Deciphering tumour tissue organization by 3D electron microscopy and machine learning.

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumour tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also found anatomical connections between the blood capillary and the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for future investigations in the emerging onconanotomy field.

Generation and characterization of rabbit polyclonal antibodies against Vasohibin-2 for determination of its intracellular localization.

Vasohibin-2 was recently identified as an important pro-angiogenesis factor in solid tumor and intracellular localization of its variants is important for elucidating the downstream mechanism(s) of its effects. Currently there are no reported antibodies affordable for intracellular localization. The aim of this study was to generate and characterize polyclonal antibodies against Vasohibin-2 and to determine the intracellular localization of Vasohibin-2. In this study, two polypeptides were synthesized and one prokaryotic Vasohibin-2 recombinant protein was custom-made. New Zealand rabbits were immunized with the polypeptide mixture and prokaryotic recombinant protein, respectively. The purified antibodies from the antiserum were validated by ELISA, western blotting (WB), immunofluorescence (IF), immunohistochemistry (IHC) and immunoprecipitation (IP). In order to determine intracellular localization, the cytoplasmic and nuclear proteins of the human liver cancer cell line HepG2 were isolated for the detection of Vasohibin-2 by western blotting. Vasohibin-2 cDNA, coding for 311 and 355 amino acid residues, fused with or without a DDK/V5 tag at the c-terminus, respectively, was cloned into the Lv-CMV-EGFP vector. Lentiviruses were successfully packaged. Vasohibin-2-overexpressing HepG2-VASH2 (355 amino acid residues) and HepG2-VASH2-V5 (311 amino acid residues fused with V5 tag at the c-terminus) human liver cancer cell lines were established. Approximately 1-2x106 HepG2, HepG2-VASH2 and HepG2-VASH2-V5 cells were injected subcutaneously into the flanks of BALB/c nude mice. Xenograft tumors were harvested for immunohistochemistry. HepG2 cells were transiently transfected with the Lv-CMV-EGFP vectors containing Vasohibin-2 cDNA (coding for 311/355 amino acid residues with a DDK tag at the c-terminal), followed by anti-DDK immunofluorescence. The antibodies obtained were able to detect human VASH2 successfully as applied in western blotting, IF, IHC and IP. Results from IF, IHC and WB (post cytoplasmic/nuclear protein isolation) showed a quite different intracellular localization of VASH2 protein. The VASH2 (with 355 amino acid residues) was located in the cytoplasm while VASH2 (with 311 amino acid residues) was located in the nucleus. The former was found to be a relatively low abundance protein. We successfully generated three rabbit anti-human Vasohibin-2 polyclonal antibodies which can be used for western blotting, IF, IP and IHC. These antibodies will provide a convenient tool for further studies on Vasohibin-2. This is the first study to report differences in the intracellular localization of the VASH2 protein and, hence, a new research direction on the study of VASH2.

Modulation of morphology and functions of human hepatoblastoma cells by nano-grooved substrata.

It is known that cellular behavior is affected by nano-patterned topography. For example, many cell types tend to align and extend along the direction of nano-grooves/ridges structures. In this study, we investigated the impact of nano-grooves/ridges on hepatocyte morphology and functions. HepG2/C3A (C3A) cells were cultured on nano-grooved silicon or polystyrene substrata with various widths (from 100 to 500 nm) and depths (from 100 to 380 nm). Nano-grooved substrates induced dramatic changes in C3A cell morphology. The cells formed spheroids on the flat substrates, while C3A cells spread and grew confluently with elongated and aligned morphology along the nano-grooves/ridges. Albumin synthesis was enhanced on the nano-grooved silicon substrates compared to the flat surface, and was decreased with increasing groove depths. Urea conversion on the shallow grooves (400 nm wide and 100 nm deep) remained at the same level of that on the flat surfaces, but was decreased on the deeper grooves. We found that the functions of hepatocytes were enhanced on the substrates with shallow grooves. The nano-grooved substrates may be applied as in vitro culture systems of hepatocytes for both diagnostic and therapeutic applications.

The expression of Bcl-XL, Bcl-XS and p27Kip1 in topotecan-induced apoptosis in hepatoblastoma HepG2 cell line.

