Biliary tract cancers (BTCs) was heterogeneous and characterized by late diagnosis and fatal outcome. To identify new biomarkers for BTCs, we performed Robust Rank Aggreg (RRA) analysis and identified that IDH1 mutation was common in ICC, while IDH1

Ovarian cancer remains the most lethal type of cancer among all gynecological malignancies. The majority of patients are diagnosed with ovarian cancer at the late stages of the disease. Therefore, there exists an imperative need for the development of early ovarian cancer diagnostic techniques. Exosomes, secreted by various cell types, play pivotal roles in intercellular communication, which emerge as promising diagnostic and prognostic biomarkers for ovarian cancer. In this study, we present for the first time, at least to the best of our knowledge, the proteomics profiling of exosomes derived from the plasma of patients with ovarian cancer via liquid chromatography tandem mass spectrometry (LC‑MS/MS) with tandem mass tagging (TMT). The exosomes enriched from patient plasma samples were characterized by nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), transmission electron microscopy (TEM) and western blot analysis. The size of the plasma exosomes fell into the range of 30 to 100 nm in diameter. The exosomal marker proteins, CD81 and TSG101, were clearly stained in the exosome samples; however, there was no staining for the endoplasmic reticulum protein, calnexin. A total of 294 proteins were identified with all exosome samples. Among these, 225 proteins were detected in both the cancerous and non‑cancerous samples. Apart from universal exosomal proteins, exosomes derived from ovarian cancer patient plasma also contained tumor‑specific proteins relevant to tumorigenesis and metastasis, particularly in epithelial ovarian carcinoma (EOC). Patients with EOC often suffer from coagulation dysfunction. The function of exosomes in coagulation was also examined. Several genes relevant to the coagulation cascade were screened out as promising diagnostic and prognostic factors that may play important roles in ovarian cancer progression and metastasis. On the whole, in this study, we successfully isolated and purified exosomes from plasma of patients with EOC, and identified a potential role of these exosomes in the coagulation cascade, as well as in the diagnosis and prognosis of patients.

PURPOSE: Multicolor flow cytometry (MFC) is widely available, fast and has an easy-to perform approach for finding neuroblastoma (NB) cells among normal bone marrow (BM) hematopoietic cells. Aim of the study was to investigate prognostic significance of initial MFC tumor cells' detection in BM of children with NB.
METHODS: 51 patients (24 boys and 27 girls) aged from 6 days to 15 years (median age 1 year 3 months) with NB were included in the study. BM samples at the time of diagnosis were obtained from 2 to 5 aspiration sites per patient. CD45(-)CD56(+)CD81(+)GD2(+)-cells were evaluated by MFC.
RESULTS: NB cells were detected in BM by FC more frequently compared to conventional cytomorphology (49.0% and 29.4% patients, respectively, р = 0.043). Patients with NB cells detected in BM by MFC had significantly worse event-free survival and cumulative incidence of relapse/progression [0.24(0.08) and 0.60(0.10), respectively] compared to children with negative result of immunophenotyping [0.85(0.07) and 0.12(0.06), respectively, p < 0.001 in both cases]. BM involvement detection by MFC maintained its prognostic significance in various patients groups. In multivariate analysis, immunophenotyping proved to be an independent prognostic factor when analyzed jointly with other NB risk factors. In 42 patients BM involvement was also studied by RQ-PCR for PHOX2B and TH genes expression. Within groups of patients divided by RQ-PCR positivity, MFC-positivity retained prognostic significance.
CONCLUSIONS: Thus flow cytometric BM involvement detection has very strong prognostic impact even stronger than RQ-PCR. It could be used in combination with other parameters for the treatment strategy choice in patients with NB.

