ABSTRACT: Increasing evidences have been recently reported on tumor cells heterogeneity within several malignancies, but data on liver tumours are still lacking. The aim of this study was to investigate and characterize tumor initiating cell (TIC) compartments within human hepatocellular carcinoma (HCC). After long term culture, we were able to identify 3 morphologically different tumor cell populations from an individual HCC specimen. These cell populations were extensively characterized by flow cytometry, fluorescence microscopy, single cell cloning, xenotransplantation in NOD/SCID IL2Rgamma -/- mice, karyotyping and microarray analyses. The 3 primary cell populations, hcc-1, -2 and -3, and the 2 clones (clone-1/7 and -1/8) generated by limiting dilution from hcc-1 showed different expression profiles for several tumor associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19. Moreover, they showed different doubling time and drug resistance. The tumorigenic potential was higher for hcc-1 and clone-1/7. Karyotyping analyses revealed a clonal evolution of cell populations and clones within the primary tumor. Importantly, we noticed that the primary tumor cell population with a higher tumorigenic potential and drug resistance was that showing more chromosomal alterations and containing different clones with both epithelial and mesenchymal features. Individual HCC can harbor different self-renewing tumorigenic cell types expressing a variety of morphology and phenotypic markers, karyotypic evolution and different gene expression profiles. This finding suggests that the therapeutic approach for HCC eradication should take into account the possible TIC heterogeneity due to intratumor clonal evolution. The aim was to analyse the whole genome expression profile of 3 HCC cell lines (Hcc-1, Hcc-2 and Hcc-3) and two clones (Clone-1/7 and Clone-1/8) cells obtained from one patient. Tumor sample was dissociated and cells cultured on collagen coated Petri dishes. When cell colonies appeared in culture, they were detached and cultured separately; clones were obtained fron HCC-1 cell lines by means of limiting dilution. Total Five independent total RNA extractions were performed for each cell line and clone and after checked the quality, concentration and integrity of the purified RNA, only samples showing the better results were chosen to be further processed. After the acquisition of the raw intensity data matrix with the corresponding annotations, probes corresponding to "predicted" genes or not associated to a Entrez ID were discarded from the analysis; moreover, different probe signals corresponding to the same Entrez ID were converted into unique intensity signal, corresponding to the median of the values for each array, in order to obtain a single signal for each Entrez ID. After this process, we generated a boxplot of 16964 intensity signals whose values were log2-transformed and quantile normalized using the aroma.light package for Bioconductor. Genes with a differential expression value of at least 2 fold (on log2 transformed scale) were considered differentially expressed.
