Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms.

The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host- or biopharmaceutical-specific product quality obstacles. In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.

Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.

Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells.

MicroRNAs with their unique ability to target hundreds of genes have been highlighted as powerful tools to improve bioprocess behavior of cells. The common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, which requires the introduction and expression of extra genetic material. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate Chinese hamster ovary (CHO) cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in controlling CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We showed that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures making it a potentially interesting target to improve bioprocess performance of CHO cells.

CRISPR/Cas9-Multiplexed Editing of Chinese Hamster Ovary B4Gal-T1, 2, 3, and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins.

In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, and -T3 with and without a disrupted B4Gal-T4 sequence resulted in only ≈1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. The authors revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ≈6% and ≈3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, the authors present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, the authors provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins.

Exploring the capabilities of fluorometric online monitoring on chinese hamster ovary cell cultivations producing a monoclonal antibody.

Online monitoring of Chinese hamster ovary fed-batch cell cultures via two-dimensional fluorescence spectroscopy (2DFS) was evaluated in this work. Particular attention was directed toward different process strategies regarding the use of nutrient-rich feed media and temperature shifts. These intentionally performed process manipulations broadened the variances in the obtained fluorescence spectra and this was suspected to hamper the generation of reliable soft sensors. Principal component analysis of the obtained fluorescence data showed that temperature shift and feeding strategy had a considerable impact on the fluorescence signals. Partial least square regression models were calculated for the prediction of glucose, lactate, monoclonal antibody (mAb), and viable cell concentrations (VCC). It was aimed to integrate all 2DFS datasets in the respective calibration models regardless of the process-strategy-dependent diversity. Contrary to the expectations, it was feasible to calibrate soft sensors for the online prediction of glucose (7 latent variables (LVs), Rcal2 = 0.97, rout mean squared error of prediction (RMSEP) = 1.1 g L-1 ), lactate (5 LV; Rcal2 = 0.96; RMSEP = 0.5 g L-1 ) and mAb concentrations (4 LV; Rcal2 = 0.99; RMSEP = 11.4 mg L-1 ). Feeding and temperature shifts had the highest impact on the VCC model (3 LV; Rcal2 = 0.94; RMSEP 3.8 × 105 mL-1 ), nevertheless the prediction of VCC from the fed-batch 2DFS data was feasible. The results strongly indicate that variances in the datasets due to the process strategy can be tolerated to some extent by the respective soft sensors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1592-1600, 2016.

Causal Modeling of Cancer-Stromal Communication Identifies PAPPA as a Novel Stroma-Secreted Factor Activating NFκB Signaling in Hepatocellular Carcinoma.

Inter-cellular communication with stromal cells is vital for cancer cells. Molecules involved in the communication are potential drug targets. To identify them systematically, we applied a systems level analysis that combined reverse network engineering with causal effect estimation. Using only observational transcriptome profiles we searched for paracrine factors sending messages from activated hepatic stellate cells (HSC) to hepatocellular carcinoma (HCC) cells. We condensed these messages to predict ten proteins that, acting in concert, cause the majority of the gene expression changes observed in HCC cells. Among the 10 paracrine factors were both known and unknown cancer promoting stromal factors, the former including Placental Growth Factor (PGF) and Periostin (POSTN), while Pregnancy-Associated Plasma Protein A (PAPPA) was among the latter. Further support for the predicted effect of PAPPA on HCC cells came from both in vitro studies that showed PAPPA to contribute to the activation of NFκB signaling, and clinical data, which linked higher expression levels of PAPPA to advanced stage HCC. In summary, this study demonstrates the potential of causal modeling in combination with a condensation step borrowed from gene set analysis [Model-based Gene Set Analysis (MGSA)] in the identification of stromal signaling molecules influencing the cancer phenotype.

In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice.