Antimicrobial peptides (AMPs) are multifunctional factors with an important role in the innate immune system. Our previous studies revealed that the human cathelicidin LL‑37 and its analog, FF/CAP18, limit the proliferation of colon cancer cell lines. In the present study, the exosomes released by HCT116 cells treated with FF/CAP18 were analyzed. After the treatment, exosomes were isolated from the culture supernatant by ultrafiltration and using the miRCURY™ Exosome Isolation Kit. Membrane vesicles 40‑100‑nm expressing CD63 and CD81 were identified before and after FF/CAP18 treatment. Exosome concentration in the culture supernatant was increased after treatment with FF/CAP18. Exosomes formed in HCT116 cells treated with FF/CAP18 induced growth suppression of the cells in a dose‑dependent manner. By contrast, the exosomes formed in non‑treated HCT116 cells did not affect cell viability. Microarray analysis of miRNA expression indicated that FF/CAP18 treatment induced increases in the expression of three miRNAs (miR‑584‑5p, miR‑1202 and miR‑3162‑5p) in both HCT116 cells and exosomes. These results suggest that FF/CAP18 treatment increases exosome formation, and that exosome‑encapsulated miRNAs suppress HCT116 cell proliferation. Exosomal miRNAs are considered to be involved in the dissemination of cell signals to control local cellular microenvironments. The present findings suggest that FF/CAP18 regulates cancer growth by modulating cell‑to‑cell communication. AMPs localize in the cytoplasm of cancer cells and enhance the expression of growth‑suppressing miRNAs. These miRNAs are also transported to other cancer cells via exosomes. Therefore, transportation of these miRNAs has the potential to suppress cancer growth. AMPs exert their effects directly by targeting cancer cells and indirectly via exosomes.

BACKGROUND CD81, a member of the tetraspanin family, is overexpressed in several tumor types, but its role in breast cancer remains unknown. The present study aimed to investigate the effects of increased CD81 expression on cell migration and proliferation in breast cancer cell lines, MDA-MB-231 and MDA-MB-435S in vitro, and the effects of increased CD81 expression in breast cancer tissue microarrays on patient prognosis. MATERIAL AND METHODS The expression of CD81 was evaluated using immunohistochemistry in a human breast cancer tissue microarray containing 140 tumor tissues and a microarray containing 77 normal breast tissues. The effects of increased CD81 expression on cell proliferation and migration in breast cancer cell lines, MDA-MB-231 and MDA-MB-435S, were evaluated by proliferation, transwell migration, and cell counting kit-8 (CCK-8) assays. CD81-expressing plasmid transfection upregulated CD81 expression, and short hairpin RNA (shRNA) lentivirus silenced CD81 expression in vitro. RESULTS CD81 expression was significantly increased in breast cancer tissues compared with normal breast tissues (P<0.05). Increased expression of CD81 was significantly associated with lymph node metastasis (P<0.05), clinical stage (P<0.05) and with reduced overall survival (OS) in patients with breast cancer (P<0.05). Increased CD81 expression in MDA-MB-231 and MDA-MB-435S cells promoted cell proliferation and migration, which were inhibited by CD81 silencing. CONCLUSIONS The findings of the study showed that CD81 might be a potential prognostic biomarker associated with poor patient prognosis in breast cancer. These findings should be investigated further with large-scale controlled prospective studies in patients with breast cancer.

INTRODUCTION: The discrimination of leukemia lymphoblasts (LB) in diagnosis and follow-up of B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) by multiparameter flow cytometry (MFC) may be difficult due to the presence of hematogones (HG). The aim of this study was to compare lymphoblasts of BCP-ALL and HG for the expression of the most discriminating antigens.
METHODS: A total of 82 bone marrow samples (39 BCP-ALL and 43 patients with HG) were analyzed using MFC. Mean fluorescence intensity (MFI) was measured for ten markers commonly used in hematology laboratories: CD45, CD19, CD10, CD34, CD38, CD20, CD22, CD58, CD81, and CD123. Statistical comparison of the MFI between LB and HG was performed. The presence on LB of aberrant expression of myeloid and/or T-cell markers was also investigated.
RESULTS: Qualitative pattern expression of antigens showed overexpression on LB of CD58, CD22, CD34, CD10 and underexpression of CD81, CD45, CD38 when compared to HG. Expression of CD123 was positive in 34% of BCP-ALL LB and always absent on HG. Aberrant antigen expression (myeloid and/or T-cell marker) including CD123 was observed in 58% of BCP-ALL patients. The use of a MFI antigen ratio of the most discriminating markers (CD81/CD58) (analysis of variance, P < 0.005) increased the distinction of LB versus HG with a high specificity and sensitivity as demonstrated by the use of ROC curve analysis (AUC of CD81/CD58: 0.995).
CONCLUSION: We demonstrate in this study that routine use of the MFI antigen ratio (CD81/CD58) in addition to the MFC evaluation using WHO classical criteria appears to be an efficient approach to discriminate LB from HG.

Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.

Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.

The notion that plasma cells (PCs) are terminally differentiated has prevented intensive research in multiple myeloma (MM) about their phenotypic plasticity and differentiation. Here, we demonstrated in healthy individuals (n=20) that the CD19-CD81 expression axis identifies three bone marrow (BM)PC subsets with distinct age-prevalence, proliferation, replication-history, immunoglobulin-production, and phenotype, consistent with progressively increased differentiation from CD19+CD81+ into CD19-CD81+ and CD19-CD81- BMPCs. Afterwards, we demonstrated in 225 newly diagnosed MM patients that, comparing to normal BMPC counterparts, 59% had fully differentiated (CD19-CD81-) clones, 38% intermediate-differentiated (CD19-CD81+) and 3% less-differentiated (CD19+CD81+) clones. The latter patients had dismal outcome, and PC differentiation emerged as an independent prognostic marker for progression-free (HR: 1.7; P=0.005) and overall survival (HR: 2.1; P=0.006). Longitudinal comparison of diagnostic vs minimal-residual-disease samples (n=40) unraveled that in 20% of patients, less-differentiated PCs subclones become enriched after therapy-induced pressure. We also revealed that CD81 expression is epigenetically regulated, that less-differentiated clonal PCs retain high expression of genes related to preceding B-cell stages (for example: PAX5), and show distinct mutation profile vs fully differentiated PC clones within individual patients. Together, we shed new light into PC plasticity and demonstrated that MM patients harbouring less-differentiated PCs have dismal survival, which might be related to higher chemoresistant potential plus different molecular and genomic profiles.

Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc.

BACKGROUND: The aim of this study was to evaluate the gene expression profiles of a set of prostate cancer-associated genes in prostate cancer cell lines, to determine their association with different cancer phenotypes and identify potential novel biomarkers for this disease.
METHODS: Quantitative real-time PCR was used to determine the expression profiles of 21 prostate cancer-associated genes in the human prostate cancer cell lines PC-3 and LNCaP, using the nontumorigenic cell line PWR-1E as control cell line. Genes evaluated were ESM-1, SERPINE2, CLU, BGN, A2M, PENK, FMOD, CD81, DCN, TSPAN8, KBTBD10, F2RL1, TMSB4X, SNCG, CXXC5, FOXQ1, PDPN, SPN, CAV1, CD24 and KLK3. A potential biomarker from this set of genes, the FMOD gene, encoding the small leucine-rich proteoglycan fibromodulin, was selected for further evaluation in clinical samples from patients diagnosed with benign or malignant prostatic disease.
RESULTS: Several of the evaluated genes showed significantly altered expression in the prostate cancer cell lines, compared with nontumorigenic PWR-1E cells. Further evaluation of FMOD transcript in prostate clinical samples from patients diagnosed with benign or malignant prostatic disease identified a significant difference in the expression levels of this proteoglycan between benign and malignant tissue (p<0.05).
CONCLUSIONS: A number of gene transcripts were differentially expressed by the cell lines assayed. Among them, FMOD was further evaluated in clinical samples and was found to be differentially expressed between benign and prostate cancer tissue. Further validation of FMOD transcript in a larger population is required to ascertain its usefulness as biomarker for prostate cancer.

Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.