BACKGROUND: To assess the efficacy of topotecan, a topoisomerase I specific inhibitor in S-phase, the reagent-induced apoptosis and cytotoxicity as well as related proteins expression, had been preliminarily investigated in human hepatoblastoma HepG2 cells. METHODS: Microculture tetrazolium assay (MTT), HE staining, transmission electron microscopy (TEM), flow cytometry (FCM), quantitative immunocytochemistry (QI), gene tranfection and RNAi technology were employed to carry out the exploration. RESULTS: Topotecan could potently kill HepG2 cells via inducing apoptosis and demonstrated strong cytotoxicity in a time, dose-dependent manner with IC50 of about 95 mu g/L. According to morphologic observation and FCM analyses, it was confirmed that the drug treatment, causing significant S-phase arrest, could trigger a typical interphase apoptosis, the main traits of which were identified as chromatin pycnosis and cytoplasm condensation. It was shown that the expression of Bcl-XL was simultaneously down-regulated with the up-regulation of Bcl-XS in cytoplasm, which was possibly a key downstream event following the topotecan-induced DNA damage in nucleus. The expression level of p27Kip1, a negative regulator in cell cycle at G1/S transient, was also elevated. Transfection of pcDNA 3.1-p27Kip1 into HepG2 cells could abrogate the cytotoxicity in a degree while silence of p27Kip1 with siRNA in drug treatments could significantly increased the chemosensitivity, strongly indicating that the up-regulation of p27Kip1 was not an apoptosis-promoting, but a self-rescue response against drug by moderate G0/G1 arrest. CONCLUSION: Topotecan had potent cytotoxicity against HepG2 cells by triggering an interphase apoptosis possibly mediated by increasing the ratio of Bcl-XS/Bcl-XL. Up-regulation of p27Kip1in TPT treatments could be a protective response for self-rescue and silence of the gene markedly augmented TPT cytotoxicity. Therefore, the experiment in vitro could provide a new idea for the clinical chemotherapy based on the combination of traditional drugs with the specific-siRNA targeted on the protective response gene.

Atomic force microscopy-based cell nanostructure for ligand-conjugated quantum dot endocytosis.

While it has been well demonstrated that quantum dots (QDs) play an important role in biological labeling both in vitro and in vivo, there is no report describing the cellular nanostructure basis of receptor-mediated endocytosis. Here, nanostructure evolution responses to the endocytosis of transferrin (Tf)-conjugated QDs were characterized by atomic force microscopy (AFM). AFM-based nanostructure analysis demonstrated that the Tf-conjugated QDs were specifically and tightly bound to the cell receptors and the nanostructure evolution is highly correlated with the cell membrane receptor-mediated transduction. Consistently, confocal microscopic and flow cytometry results have demonstrated the specificity and dynamic property of Tf-QD binding and internalization. We found that the internalization of Tf-QD is linearly related to time. Moreover, while the nanoparticles on the cell membrane increased, the endocytosis was still very active, suggesting that QD nanoparticles did not interfere sterically with the binding and function of receptors. Therefore, ligand-conjugated QDs are potentially useful in biological labeling of cells at a nanometer scale.

Fine needle aspiration cytology of hepatoblastoma. Recognition of subtypes on cytomorphology.