Oxidative stress contributes to the progression of acute liver failure (ALF). Transcription factor nuclear factor-erythroid 2-related factor (Nrf2) serves as an endogenous regulator by which cells combat oxidative stress. We have investigated liver damage and the balance between death and survival signaling pathways in concanavalin A (ConA)-mediated ALF using in vivo siRNA delivery targeting Keap1 in hepatocytes. For that goal, mice were injected with Keap1- or luciferase-siRNA-containing liposomes via the tail vein. After 48 hours, ALF was induced by ConA. Liver histology, pro-inflammatory mediators, antioxidant responses, cellular death, and stress and survival signaling were assessed. Keap1 mRNA and protein levels significantly decreased in livers of Keap1-siRNA-injected mice. In these animals, histological liver damage was less evident than in control mice when challenged with ConA. Likewise, markers of cellular death (FasL and caspases 8, 3 and 1) decreased at 4 and 8 hours post-injection. Nuclear Nrf2 and its target, hemoxygenase 1 (HO1), were elevated in Keap1-siRNA-injected mice compared with control animals, resulting in reduced oxidative stress in the liver. Similarly, mRNA levels of pro-inflammatory cytokines were reduced in livers from Keap1-siRNA-injected mice. At the molecular level, activation of c-jun (NH2) terminal kinase (JNK) was ameliorated, whereas the insulin-like growth factor I receptor (IGFIR) survival pathway was maintained upon ConA injection in Keap1-siRNA-treated mice. In conclusion, our results have revealed a potential therapeutic use of in vivo siRNA technology targeted to Keap1 to combat oxidative stress by modulating Nrf2-mediated antioxidant responses and IGFIR survival signaling during the progression of ALF.

Loss of anterior gradient-2 expression is an independent prognostic factor in colorectal carcinomas.

AIMS: The human Anterior Gradient-2 (AGR2) protein is strongly expressed in various human cancers, and it has been described to promote aggressive tumour features in some entities. So far, a comprehensive analysis of AGR2 expression in colorectal carcinomas has not been described. METHODS: Normal intestinal cells and colorectal carcinoma cell lines were analysed for AGR2 expression. AGR2 protein expression was immunohistochemically analysed in 28 normal tissue samples and 1068 tissue samples of clinically well characterised colorectal carcinomas. For statistical analysis, chi square test, spearman rank correlations, Kaplan-Meier estimates (Log rank test) and Cox regression were applied to test for diagnostic or prognostic associations. RESULTS: In the normal intestinal cell line and in normal colon mucosa AGR2 was found in all cases (n=28). In contrast, loss of AGR2 was found in all six analysed colorectal cancer cell lines and in 833/1068 (78%) of the colorectal carcinoma tissue samples analysed, and it was significantly associated with a higher tumour grade and tumour localisation in the left-sided colon. In addition to the conventional prognostic tumour parameters pT category, nodal status, metastasis and histological tumour grade the loss of AGR2 expression was significantly associated with reduced overall survival times in univariate and multivariate analyses, thus suggesting AGR2 as an independent prognostic factor in primary colorectal carcinoma. CONCLUSIONS: AGR2 is frequently lost in colorectal carcinomas and might be a novel independent prognostic factor for overall patient survival.

Targeting melanoma metastasis and immunosuppression with a new mode of melanoma inhibitory activity (MIA) protein inhibition.

Melanoma is the most aggressive form of skin cancer, with fast progression and early dissemination mediated by the melanoma inhibitory activity (MIA) protein. Here, we discovered that dimerization of MIA is required for functional activity through mutagenesis of MIA which showed the correlation between dimerization and functional activity. We subsequently identified the dodecapeptide AR71, which prevents MIA dimerization and thereby acts as a MIA inhibitor. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy demonstrated the binding of AR71 to the MIA dimerization domain, in agreement with in vitro and in vivo data revealing reduced cell migration, reduced formation of metastases and increased immune response after AR71 treatment. We believe AR71 is a lead structure for MIA inhibitors. More generally, inhibiting MIA dimerization is a novel therapeutic concept in melanoma therapy.

Adiponectin reduces connective tissue growth factor in human hepatocytes which is already induced in non-fibrotic non-alcoholic steatohepatitis.