In recent years, several studies on large series of multiple myeloma (MM) patients have demonstrated the clinical utility of flow cytometry monitoring of minimal residual disease (flow-MRD) in bone marrow (BM), for improved assessment of response to therapy and prognostication. However, disturbing levels of variability exist regarding the specific protocols and antibody panels used in individual laboratories. Overall, consensus exists about the utility of combined assessment of CD38 and CD138 for the identification of BM plasma cells (PC); in contrast, more heterogeneous lists of markers are used to further distinguish between normal/reactive PCs and myeloma PCs in the MRD settings. Among the later markers, CD19, CD45, CD27, and CD81, together with CD56, CD117, CD200, and CD307, have emerged as particularly informative; however, no single marker provides enough specificity for clear discrimination between clonal PCs and normal PCs. Accordingly, multivariate analyses of single PCs from large series of normal/reactive vs. myeloma BM samples have shown that combined assessment of CD138 and CD38, together with CD45, CD19, CD56, CD27, CD81, and CD117 would be ideally suited for MRD monitoring in virtually every MM patient. However, the specific antibody clones, fluorochrome conjugates and sources of the individual markers determines its optimal (vs. suboptimal or poor) performance in an eight-color staining. Assessment of clonality, via additional cytoplasmic immunoglobulin (CyIg) κ vs. CyIgλ evaluation, may contribute to further establish the normal/reactive vs. clonal nature of small suspicious PC populations at high sensitivity levels, provided that enough cells are evaluated.

Cancer recognized as one of the leading irrepressible health issues is contributing to increasing mortality-rate day-by-day. The tumor microenvironment is an important field of cancer to understand the detection, treatment and prevention of cancer. Recently, cancer stem cell (CSC) research has shown promising results aiming towards cancer diagnostics and treatment. Here, we found that prostate and breast cancer stem cells secreted vesicles of endosomal origin, called exosomes showed strong connection between autophagy and exosomes released from CSCs. Exosomes may serve as vesicles to communicate with neoplastic cells (autocrine and paracrine manner) and normal cells (paracrine and endocrine manner) and thereby suppress immune systems and regulate neoplastic growth, and metastasis. They can also be used as biomarkers for various cancers. We detected tetraspanin proteins (CD9, CD63, CD81), Alix and tumor susceptibility gene-101 (TSG101) of exosomal markers from rotenone treated CSCs. We have also detected the induction of autophagy genes, Atg7 and conversion of autophagy marker (LC3-I to LC3-II), and tetraspanin proteins (CD9, CD63, CD81) in rotenone treated CSCs by western blotting. The mRNA expression of CD9, CD63, CD81 and TSG101 analyzed by qRT-PCR showed that the rotenone induced the expression of CD9, CD63, CD81 and TSG101 in CSCs. Electron microscopy of rotenone treated CSCs showed the mitochondrial damage of CSCs as confirmed by the release of exosomes from CSCs. The constituents of exosomes may be useful to understand the mechanism of exosomes formation, release and function, and also serve as a useful biomarker and provide novel therapeutic strategies for the treatment and prevention of cancer.

BACKGROUND: Metastasis is a major reason for poor prognosis in patients with cancer, including hepatocellular carcinoma (HCC). A salient feature is the ability of cancer cells to colonize different organs. Long non-coding RNAs (lncRNAs) play important roles in numerous cellular processes, including metastasis.
MATERIALS AND METHODS: In this study, the lncRNA expression profiles of two HCC cell lines, one with high potential for metastasis to the lung (HCCLM3) and the other to lymph nodes (HCCLYM-H2) were assessed using the Arraystar Human LncRNA Array v2.0, which contains 33,045 lncRNAs and 30,215 mRNAs. Coding-non-coding gene co-expression (CNC) networks were constructed and gene set enrichment analysis (GSEA) was performed to identify lncRNAs with potential functions in organ-specific metastasis. Levels of two representative lncRNAs and one representative mRNA, RP5-1014O16.1, lincRNA-TSPAN8 and TSPAN8, were further detected in HCC cell lines with differing metastasis potential by qRT-PCR.
RESULTS: Using microarray data, we identified 1,482 lncRNAs and 1,629 mRNAs that were differentially expressed (≥1.5 fold-change) between the two HCC cell lines. The most upregulated lncRNAs in H2 were RP11-672F9.1, RP5-1014O16.1, and RP11-501G6.1, while the most downregulated ones were lincRNA-TSPAN8, lincRNA-CALCA, C14orf132, NCRNA00173, and CR613944. The most upregulated mRNAs in H2 were C15orf48, PSG2, and PSG8, while the most downregulated ones were CALCB, CD81, CD24, TSPAN8, and SOST. Among them, lincRNA-TSPAN8 and TSPAN8 were found highly expressed in high lung metastatic potential HCC cells, while lowly expressed in no or low lung metastatic potential HCC cells. RP5- 1014O16.1 was highly expressed in high lymphatic metastatic potential HCC cell lines, while lowly expressed in no lymphatic metastatic potential HCC cell lines.
CONCLUSIONS: We provide the first detailed description of lncRNA expression profiles related to organ-specific metastasis in HCC. We demonstrated that a large number of lncRNAs may play important roles in driving HCC cells to metastasize to different sites; these lncRNAs may provide novel molecular biomarkers and offer a new basis for combating metastasis in HCC cases.

