2ml of PBS The mice were observed for 2 months for the appearanc

2ml of PBS. The mice were observed for 2 months for the appearance of tumour development. Matrigel assay Colospheres and spheroids were added to Matrigel solution (3mgml?1) (BD Biosciences, Le Pont de Claix, France) in 24-well plates before polymerisation of the lattice. Culture medium was added to the Matrigel such information layer. Gelatin zymography Gelatin zymography assays were performed with a Bio-Rad laboratories kit (Marnes La Coquette, France), according to the manufacturer’s instructions. Colospheres, spheroids and xenograft tissue were lysed with zymogram sample buffer and the amount of protein was estimated using the Bradford method (Biorad Dc Protein Assay; Bio-Rad Laboratories). Twenty micrograms of protein from each sample was separated on polyacrylamide gels containing 10% gelatin.

After electrophoresis, the gels were washed in renaturation buffer for 30min, and incubated overnight at 37��C in the development buffer. Gels were stained with a Coomassie blue R250 0.5% solution and destained with 3 �� destaining solution. Flow cytometric analysis Disaggregated colospheres, spheroids and xenografts were assessed using an Epics XL cytometre (Coulter, Villepinte, France) with the following antibodies: anti-human CD133/2 (clone 293C3) phycoerythrin, anti-human EpCAM (clone HEA-125) fluorescein isothiocyanate (Miltenyi-Biotec SAS, Paris, France) and anti-human CD44-FITC (clone G44-26; BD Biosciences), at appropriate dilutions. As for xenograft tissue, human colon cancer cells were magnetically labelled and separated from mouse stroma cells using human EpCAM microbeads (Miltenyi-Biotec SAS) according to the manufacturer’s instructions, before assessment by flow cytometry.

Confocal microscopy About 30 colospheres in suspension were fixed for 3h at 4��C in PBS containing 4% PFA, followed by washes in PBS. Colospheres were then permeabilised in 1% Triton X-100 in PBS for 1h and washed with PBS. After a 1h incubation in PBS/NH4Cl (50mmoll?1), followed by washes in PBS, colospheres were saturated overnight with 3% BSA in PBSt (0.1% Triton X-100 in PBS) at 4��C and washed in PBSt. Colospheres were incubated for 24h at 4��C with anti-human EpCAM-FITC (clone HEA-125) in PBSt (dilution 1:50) and rinsed with PBSt. The DNA marker, TOPRO-3 (Invitrogen-Molecular Probes, Cergy Pontoise, France), was then applied for 20min at room temperature.

Colospheres were mounted in glycerol/PBS (90/10: v/v). Images were recorded on a Leica TCS SP2 confocal microscope. Results Observation of colospheres within ex vivo dissociated colon primary tumours Dissociation of 203 fresh colon primary tumour tissues directly harvested from human patients undergoing surgery led to the formation of spherical organoid structures, Cilengitide which we named colospheres (Figure 1A), in 98 out of the 203 specimens (47%) in just 1 day. Colospheres displayed a diameter comprised between 20 and 200��m and were easily identifiable because of their smooth refringent outline.

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