Connective tissue growth factor (CTGF) is induced in liver fibrosis and enhances the activity of transforming growth factor ß (TGFß). Recently we have shown that the hepatoprotective adipokine adiponectin downregulates CTGF in primary human hepatocytes (PHH). In the current study, the mechanisms mediating suppression of CTGF by adiponectin and the well described downstream effector of adiponectin receptor 2 (AdipoR2), peroxisome proliferator activated receptor α (PPARα), were analyzed in more detail. Adiponectin downregulated CTGF mRNA and protein in primary human hepatocytes (PHH) and suppression was blocked by a PPARα antagonist indicating that AdipoR2 is involved. The PPARα agonists fenofibrate and WY14643 also reduced CTGF protein in these cells. Adiponectin further impaired TGFß-mediated upregulation of CTGF. Phosphorylation of the TGFß downstream effectors SMAD2 and -3 was reduced in PHH incubated with adiponectin or PPARα agonists suggesting that early steps in TGFß signal transduction are impaired. CTGF and TGFß mRNA levels were increased in human non-fibrotic non-alcoholic steatohepatitis (NASH), and here AdipoR2 expression was significantly reduced. Current data show that CTGF and TGFß are already induced in non-fibrotic NASH and this may be partly explained by low adiponectin bioactivity which interferes with TGFß signaling by reducing phosphorylation of SMAD2/3 and by downregulating CTGF.

Analysis of a promoter polymorphism of the GLUT1 gene in patients with hepatocellular carcinoma.

The glucose transporter isoform 1 (GLUT1) is a key rate-limiting factor in the transport and metabolism of glucose in cancer cells. Recently, we found that GLUT1 expression is increased in hepatocellular carcinoma (HCC) and promotes tumorigenicity of HCC cells. Hypoxia further increased GLUT1 expression in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor (HIF)-1alpha. The promoter region of the GLUT1 gene harbors a single nucleotide polymorphism (SNP; Rs710218; A to T at -2841) closely positioned to a putative HIF-1alpha binding site, and recently, this SNP was found to be more frequent in patients with renal cell carcinoma. In the present study, the A-2841T genotype distribution did not differ significantly between HCC patients (n = 95; AA: 60%; AT 36% and TT: 4%) and healthy controls (n = 127; AA: 50%; AT 41% and TT: 9%). However and noteworthy, non-carriers of the T allele had higher GLUT1 expression levels in cancerous hepatic tissue, and tended to reveal a more aggressive tumour growth. These data indicate that the SNP Rs710218 is not associated with a higher risk for HCC but rather for HCC progression, potentially via HIF-1alpha mediated increased GLUT1 expression.

FHL2 suppresses growth and differentiation of the colon cancer cell line HT-29.

Four and a half LIM domain protein 2 (FHL2) can interact with many proteins and regulates different cellular processes, including proliferation and differentiation. FHL2 expression is often deregulated in cancer and may act as both tumor-promoter or tumor-suppressor depending on the type of cancer. Thus, a previous study found that increased FHL2 expression in colon cancer and suppression of FHL2 in a colon cancer cell line with endogenously high FHL2 expression inhibited tumor growth. We applied the opposite strategy, an FHL2 expression plasmid was stably transfected into HT-29 cells, a colon carcinoma cell line which exhibits very low basal levels of FHL2. Stable expression of FHL2 in HT-29 cells induced a G2/M arrest and inhibited anchorage-dependent and -independent growth in vitro. Further, FHL2 expressing HT-29 cell clones revealed significantly higher expression of the differentiation marker E-cadherin but reduced activity of the transcription factor NF-kappaB, which is known to promote colon cancer progression. These findings further underscore the complex role of FHL2 in tumorigenicity, with even different effects on cellular functions of cancer cell lines derived from the same type of tumor and distinctly suggest caution regarding therapeutic strategies targeting FHL2 to treat (colon) cancer.

Reduced expression of fibroblast growth factor receptor 2IIIb in hepatocellular carcinoma induces a more aggressive growth.

