Project description:To demonstrate CD133+CD44+ and CD133+CD44- subpopulations of hepatocellular carcinoma as distinct subgroups, we have employed whole genome microarray expression profiling as a discovery platform to reveal the gene profiles of different subgroups and identify genes responsible for the enhanced metastatic potentials of CD133+CD44+ tumor cells. CD133+CD44+ and CD133+CD44- tumor cells were isolated from three human metastatic hepatocellular carcinoma specimens. A 76-gene consensus signature was identified that distinguished between CD133+CD44+ and CD133+CD44- subgroups. CD133+CD44+ and CD133+CD44- subgroups from different patients were well clustered as two distinct classes according to this signature, and many genes in this signature were reported involved in tumor metastasis. Expression of four genes (CCL4, DKK3, CCR5 and MMP12) from this signature was confirmed in another three metastatic HCC specimens by real-time PCR. CD133+CD44+ and CD133+CD44- subpopulations of hepatocellular carcinoma were isolated from three metastatic hepatocellular carcinoma specimens by flow cytometry. A total of 30K to 50K cells for each subgroup was obtained for each microarray.
Project description:We devised a novel insertional mutagenesis approach based on lentiviral vectors to induce hepatocellular carcinoma in three mouse models and identified four novel cancer initiating genes. Two genes are the well characterized Braf and Sos1, while the other two are Fign, encoding an AAA ATPase whose functions are poorly understood, and the paternally expressed gene Rtl1 within the complex Dlk1-Dio3 imprinted region recently involved in stemness. Interestingly, Fign and Braf regulate the expression of the maternal genes of the Dlk1-Dio3 imprinted region, suggesting that both maternally and paternally expressed genes of this region play a role in hepatocarcinogenesis. Moreover, all the genes identified are upregulated and/or amplified/deleted in human hepatocellular carcinoma and play a relevant role in human hepatocarcinogenesis, as their expression levels and/or transcriptional signatures induced by their deregulation predict a different clinical outcome in hepatocellular carcinoma patients. Primary human hepatocytes were transduced with SINLV.ET.trBRAF, SINLV.PGK.GFP or mock treated. RNA was collected at 5 different timepoints post-transduction: 24h, 36h, 48h, 72h, 144h (6d). Each experimental point was done in triplicate (A,B,C)
Project description:To demonstrate CD133+CD44+ and CD133+CD44- subpopulations of hepatocellular carcinoma as distinct subgroups, we have employed whole genome microarray expression profiling as a discovery platform to reveal the gene profiles of different subgroups and identify genes responsible for the enhanced metastatic potentials of CD133+CD44+ tumor cells. CD133+CD44+ and CD133+CD44- tumor cells were isolated from three human metastatic hepatocellular carcinoma specimens. A 76-gene consensus signature was identified that distinguished between CD133+CD44+ and CD133+CD44- subgroups. CD133+CD44+ and CD133+CD44- subgroups from different patients were well clustered as two distinct classes according to this signature, and many genes in this signature were reported involved in tumor metastasis. Expression of four genes (CCL4, DKK3, CCR5 and MMP12) from this signature was confirmed in another three metastatic HCC specimens by real-time PCR.
Project description:Tumor cells were microdissected from FFPE sections of hepatocellular carcinoma (HCC) samples. Samples were compared between different localisations either within or around the tumor. miRNA was labeled with the Affymetrix FlashTag Biotin HSR RNA Labeling Kit 6 microdissected hepatocellular carcinoma samples were compared to four different tumor localisations (LC: cirrhotic septa of the tumor-adjacent liver; LP: tumor-adjacent liver parenchyma;TC: fibrinous capsule of the tumor; TP: tumor parenchyma)
Project description:We devised a novel insertional mutagenesis approach based on lentiviral vectors to induce hepatocellular carcinoma in three mouse models and identified four novel cancer initiating genes. Two genes are the well characterized Braf and Sos1, while the other two are Fign, encoding an AAA ATPase whose functions are poorly understood, and the complex Dlk1-Dio3 imprinted region which has been recently implicated in cancer and stemness. Activation of Fign or Braf and upregulation of the Dlk1-Dio3 imprinted region are functionally interconnected and may altogether control cell transformation, stemness and energy metabolism. Moreover, all the genes identified play a relevant role in human hepatocarcinogenesis as their expression levels and/or transcriptional signatures induced by their deregulation predict a different clinical outcome in hepatocellular carcinoma patients. These series consists of mRNA expression microarray data (The GeneChip® Mouse Gene 1.0 ST Array, Affymetrix) from 8 non-tumoral liver and 21 hepatocellular carcinoma induced by insertional mutagenesis. LV.ET.LTR was administered to newborn mice with 3 different genetic backgrounds. Hepatocellular carcinomas were induced in all the mouse models. Vector integrations were retrieved form each single liver mass. From 21 liver masses, RNA was collected to perform whole transcriptome analysis.
Project description:To explore the target genes of long noncoding RNA lncTCF7, we established lncTCF7-silenced HCC primary CSC cells and conducted transcriptome microarray analysis. We used microarrays to identify distinct gene expression underlying shCtrl and shlncTCF7 of hepatocellular carcinoma sample stem cells. We cultured shlncTCF7 and shCtrl cells from hepatocellular carcinoma (HCC) clinical sample, then hybridized on Affymetrix microarrays. We sought to identify distinct target genes of lncTCF7 in liver cancer stem cells (CSCs).
Project description:We devised a novel insertional mutagenesis approach based on lentiviral vectors to induce hepatocellular carcinoma in three mouse models and identified four novel cancer initiating genes. Two genes are the well characterized Braf and Sos1, while the other two are Fign, encoding an AAA ATPase whose functions are poorly understood, and the paternally expressed gene Rtl1 within the complex Dlk1-Dio3 imprinted region recently involved in stemness. Interestingly, Fign and Braf regulate the expression of the maternal genes of the Dlk1-Dio3 imprinted region, suggesting that both maternally and paternally expressed genes of this region play a role in hepatocarcinogenesis. Moreover, all the genes identified are upregulated and/or amplified/deleted in human hepatocellular carcinoma and play a relevant role in human hepatocarcinogenesis, as their expression levels and/or transcriptional signatures induced by their deregulation predict a different clinical outcome in hepatocellular carcinoma patients.
Project description: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.