyaluronan Receptors
  • MUC2 protein, human
  • Mucin-2
  • Nanog Homeobox Protein
  • Green Fluorescent Proteins