Fibroblast growth factor receptor 2 isoform b (FGFR2-IIIb) is highly expressed in hepatocytes and plays an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR2-IIIb in hepatocellular carcinoma (HCC). FGFR2-IIIb expression in HCC tissues and cell lines was lower than in primary human hepatocytes and nontumorous tissue. FGFR2-IIIb-negative HCCs showed a significantly higher Ki-67 labeling index, and loss of FGFR2-IIIb expression correlated significantly with vascular invasion and more advanced tumor stages. A decrease in FGFR-2IIIb expression in HCC cell lines was not related to promoter hypermethylation. However, PCR analysis indicated that chromosomal deletion at 10q accounted for the loss of FGFR2 expression in a subset of HCC cells. FGFR2-IIIb re-expression in stable transfected HCC cell lines induced a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential in vitro. In nude mice, FGFR2-IIIb re-expressing HCC cells grew significantly slower, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed higher apoptosis rates. The antitumorigenic effects of FGFR2-IIIb expression in HCC cells were not affected by keratinocyte growth factor or an inhibitor of FGFR-phosphorylation, indicating that they are independent of tyrosine kinase activation. In conclusion, our data indicate that FGFR2-IIIb inhibits tumorigenicity of HCC cells. Identification of the molecular mechanisms promoting regeneration in normal tissue while suppressing malignancy may lead to novel therapeutic targets of this highly aggressive tumor.

GLUT1 as a therapeutic target in hepatocellular carcinoma.

Primary hepatocellular carcinoma (HCC) is one of the most fatal cancers in humans with rising incidence in many regions around the world. Currently, no satisfactory curative pharmacological treatment is available, and the outcome is mostly poor. Recently, we have shown that the glucose transporter GLUT1 is increased in a subset of patients with HCC and functionally affects tumorigenicity. GLUT1 is a rate-limiting transporter for glucose uptake, and its expression correlates with anaerobic glycolysis. This phenomenon is also known as the Warburg effect and recently became of great interest, since it affects not only glucose uptake and utilization but also has an influence on tumorigenic features like metastasis, chemoresistance and escape from immune surveillance. Consistent with this, RNA-interference-mediated inhibition of GLUT1 expression in HCC cells resulted in reduced tumorigenicity. Together, these findings indicate that GLUT1 is a novel and attractive therapeutic target for HCC. This review summarizes our current knowledge on the expression and function of GLUT1 in HCC, available drugs/strategies to inhibit GLUT1 expression or function, and potential side effects of such therapeutic strategies.

Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells.

Despite the initial belief that non-alcoholic fatty liver disease is a benign disorder, it is now recognized that fibrosis progression occurs in a significant number of patients. Furthermore, hepatic steatosis has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of other liver diseases. Here, we established an in vitro model to study the effect of hepatic lipid accumulation on hepatic stellate cells (HSCs), the central mediators of liver fibrogenesis. Primary human hepatocytes were incubated with the saturated fatty acid palmitate to induce intracellular lipid accumulation. Subsequently, human HSCs were incubated with conditioned media (CM) from steatotic or control hepatocytes. Lipid accumulation in hepatocytes induced the release of factors that accelerated the activation and proliferation of HSC, and enhanced their resistance to apoptosis, largely mediated via activation of the PI-3-kinase pathway. Furthermore, CM from steatotic hepatocytes induced the expression of the profibrogenic genes TGF-beta, tissue inhibitor of metallo-proteinase-1 (TIMP-1), TIMP-2 and matrix-metallo-proteinase-2, as well as nuclear-factor kappaB-dependent MCP-1 expression in HSC. In summary, our in vitro data indicate a potential mechanism for the pathophysiological link between hepatic steatosis and fibrogenesis in vivo. Herewith, this study provides an attractive in vitro model to study the molecular mechanisms of steatosis-induced fibrogenesis, and to identify and test novel targets for antifibrotic therapies in fatty liver disease.

Bone morphogenetic protein 4 is induced in hepatocellular carcinoma by hypoxia and promotes tumour progression.

