Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system

Exp Cell Res. 2012 Aug 1;318(13):1577-85. doi: 10.1016/j.yexcr.2012.03.026. Epub 2012 Apr 2.


Clinically relevant in vitro methods are needed to identify new cancer drugs for solid tumors. We report on a new 3-D spheroid cell culture system aimed to mimic the properties of solid tumors in vivo. The colon cancer cell lines HCT-116 wt and HCT-116 wt/GFP were grown as monolayers and for 3 or 6 days on 96-well NanoCulture® plates to form spheroids. Expression of surface markers, genes and hypoxia were assessed to characterize the spheroids and drug induced cytotoxicity was evaluated based on fluorescein diacetate (FDA) conversion by viable cells to fluorescent fluorescein or by direct measurement of fluorescence of GFP marked cells after a 72 h drug incubation. The cells reproducibly formed spheroids in the NanoCulture® plates with tight cell-attachment after 6 days. Cells in spheroids showed geno- and phenotypical properties reminiscent of hypoxic stem cells. Monolayer cultured cells were sensitive to standard and investigational drugs, whereas the spheroids gradually turned resistant. Similar results for cytotoxicity were observed using simplified direct measurement of fluorescence of GFP marked cells compared with FDA incubation. In conclusion, this new 3-D spheroid cell culture system provides a convenient and clinically relevant model for the identification and characterization of cancer drugs for solid tumors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Cell Culture Techniques / methods
  • Cell Hypoxia / genetics
  • Cell Survival / drug effects
  • Colonic Neoplasms / drug therapy*
  • Colonic Neoplasms / genetics
  • Colonic Neoplasms / metabolism
  • Colonic Neoplasms / pathology*
  • Drug Resistance, Neoplasm*
  • Drug Screening Assays, Antitumor / methods
  • Gene Expression
  • Green Fluorescent Proteins / genetics
  • HCT116 Cells
  • Humans
  • Immunohistochemistry
  • Spheroids, Cellular / drug effects
  • Spheroids, Cellular / metabolism
  • Spheroids, Cellular / pathology


  • Antineoplastic Agents
  • Green Fluorescent Proteins