OBJECTIVE: To delineate the cytomorphologic appearances of hepatoblastoma (HBL) in the largest series to date and to evaluate the feasibility of subtyping on fine needle aspiration cytology (FNAC). STUDY DESIGN: Papanicolaou- and May-Grünwald-Giemsa-stained smears of aspirates from 26 cases of HBL were analyzed by 2 observers. Histologic material, available in 15 cases, was correlated. A cytology grouping system was proposed on the basis of which all cases were classified. RESULTS: The ages of the patients ranged from 4 months to 9 years. Twenty-five cases were categorized as epithelial HBL, with epithelial fragments showing a trabecular arrangement and acinar formation in all, and extramedullary hemopoiesis in 20 cases. It was possible to differentiate fetal and embryonal areas on FNAC. Six cases showed only fetal elements (cytology group F), characterized by cells with abundant cytoplasm and a small, rounded nucleus resembling a normal fetal hepatocyte. The chromatin was finely granular, with a single, central nucleolus. Pleomorphism and mitoses were not seen, and the nuclear/cytoplasmic ratio was < or = 1/3. Fourteen cases showed, in addition to fetal elements, an embryonal component characterized by cells with scant cytoplasm, a pleomorphic nucleus, N/C ratio of > or = 3/1, coarsely granular chromatin and 2-4 angulated nucleoli. Mitoses were seen in these cells (1-4/1,000 cells). Of these 14 cases, 6 showed predominantly fetal and scant embryonal cells, while 8 cases showed fetal and embryonal components in equal amounts (cytology groups Fe and FE, respectively). Four cases showed predominantly embryonal cells (cytology group E). One case was unclassifiable (U). On histology, 8 of 14 cases were of mixed epithelial and mesenchymal type, but mesenchymal tissue was not seen on the corresponding cytology. The cytology grouping system correlated well with histology. One case was small cell undifferentiated HBL and resembled a round cell tumor without differentiation. Macrotrabecular arrangement was not seen on cytology but was seen on histology in 1 case. CONCLUSION: Epithelial HBL can be easily diagnosed in aspirates further classified into fetal and embryonal subtypes, which may be of prognostic relevance. The proposed cytology grouping system is effective in semiquantification of the observed subtypes.

Effects of P-glycoprotein modulation on the chemotherapy of xenotransplanted human hepatoblastoma.

Multidrug resistance (MDR) contributes to limited treatment results in human hepatoblastoma (HB). The MDR1 gene and its product P-glycoprotein (P-gP) has been identified as important factor in this development. In other tumors, P-gP modulation leads to a restored chemosensitivity of the cells. The aim of this study was to analyze the P-gP-modulating effects of PSC 833, a cyclosporine derivate, and verapamil on the chemotherapy of HB in vivo. HB from 2 patients were transplanted subcutaneously into nude mice NMRI (nu/nu). Animals were divided into 7 groups: Group 1 (Control); Group 2 (CDDP); Group 3 (DOXO); Group 4 (DOXO + verapamil); Group 5 (DOXO + PSC 833); Group 6 (CDDP + verapamil); and Group 7 (CDDP + PSC 833). If DOXO was administered (regardless of the combination), the dose was two times 60 mg/m2. If CDDP was administered, the dose was two times 27 mg/m2. When the chemosensitizers were administered, the doses for PSC 833 and for verapamil were four times 5 mg/kg body-weight. In the combined treatment groups the chemosensitizers were given ten minutes prior to CDDP and DOXO. Tumor volume developments and a-fetoprotein (AFP) alterations were assessed. Relative expression levels of the MDR1 gene after treatment were determined using a semiquantitative rT-PCR approach. In a mixed HB, both chemosensitizers combined with DOXO or CDDP produced a significant reduction of tumor growth (p = .0001-.00063) and AFP levels (p = .0006-.0128) compared to tumors treated with DOXO or CDDP only. Treatment results were identical to those in a less differentiated pure embryonal HB, but only in one case (DOXO + PSC 833, p = .031) significant. The chemosensitizers had no influence on the MDR1 gene expression. MDR1 modulators improve the efficiency of DOXO and CDDP treatment in xenotransplanted HB. They do not induce a further increase of drug resistance in the tumors. The data provide evidence that chemosensitizers might improve treatment results in patients with advanced or relapsed HB.

Membranes for biohybrid liver support: the behaviour of C3A hepatoblastoma cells is dependent on the composition of acrylonitrile copolymers.

Co-polymers based on acrylonitrile, N-vinylpyrrolidone, aminoethylmethacrylate and sodium methallylsulfonate were used to prepare flat membranes by phase inversion. The surface properties of membranes were characterised by water contact angle measurements, atomic force microscopy and X-ray photoelectron spectroscopy (XPS). Membrane permeability was estimated by porosity measurements with water as test liquid. Human C3A hepatoblastoma cells were plated on these materials. Cell-material interaction was characterised by overall cell morphology, formation of focal adhesion contacts and intercellular junctions. Furthermore, cell proliferation was measured and compared with the functional activity of cells as indicated by 7-ethoxycoumarin-O-deethylation. More hydrophilic materials reduced spreading of cells, formation of focal adhesion and subsequent proliferation while homotypic cell adhesion was facilitated in correlation with stronger expressions of intercellular junctions and improved functional activity. In contrast, membranes with stronger adhesivity enhanced cell proliferation but reduced the functional activity of cells. It was concluded that the co-polymerisation of acrylonitrile with hydrophilic co-monomers, such as N-vinylpyrrolidone, could be used to tailor membrane materials for the application in biohybrid liver support systems.

