More specifically, three distinct and stable primary tumour propagating cell lineages obtained from a 74-year-old male patient with advanced HCC were expanded and extensively characterised by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rc2/2 mice. The results indicate that the clonal evolution of TPCs is a driver of intra-HCC heterogeneity. After appropriate samples had been taken for histological purposes, a liver tumour tissue specimen was collected and dissociated as previously described . The cell suspensions obtained from the tumoral tissue were cultured onto order Kinase Inhibitor Library collagen-coated Petri dishes at a density of 56105 cells/cm2 in IMDM, supplemented with 20% FBS, 1% nonessential amino acids, 1% glutamine, and 1% penicillin/streptomycin . The medium was changed 24 hours after seeding in order to remove dead cells and debris, and was then replaced twice a week; the cells were maintained at 37uC in a humidified 5% CO2 incubator. After the appearance of colonies of 50�C100 cells , the cells were replated in plastic flasks. Colonies with different cell morphologies were picked up separately and replated in different flasks. Confluent cells were detached using trypsin/EDTA , counted and replated 1:3 at every split in order to FTY720 chemical information determine their growth kinetics. Three morphologically different cell colonies were raised in culture from a single specimen of about 15 grams taken from a male patient with trabecular HCC who was HBsAg and anti-HCV negative and had no clinical or histological evidence of liver cirrhosis. In order to determine whether heterogeneity was an intrinsic property of the three cell populations, each one was plated as a single cell by means of limiting dilution in 96-well plates. Clones with .50 cells were scored after three weeks, picked up and plated alone in flasks; cloning efficiency was defined as the percentage of cells developing a clone. The clones were characterised by flow cytometry at the first and fifth culture passages, after which only those whose morphological and/or phenotypical profile were different from the original mother population were further characterised. Moreover, once we had evaluated the tumorigenicity in vivo of the three HCC cell lineages and since previous reports support the evidence that in vitro clonogenicity is related to in vivo tumorigenicity ,