INTRODUCTION: Gelatinous Heberden's nodes (HNs), also termed synovial cysts, are a common form of generalized osteoarthritis (OA). We sought to determine whether HN cases at clinical presentation contained multipotential stromal cells (MSCs) and to explore whether such cells were more closely related to bone marrow (BM) or synovial fluid (SF) MSCs by transcriptional analysis.
METHODS: At clinical presentation, gelatinous material was extracted/extruded from the distal phalangeal joint of OA patients with HNs. From this, plastic adherent cells were culture-expanded for phenotypic and functional characterization and comparison with BM- and SF-MSCs. Mesenchymal related gene expression was studied by using a custom-designed TaqMan Low Density Array to determine transcriptional similarities between different MSC groups and skin fibroblasts.
RESULTS: In all cases, HN material produced MSC-like colonies. Adherent cultures displayed an MSC phenotype (CD29(+), CD44(+), CD73(+), CD81(+), and CD90(+) and CD14(-) CD19(-), CD31(-), CD34(-), CD45(-), and HLADR(-)) and exhibited osteogenic, chondrogenic lineage differentiation but weak adipogenesis. Gene cluster analysis showed that HN-MSCs were more closely related to SF- than normal or OA BM-MSCs with significantly higher expression of synovium-related gene markers such as bone morphogenic protein 4 (BMP4), bone morphogenetic protein receptor type 1A (BMPR1A), protein/leucine-rich end leucine-rich repeat protein (PRELP), secreted frizzled-related protein 4 (SFRP4), and tumor necrosis factor alpha-induced protein 6 (TNFAIP6) (P <0.05).
CONCLUSIONS: Gelatinous HNs derived from hand OA at clinical presentation contain a population of MSCs that share transcriptional similarities with SF-derived MSCs. Their aberrant entrapment within the synovial cysts may impact on their normal role in joint homeostasis.

UNLABELLED: Hepatitis C virus (HCV) exposure leads to persistent life-long infections characterized by chronic inflammation often developing into cirrhosis and hepatocellular carcinoma. The mechanism by which HCV remains in the liver while inducing an inflammatory and antiviral response remains unclear. Though the innate immune response to HCV in patients seems to be quite active, HCV has been shown in cell culture to employ a diverse array of innate immune antagonists, which suggests that current model systems to study interactions between HCV and the innate immune system are not representative of what happens in vivo. We recently showed that hepatoma-derived HepG2 cells support the entire HCV life cycle if the liver-specific microRNA, miR-122, is expressed along with the entry factor, CD81 (termed HepG2-HFL cells). We found that there was a striking difference in these cells' ability to sustain HCV infection and spread when compared with Huh-7 and Huh-7.5 cells. Additionally, HepG2-HFL cells exhibited a more robust antiviral response when challenged with other RNA viruses and viral mimetics than Huh-7 and Huh-7.5 cells. HCV infection elicited a potent interferon-lambda (IFN-λ), IFN-stimulated gene, and cytokine response in HepG2-HFL cells, but not in Huh-7 cells, suggesting that HepG2-HFL cells more faithfully recapitulate the innate immune response to HCV infection in vivo. Using this model, we found that blocking the retinoic acid-inducible gene I (RIG-I)-like receptor pathway or the IFN-λ-signaling pathway promoted HCV infection and spread in HepG2-HFL cells.
CONCLUSION: HepG2-HFL cells represent a new system to study the interaction between HCV and the innate immune system, solidifying the importance of IFN-λ in hepatic response to HCV infection and revealing non-redundant roles of RIG-I and melanoma differentiation-associated protein 5 in HCV recognition and repression of infection.