Stem-like cells in hepatoblastoma.

BACKGROUND: The principles of tumor biology suggest that hepatoblastoma is derived from a pluripotent stem cell. Our studies were undertaken to investigate this tumor for the presence of cells with morphologic and immunophenotypic features of the oval cells of rodents that are thought to be closely related to hepatic stem cells. PROCEDURE: Hepatoblastomas of various subtypes were investigated by electron microscopy and immunoelectron microscopy with antibodies against cytokeratin 7, in the liver a marker of biliary differentiation, and albumin, a marker of hepatocytic differentiation. Immunohistochemical investigations were performed with the antibodies OV-1 and OV-6, which recognize antigens associated with oval cells. RESULTS: OV-1 stained scattered cells in seven of 12 tumors investigated and OV-6 in nine. Small epithelial cells (SEC) with the ultrastructural features of the oval cells were found by electron microscopy. They were characterized by small size (7-18 microm), often an oval shape, tonofilament bundles, and tight junctions or desmosome-like junctions. SEC were found in small numbers in areas of fetal differentiation and in moderate numbers in areas of embryonal differentiation. In small cell hepatoblastoma, nearly all the tumor cells exhibited SEC-like features. Immunoelectron microscopy revealed coexpression of cytokeratin 7 and albumin by SEC. CONCLUSIONS: SEC with ultrastructural and immunophenotypic features exhibited by oval cells, i.e., hepatic stem-like cells, are found in hepatoblastoma. Their numbers vary with the differing degrees of differentiation seen in the various subtypes. The findings further support the hypothesis that hepatoblastoma is derived from a pluripotent stem cell.

Paclitaxel: an effective antineoplastic agent in the treatment of xenotransplanted hepatoblastoma.

BACKGROUND: Hepatoblastoma is an uncommon liver tumor of infancy and early childhood. Though most patients with nonmetastatic hepatoblastomas can be cured by defining surgical strategies and chemotherapy regimes, new drugs are needed for children with advanced hepatoblastomas. The activity of paclitaxel as a new antineoplastic agent with limited experience in pediatric oncology was studied in a xenograft model. PROCEDURE: Hepatoblastoma cell suspensions from three children were transplanted subcutaneously into nude mice NMRI (nu/nu). One of the primary tumors was an embryonal multifocal hepatoblastoma, whereas the other tumors were embryonal/fetal hepatoblastomas localized on a liver lobe. After 4 weeks, xenografted tumor sizes reached 50-100 mm3. The xenografted tumors resembled their originals histologically and produced high levels of alpha-fetoprotein. The efficiency of paclitaxel at equitoxic doses was analyzed. RESULTS: Paclitaxel produced an effect in all three hepatoblastomas. There was a significant reduction of tumor volume (P < 0.001) and alpha-fetoprotein levels after chemotherapy (P < 0.0001). The proliferation activity of the tumor cells corresponded with these results. Histologically, after treatment with paclitaxel the tumor regression was 35%-49%. The mechanism of paclitaxel action could be demonstrated by light microscopy immunohistochemistry and electron microscopy. CONCLUSIONS: The preliminary results in phase I trials of solid tumors in children and the results of this study suggest that paclitaxel in phase II studies can now be entertained for patients with hepatoblastoma.

IP6 in treatment of liver cancer. I. IP6 inhibits growth and reverses transformed phenotype in HepG2 human liver cancer cell line.