Striking similarities exist between molecular mechanisms driving embryonic liver development and progression of hepatocellular carcinoma (HCC). Bone morphogenetic proteins (BMPs), particularly BMP4, have been proposed to regulate embryonic hepatic development. BMP expression has been observed in neoplasia but the expression and biological role of BMP4 in human HCC are unknown. We found increased BMP4 mRNA and protein in HCC cell lines and tissue samples compared to primary human hepatocytes and corresponding non-tumourous tissue. Hypoxia further induced BMP4 expression in HCC cells, which was abolished by transfection of a dominant negative form of HIF-1 alpha (dnHIF-1 alpha). However, gel shift assays revealed only minor binding activity in nuclear extracts from (hypoxic) HCC cells to a putative hypoxia-response element in the BMP4 promoter. Sequence analysis of the BMP4 promoter revealed two Ets-1 binding sites, and Ets-1 activity was increased in HCC cells under hypoxic conditions. Transfection of dnHIF-1 alpha completely abrogated hypoxia-induced Ets-1 activity as well as BMP4 expression. Overexpression of Ets-1 markedly enhanced BMP4 promoter activity, while antisense Ets-1 almost completely abolished basal as well as hypoxia-induced BMP4 expression. These data demonstrate that Ets-1 activity contributes to baseline expression of the BMP4 gene and is the predominant mediator of the HIF-dependent BMP4 induction under hypoxic conditions. To determine the functional relevance of BMP4 expression, HCC cell lines were treated with antisense BMP4 constructs or siRNA against BMP4. BMP4 suppression resulted in a strong reduction of the migratory and invasive potential and anchorage-independent growth. Furthermore, tube formation assays indicated that BMP4 expressed by HCC cells promotes vasculogenesis. Our findings demonstrate that BMP4 is increased in HCC and promotes HCC progression. Therefore, BMP4 expression may have clinical relevance, and interfering with BMP4 signalling appears as an attractive therapeutic target for this highly aggressive tumour.

GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis.

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma.

Liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells (HSC) are the effector cells of hepatic fibrosis and also infiltrate the HCC stroma where they might play a critical role in HCC progression. Here we aimed to analyze the effects of activated HSC on the proliferation and growth of HCC cell lines in vitro and in vivo. Conditioned media (CM) collected from HSC significantly induced proliferation and migration of HCC cells cultured in monolayers. In a 3-dimensional spheroid coculture system, HSC promoted HCC growth and diminished the extent of central necrosis. In accordance, in vivo simultaneous implantation of HSC and HCC cells into nude mice promoted tumor growth and invasiveness, and inhibited necrosis formation. As potential mechanism of the tumorigenic effects of HSC we identified activation of NFkappaB and extracellular-regulated kinase (ERK) in HCC cells, two signaling cascades that play a crucial role in HCC progression. In summary, our data indicate that stromal HSC promotes HCC progression and suggest the HSC-HCC interaction as an interesting tumor differentiation-independent target for therapy of this highly aggressive cancer.

New insights into the lifestyle of the cold-loving SM1 euryarchaeon: natural growth as a monospecies biofilm in the subsurface.

In the surface waters of sulfidic springs near Regensburg, Bavaria, Germany, the SM1 euryarchaeon, together with filamentous bacteria, forms the recently described unique string-of-pearls community. In addition to naturally occurring string-of-pearls communities, the growth of these communities was also observed on polyethylene nets provided as an artificial attachment material in the streamlets of springs. In order to learn more about the distribution and origin of the SM1 euryarchaeon and its possible occurrence in the subsurface, polyethylene nets were incubated as deeply as possible in different spring holes. After a short residence time, slime-like, milky drops, almost completely composed of SM1 euryarchaeon, were attached to the nets, indicating that this organism grows independent of a partner in deeper earth layers. A newly designed in situ biofilm trapping system allowed the quantitative harvesting of organisms exhibiting this newly discovered lifestyle of the SM1 euryarchaeon for detailed biological studies. The discovery of naturally occurring archaeal biofilms extends our knowledge of the biology and ecological significance of archaea in their environments.