Despite the importance of multiple tetraspanin proteins in cancer invasion and metastasis, little is known about the role and significance of tetraspanin CD81 in these processes. In the present study, we examined CD81 effects on melanoma cell invasiveness and metastasis. Transfection of CD81 into melanoma cells lacking endogenous CD81 expression significantly enhanced the migrating, invasive, and metastatic abilities of melanoma cells. Interestingly, membrane type 1 matrix metalloproteinase (MT1-MMP) expression was found in CD81-expressing melanoma cells but not in CD81-deficient cells. siRNA knockdown of CD81 in melanoma cells with endogenous CD81 demonstrated decreased MT1-MMP levels and cell motility. Notably, CD81-induced cell migration was abrogated by antibody blocking and siRNA knockdown of MT1-MMP, indicating that MT1-MMP is responsible for CD81-stimulated melanoma cell migration. Promoter analysis revealed an essential role of the Sp1 transcription factor in CD81-induced MT1-MMP transcription. We also demonstrate that the Sp1-activating Akt pathway is involved in adhesion-dependent CD81 signaling to induce MT1-MMP expression and cell motility. Importantly, human skin cancer tissue specimens displayed a positive correlation of CD81 with MT1-MMP expression levels and a close association of CD81 with malignant melanomas. Taken together, these results strongly suggest that CD81 stimulates melanoma cell motility by inducing MT1-MMP expression through the Akt-dependent Sp1 activation signaling pathway, leading to increased melanoma invasion and metastasis.

BACKGROUND: Pleomorphic xanthoastrocytoma (PXA) is a rare WHO grade II tumor accounting for less than 1% of all astrocytomas. Malignant transformation into PXA with anaplastic features, is unusual and correlates with poorer outcome of the patients.
METHODS: Using a DNA methylation custom array, we have quantified the DNA methylation level on the promoter sequence of 807 cancer-related genes of WHO grade II (n = 11) and III PXA (n = 2) and compared to normal brain tissue (n = 10) and glioblastoma (n = 87) samples. DNA methylation levels were further confirmed on independent samples by pyrosequencing of the promoter sequences.
RESULTS: Increasing DNA promoter hypermethylation events were observed in anaplastic PXA as compared with grade II samples. We further validated differential hypermethylation of CD81, HCK, HOXA5, ASCL2 and TES on anaplastic PXA and grade II tumors. Moreover, these epigenetic alterations overlap those described in glioblastoma patients, suggesting common mechanisms of tumorigenesis.
CONCLUSIONS: Even taking into consideration the small size of our patient populations, our data strongly suggest that epigenome-wide profiling of PXA is a valuable tool to identify methylated genes, which may play a role in the malignant progression of PXA. These methylation alterations may provide useful biomarkers for decision-making in those patients with low-grade PXA displaying a high risk of malignant transformation.