Hepatocellular carcinoma (HCC) is a common tumor world-wide with extremely poor prognosis. Recent studies have shown that inositol hexaphosphate (IP6), a naturally occurring carbohydrate, has novel anti-cancer function in various in vitro and in vivo models. The aim of this study was to assess whether IP6 could inhibit the growth of human hepatocellular carcinoma. We treated HepG2, a human liver cancer cell line in vitro with IP6 and evaluated its effect on growth and differentiation. IP6 treatment of HepG2 cells caused a dose-dependent growth inhibition. Compared to other cancer cell lines, HepG2 cells were quite sensitive to IP6, IC50 (50% inhibition of cell growth) of IP6 being < 1.0 mM (0.338 mM). Treatment with IP6 decreased the ability of HepG2 cells to form colonies, as assessed in the plating efficiency assay. Morphological changes induced by IP6 were consistent with differentiation of HepG2 cells. Exposure of HepG2 cells to IP6 drastically decreased the rate of production of alpha-fetoprotein (AFP), a tumor marker of HCC, indicating also that IP6 treatment leads to differentiation of malignant liver cells. Further, IP6 treatment caused a decreased expression of mutant p53 protein in HepG2 cells, with no significant change in the expression of wild-type p53. The expression of p21WAF1 protein was increased by 1.5 fold, as determined by immunocytochemical staining and ELISA assay. These data demonstrate that IP6 inhibits the growth, and induces differentiation, and a less aggressive phenotype of HepG2 cells, suggesting a role of IP6 in the treatment of HCC.

Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6.

AIMS: In a recent study we described a population of small epithelial cells (SEC) in human hepatoblastoma that exhibit ultrastructural features of the oval cell of rodents. Both SEC and oval cells are immunoreactive for cytokeratin 7, a marker of biliary differentiation, and it was postulated that SEC, like oval cells, are closely related to hepatic stem cells. This study was undertaken to investigate whether SEC also exhibit immunolabelling for albumin, a marker of hepatocytic differentiation, and to determine whether other antigens typical of oval cells are detectable in hepatoblastoma. METHODS AND RESULTS: Hepatoblastomas of various subtypes were investigated by electron microscopy, and by immunohistochemistry with the monoclonal antibodies OV-1 and OV-6, which recognize antigens associated with oval cells. Double-labelling for cytokeratin 7 and albumin was carried out by immuno-electron microscopy. OV-1 stained scattered cells in seven of 12 tumours investigated and OV-6 in nine. On immunoelectron-microscopic investigation, SEC exhibited labelling for both cytokeratin 7 and albumin. CONCLUSIONS: The results demonstrate that antigens associated with oval cells are found in certain cells in hepatoblastoma. SEC, like oval cells, co-express markers for hepatocytic and biliary differentiation. The findings further support the hypothesis that SEC are closely related to the putative bipotent hepatic stem cell, which, by definition, gives rise to both hepatocytes and biliary epithelial cells.

Peroxisome proliferation and beta-oxidation in Fao and MH1C1 rat hepatoma cells, HepG2 human hepatoblastoma cells and cultured human hepatocytes: effect of ciprofibrate.

Human HepG2, rat Fao and MH1C1 hepatoma cell lines have been examined for their response to ciprofibrate, a potent peroxisome proliferator. Changes in the morphological characteristics of peroxisomes, the inductibility of their proliferation and of their beta-oxidation enzymes, palmitoyl-CoA oxidase and bifunctional enzyme, were studied in control and treated cells. In Fao cells, peroxisomes are less numerous and smaller than in rat liver, but they increase in size and number under the effect of ciprofibrate, similarly to those of treated rat liver. The high peroxisome proliferation is accompanied by a strong induction of beta-oxidation enzymes as in vivo. In MH1C1 cells, peroxisomes are seen in irregular clusters in the cytoplasm, small with rounded to tubular forms, suggesting rapid peroxisomal growth. A striking observation is the particularly elongated, worm-like form of many of the peroxisomes. Under the effect of ciprofibrate, the proliferation is low, as is the induction of beta-oxidation enzymes. HepG2 cells contain few, small peroxisomes with a heterogeneity of forms, from spherical to elongated. The only peroxisomal response to ciprofibrate in these cells seemed to be a morphological reorganization. There is little or no induction of beta-oxidation enzymes by ciprofibrate in HepG2 cells, as in cultured human hepatocytes. Therefore, on the one hand, Fao and MH1C1 cells are complementary tools in the investigation of the regulation of the hepatic response to peroxisome proliferators in the rat, on the other hand, HepG2 and Fao cells are useful in the study of the species specificity of the response.

