Figure 2
Colospheres are kept as a viable short-term 3D culture model in non-adherent conditions and resist anoikis as well-organised aggregates. (A) Colospheres maintained on tissue-culture-treated plastic form adherent cell sheets. Morphological changes were documented at day 3 (D3)–D10 after ex vivo dissociation. Bar: 200 μm. (B and C) Colospheres collected at D3 after tumour tissue dissociation are cultured for additional days on agarose to prevent adhesion to the plastic. (B) Haemalun–eosin–safran and anti-Ki-67 staining at D3 and D17. Magnification × 100. (C) Cell cycle analysis by propidium iodide in XenoCT320 colosphere-forming cells at D3 (left) and D18 (right). The percentages for each cell cycle phase are presented as the mean±s.d. of at least three independent experiments. (D) Three days after tumour tissue dissociation, the non-forming colosphere cells are collected. Haemalun–eosin–safran (top) and pancytokeratin (bottom) staining show that these single epithelial cells are massively dead. Magnification × 200. (E and F) Overnight incubated with anti-human E-cadherin or with EDTA lead to colosphere dispersion (E) and significant increase in the caspase-3 activity (F, *P<0.05, Student’s t-test). Expression of caspase-3 activity as relative light units. HT29 monolayer is used to control the non-toxicity of EDTA concentration.