Recombinant Newcastle Disease Virus (rNDV) has shown oncolytic therapeutic effect in preclinical studies. Previous data indicate that rNDV carrying IL2 has shown promise in cancer therapy. Due to the significant side effects of IL2, IL15 has been introduced into cancer therapy. A number of studies have suggested that IL15 efficiently enhances the activities of CTL and NK cells and inhibits the tumor recurrence and metastasis. Furthermore, IL15 is less toxic than IL2. Therefore, we hypothesize that a recombinant NDV expressing IL15 would be a promising agent for the treatment of malignant tumors. The human IL15 gene or IL2 gene was incorporated into the genome of lentogenic LaSota strain at the position between the HN and L genes (namely rNDV-IL15 or rNDV-IL2). The two viruses efficiently infected tumor cells and expressed IL15 or IL2 protein. Melanoma tumor-bearing mice were treated by intra-tumoral (i.t.) injection of rNDV-IL15 or rNDV-IL2. Both rNDV-IL15 and rNDV-IL2 effectively suppressed tumor growth compared with rNDV. The 120-day survival rate of rNDV-IL15- treated group was 12.5% higher than that of rNDV-IL2 group, although the difference was not statistically significant, both recombinant viruses had strong abilities to induce CD41 T cell and CTL cell responses. However, rNDV-IL15 significantly induced more IFN-γ release and stimulated more CD81 T cells infiltration in the tumor sites compared with rNDV-IL2. In the tumor re-challenged experiment, the survival rates of rNDV-IL15 group and rNDV-IL2 group were statistically higher than that of PBS group. The survival rate of rNDV-IL15 group was 26.67% higher than that of rNDV-IL2 group although the difference was not statistically significant. In conclusion, rNDV-IL15 is a promising antitumor agent against melanoma.

AIMS: Limited information exists about the impact of cytogenetic alterations on the protein expression profiles of individual meningioma cells and their association with the clinicohistopathological characteristics of the disease. The aim of this study is to investigate the potential association between the immunophenotypic profile of single meningioma cells and the most relevant features of the tumour.
METHODS: Multiparameter flow cytometry (MFC) was used to evaluate the immunophenotypic profile of tumour cells (n = 51 patients) and the Affymetrix U133A chip was applied for the analysis of the gene expression profile (n = 40) of meningioma samples, cytogenetically characterized by interphase fluorescence in situ hybridization.
RESULTS: Overall, a close association between the pattern of protein expression and the cytogenetic profile of tumour cells was found. Thus, diploid tumours displayed higher levels of expression of the CD55 complement regulatory protein, tumours carrying isolated monosomy 22/del(22q) showed greater levels of bcl2 and PDGFRβ and meningiomas carrying complex karyotypes displayed a greater proliferation index and decreased expression of the CD13 ectoenzyme, the CD9 and CD81 tetraspanins, and the Her2/neu growth factor receptor. From the clinical point of view, higher expression of CD53 and CD44 was associated with a poorer outcome.
CONCLUSIONS: Here we show that the protein expression profile of individual meningioma cells is closely associated with tumour cytogenetics, which may reflect the involvement of different signalling pathways in the distinct cytogenetic subgroups of meningiomas, with specific immunophenotypic profiles also translating into a different tumour clinical behaviour.

Flow cytometric (FC) enumeration of abnormal plasma cells (APCs) for diagnosis and prognostication of plasma cell dyscrasias (PCD) is challenging. We studied antigen expression in normal plasma cells (NPC) (N = 34) and APC in a series of unselected PCD (N = 59). NPC subpopulations often demonstrated CD19(-), CD20(+), CD45(-) or dim and CD56(+), an immunophenotype observed in PCD. However abnormal CD81 was only observed in APCs (APC detection sensitivity 95%; specificity 100%). We evaluated differences in antigen expression patterns among MGUS (N = 14), SMM (N = 35) and MM (N = 10), finding the combination of CD45 and CD56 helpful in differentiating MGUS from SMM and MM (p = 0.0002).

Chronic lymphocytic leukemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20-30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging system, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 overexpression, cytogenetic abnormalities (13q-, + 12, 11q-, 17p-) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all cases of CLL at risk. In a recent review, we summarized those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan(™) CLL antibody microarray (Version 3; 182 CD antibodies), and with refined analysis of purified CD19 + B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage patients with CLL according to probability of clinical progression and potential earlier requirement for treatment.