Characterization of the continuous cell line HepT1 derived from a human hepatoblastoma.

Hepatoblastoma is the most common malignant pediatric liver tumor. The molecular mechanisms involved in the pathogenesis of hepatoblastoma are unknown. Cell lines can be valuable tools in the study of tumor biology, but only few hepatoblastoma cell lines have been established. We explanted tumor tissue from human hepatoblastomas to generate cell lines. A continuous cell line (HepT1) was established from a human hepatoblastoma with predominant embryonal differentiation. The HepT1 cell line was characterized by immunohistochemistry, electron microscopy, cytogenetics, and molecular genetic analysis. In addition, the cultured cells were xenografted into nude mice and the resulting tumors compared with the original tumor. The cells grew in epithelial clusters, and expressed cytokeratins and alpha-fetoprotein. Injection of HepT1 cells into nude mice gave rise to serially transplantable subcutaneous tumors. The cell line as well as the xenotransplants displayed the phenotypic and genotypic characteristics of the primary tumor. Ultrastructural analysis demonstrated desmosomal junctions and the formation of bile canaliculi. Cytogenetic analysis showed a near tetraploid karyotype with structural and numerical aberrations of chromosomes 1p, 6, 9, 11q, 13q, 15p, and 20 and both single and double minute chromosomes. In PCR-based microsatellite analysis of chromosome arm 11p, a loss of heterozygosity at all informative loci including the WT-1 and IGF2 genes was detected. Intratumoral erythropoiesis, a characteristic feature of hepatoblastomas, was present in the primary tumor as well as in HepT1 xenotransplants. We therefore studied the expression of erythropoietic cytokines in these cells and found both erythropoietin and stem cell factor. The HepT1 cell line displays characteristic cellular and molecular features of hepatoblastoma and we believe it will be a valuable tool for studies on the biology and pathogenesis of hepatoblastoma as well as on the differentiation of hepatocyte progenitor cells.

Small epithelial cells and the histogenesis of hepatoblastoma. Electron microscopic, immunoelectron microscopic, and immunohistochemical findings.

The wide range of epithelial and mesenchymal lines of differentiation seen in hepatoblastoma suggests that this tumor derives from a pluripotent stem cell. To test this hypothesis, seven hepatoblastomas of various subtypes were investigated for the presence of cells with the features of the oval cells found during hepatocarcinogenesis in rodents that are thought to be closely related to hepatic stem cells. Because similar cells, referred to as "small cells," have been described in human liver disease with chronic ductular reaction, five liver biopsies from infants with biliary atresia were also investigated. The specimens were investigated by electron microscopy, immunoelectron microscopy, and immunostaining for cytokeratins 7, 8, 18, and 19. Small epithelial cells (SEC) corresponding to the oval cells of the rat and the "small cells" in humans were found in both biliary atresia and hepatoblastoma. These cells were oval and exhibited intercellular junctions, tonofilament bundles, and a biliary epithelium-type cytokeratin profile. SEC were found in small numbers in fetal hepatoblastoma and in moderate numbers in embryonal hepatoblastoma. In small cell hepatoblastoma, nearly all the tumor cells exhibited SEC-like ultrastructural features and a corresponding cytokeratin profile. Thus, cells exhibiting morphological and immunophenotypic features of hepatic stem cells are detectable in hepatoblastoma. Their numbers vary according to the subtype, reflecting the differing degrees of differentiation of the various subtypes, consistent with the theory propounded in the literature that embryonal and, with further differentiation, fetal tumor cells derive from precursor small cells. The findings support the hypothesis that hepatoblastoma derives from a pluripotent, probably entodermal or even less committed, stem cell.

[Histogenesis of hepatoblastoma. Morphological, immunoelectron microscopic and immunohistochemical findings]. / Zur Histogenese des Hepatoblastoms. Morphologische, immunelektronenmikroskopische und immunhistochemische Befunde.

