The ��Ct neverless value of the sample is determined by subtracting the Ct value of the murine TBP gene from the Ct value of the total (mouse+human) TBP gene. The NMm-TBP values of the samples are subsequently normalised such that the median of the NMm-TBP values of four mouse tissues was 100. As TBP is a ubiquitously expressed housekeeping gene, which encodes the TATA box-binding protein, a component of the DNA-binding protein complex TFIID, and shows a similar expression in our human and mouse tissues (Ct=27 for 5ng cDNA), the final result (normalised NMm-TBP value) determines the proportion of mouse cell contamination for a given sample. Electron microscopy After harvesting, colospheres and spheroids were analysed using standard techniques for transmission electron microscopy (Ribadeau Dumas et al, 2004).

Ultrathin sections were examined at 80kV using a JEOL JEM-1005 electron microscope (JEOL S.A., Croissy-sur-Seine, France). For scanning electron microscopy (SEM), cells were critical-point dried in hexamethyldisilizane (Sigma-Aldrich, Saint-Quentin, France). Samples were mounted on specimen stubs, sputter coated with gold using JEOL JCF100 and examined at 16kV using JEOL ISM-35CF. At least six samples for CT320X6 and CT320 spheroids and for XenoCT320 colospheres were submitted to electron microscopy examination. In vivo tumorigenicity assay The tumorigenicity of XenoCT320 colospheres, CT320X6 spheroids and CT320X6 single cells was compared in a subrenal capsule assay in nude mice.

Three days after xenograft tissue dissociation, colospheres of 100�C150��m diameter were manually collected under the microscope and an aliquot was trypsinised to estimate the number of viable cells in the colospheres. The concentration was adjusted to have a number of colospheres equivalent to 4 �� 104 cells per 10��l for intrarenal injection. Similarly, eight spheroids formed by seeding 5 �� 103 cells per microwell were injected in 10��l. As spheroids from this cell line grow slowly, spheroids collected 3 days after initiation contained about 5000 cells per spheroid Brefeldin_A for a diameter of about 150��m. An oblique incision was made on the skin parallel to the long axis of the right kidney in anaesthetised mice (xylazin/ketamin protocol). Injections of cells equivalent to 4 �� 104 cells were administered with a 27G needle in the subcapsular space in the right kidney. The internal diameter of the 27G needles is 200��m at the minimum, which would avoid any damage on spheroids and colospheres. After cell injection, the kidney was then returned to the peritoneal space and the skin was closed with surgical staples. The recipient mice were necropsied at 14 weeks after injection and kidneys, spleen, lung and liver were removed for histological examination.