Xenograft and spheroid culture XenoCT320 xenograft, CT320 and CT320X6 cell lines have been previously described (Dangles-Marie et al, 2007). For xenograft passage, tumour fragments were subcutaneously thoroughly grafted in the interscapular region into 5-week-old athymic nude female mice (Harlan, Winkelmann, Germany) bred and maintained in specified pathogen-free conditions (protocol approval no P2.VDM.026.07, local ethical committee on animal experiments, CREEA Ren�� Descartes, Paris, France). For spheroid culture, tumour cells grown as a monolayer were resuspended with trypsin, and 5 �� 103 cells were seeded in microwells coated with 1% agarose so as to obtain, after 3 days, a single spheroid per well. 2D multipositioning light videomicroscopy Colosphere and spheroid development was monitored by time-lapse video microscopy for 65h at a 4min interval.
Dynamic sequences were obtained on a DM IRBE stand equipped with a motorised stage (Leica, Mannheim, Germany) using a 37��C 8% CO2-humidified stage-top incubator (Life Imaging Services, Basel, Switzerland). Histological characterisation Colospheres and spheroids were embedded using the Cytoblock method (Briffod et al, 2000) and the Shandon kit (Thermo electron corporation, Saint Herblay, France). Immunostaining was performed on the resulting paraffin sections using an automated immunostainer (Ventana, Strasbourg, France) with mAb to Ki67 antigen (MIB1 clone; Dako, Trappes, France) and to E-cadherin (4A2C7 clone; Zymed, Montrouge, France). At least eight independent samples were collected for the specific analysis of colospheres and spheroids.
TBP gene expression Specific mouse TBP gene expression and the expression of both the mouse and the human TBP genes were studied by real-time quantitative RT�CPCR (L��vy et al, 2004) to determine the quantity of mouse cells in human xenografts and colospheres. With the assistance of the computer program Oligo 5.0 (National Biosciences, Plymouth, MN, USA), the murine Tbp primer pair was selected to be mouse specific when compared with the sequence of the human TBP gene, whereas the total TBP primer pair was selected to amplify both the mouse and the human TBP genes. BLASTN searches against dbEST and nr (the non-redundant set of the GenBank, EMBL and DDBJ database sequences) were conducted to confirm the total gene specificity of the nucleotide sequences chosen for the primers and for the absence of DNA polymorphisms.
The nucleotide sequences of the primers used were the following: Mm-TBP-U (5��-CCCTTGTACCCTTCACCAATGAC-3��) and Mm-TBP-L (5��-TCACGGTAGATACAATATTTTGAAGCTG-3��) and Total-TBP-U (5��-TGCACAGGAGCCAAGAGTGAA-3��) and Total-TBP-L (5��-CACATCACAGCTCCCCACCA-3��). The thermal Entinostat cycling conditions comprised an initial denaturation step at 95��C for 10min, and 50 cycles at 95��C for 15s and 65��C for 1min.