The wide range of epithelial and mesenchymal lines of differentiation seen in hepatoblastoma suggests that this tumor derives from a pluripotent stem cell. In order to test this hypothesis, seven hepatoblastomas of various subtypes were investigated for the presence of cells with the features of the oval cells found during hepatocarcinogenesis in rodents that are thought to be closely related to hepatic stem cells. The specimens were investigated by electron microscopy, electron microscopic immunocytochemistry, and immunohistochemical staining for cytokeratins nos. 7, 8, 18, and 19. Small epithelial cells (SEC) corresponding to the oval cells of the rat and the "small cells" found in human liver disease with chronic ductular reaction, both of which are thought to be related to hepatic stem cells, were found. The SEC were oval and exhibited intercellular junctions, tonofilament bundles, and a biliary epithelium-type cytokeratin profile. They were found in small numbers in fetal hepatoblastoma and in moderate numbers in embryonal hepatoblastoma. In small cell hepatoblastoma, nearly all the tumor cells exhibited SEC-like ultrastructural features and a corresponding cytokeratin profile. Thus, cells with the features of hepatic stem cells are detectable in hepatoblastoma. Their numbers vary according to the subtype, reflecting the differing degrees of differentiation of the various subtypes, consistent with the theory propounded in the literature that embryonal and, with further differentiation, fetal tumor cells derive from precursor small cells. The findings support the hypothesis that hepatoblastoma derives from a pluripotent, probably entodermal or even less committed, stem cell.

Heterogeneity of peroxisomes in human hepatoblastoma cell line HepG2. Evidence of distinct subpopulations.

Peroxisomes in human hepatoblastoma cell line HepG2 have been studied using immunocytochemical, ultrastructural and biochemical techniques. By immunofluorescence, small spherical peroxisomes were seen next to rod-shaped and elongated forms. By electron microscopy and catalase cytochemistry, small particles with a diameter of 90 to 100 nm were found next to larger ones measuring up to 300 nm and some exhibiting tail-like extensions. Both the intensity of DAB-staining and the immunolabeling density for catalase were heterogenous in different peroxisomes. Contrary to a recent biochemical study, the enzyme alanine-glyoxylate-aminotransferase was found by double immunofluorescence and immunoelectron microscopy to be localized exclusively in peroxisomes of HepG2 cells. By Metrizamide density gradient centrifugation two populations of peroxisomes were isolated: a regular fraction banding at 1.20 g/cm3 with a mean diameter of 222 nm and a lighter peroxisome fraction banding at 1.14 g/cm3. The ratio of lipid beta-oxidation enzymes to catalase was significantly higher in peroxisomes with lower density than in the regular ones. These observations show clearly the existence of heterogenous populations of peroxisomes in HepG2 cells which may provide a useful model system for the investigation of biogenesis and metabolism of peroxisomes of human origin.

Hepatoblastoma. Report of a case with cytologic, histologic and ultrastructural findings.

Hepatoblastoma, although rare, is the most common primary malignant neoplasm of the liver in children. In this paper we describe a case of hepatoblastoma with unusual cytologic features and present the histologic, immunocytochemical and ultrastructural features of this neoplasm. A 7-month-old girl presented with a large hepatic mass and metastatic nodules in both lungs. Intraoperative biopsy revealed a hepatoblastoma. Aspiration biopsy yielded a highly cellular aspirate with cords of pleomorphic cells embedded in a mucoid matrix. Histologic sections showed a diffusely infiltrative neoplasm composed of sheets and cords of highly pleomorphic cells. The neoplastic cells stained strongly positive for cytokeratin CAM 5.2 and AE1 and focally positive for alpha-fetoprotein, ferritin, carcinoembryonic antigen and vimentin. Ultrastructurally, the neoplastic cells had abundant intercellular junctions and intracytoplasmic aggregates of intermediate filaments. A mucoid matrix, to our knowledge, has not been reported as a finding on aspiration biopsy. This patient presented with pulmonary metastases, and thus we think the mucoid matrix may be a marker of a more aggressive variant of hepatoblastoma. This case illustrates additional cytologic features of hepatoblastoma and the usefulness of aspiration biopsy in the rapid diagnosis of this rare tumor.