Normal and diseased cells release bilayered membrane-bound nanovesicles into interstitial spaces and into bodily fluids. A subgroup of such microvesicles is called exosomes and is described in blood as 30 to 100 nm in diameter and as spherical to cup-shaped nanoparticles with specific surface molecular characteristics (eg, expression of the tetraspanins CD9, CD81, and CD63). Extracellular microvesicles provide local signals (eg, autocrine and paracrine) and distant endocrine signals to cells via the transfer of their contents, which include signal proteins, lipids, miRNAs, and functional mRNAs. Exosomes and related microvesicles also aid cells in exporting less-needed molecules and potentially harmful molecules, including drugs; in the case of neoplasia, the export of chemotherapeutic drugs may facilitate cellular chemoresistance. Cancers have adapted the exosome and related microvesicles as a pathway by which neoplastic cells communicate with each other (autocrine) and with nonneoplastic cells (paracrine and endocrine); via this pathway, cancer suppresses the immune system and establishes a fertile local and distant environment to support neoplastic growth, invasion, and metastases. Because exosomes mirror and bind to the cells from which they arise, they can be used for delivery of drugs, vaccines, and gene therapy, as biomarkers and targets. We review how exosomes and related extracellular microvesicles facilitate the progression and metastases of cancers and describe how these microvesicles may affect clinical care.

Integrin α3β1 potently promotes cell motility on its ligands, laminin-332 and laminin-511, and this may help to explain why α3β1 has repeatedly been linked to breast carcinoma progression and metastasis. The pro-migratory functions of α3β1 depend strongly on lateral interactions with cell surface tetraspanin proteins. Tetraspanin CD151 interacts directly with the α3 integrin subunit and links α3β1 integrin to other tetraspanins, including CD9 and CD81. Loss of CD151 disrupts α3β1 association with other tetraspanins and impairs α3β1-dependent motility. However, the extent to which tetraspanins other than CD151 are required for specific α3β1 functions is unclear. To begin to clarify which aspects of α3β1 function require which tetraspanins, we created breast carcinoma cells depleted of both CD9 and CD81 by RNA interference. Silencing both of these closely related tetraspanins was required to uncover their contributions to α3β1 function. We then directly compared our CD9/CD81-silenced cells to CD151-silenced cells. Both CD9/CD81-silenced cells and CD151-silenced cells showed delayed α3β1-dependent cell spreading on laminin-332. Surprisingly, however, once fully spread, CD9/CD81-silenced cells, but not CD151-silenced cells, displayed impaired α3β1-dependent directed motility and altered front-rear cell morphology. Also unexpectedly, the CD9/CD81 complex, but not CD151, was required to promote α3β1 association with PKCα in breast carcinoma cells, and a PKC inhibitor mimicked aspects of the CD9/CD81-silenced cell motility defect. Our data reveal overlapping, but surprisingly distinct contributions of specific tetraspanins to α3β1 integrin function. Importantly, some of CD9/CD81's α3β1 regulatory functions may not require CD9/CD81 to be physically linked to α3β1 by CD151.

Bladder cancer is the 4(th) most common cancer among men in the U.S. We analyzed variant genotypes hypothesized to modify major biological processes involved in bladder carcinogenesis, including hormone regulation, apoptosis, DNA repair, immune surveillance, metabolism, proliferation, and telomere maintenance. Logistic regression was used to assess the relationship between genetic variation affecting these processes and susceptibility in 563 genotyped urothelial cell carcinoma cases and 863 controls enrolled in a case-control study of incident bladder cancer conducted in New Hampshire, U.S. We evaluated gene-gene interactions using Multifactor Dimensionality Reduction (MDR) and Statistical Epistasis Network analysis. The 3'UTR flanking variant form of the hormone regulation gene HSD3B2 was associated with increased bladder cancer risk in the New Hampshire population (adjusted OR 1.85 95%CI 1.31-2.62). This finding was successfully replicated in the Texas Bladder Cancer Study with 957 controls, 497 cases (adjusted OR 3.66 95%CI 1.06-12.63). The effect of this prevalent SNP was stronger among males (OR 2.13 95%CI 1.40-3.25) than females (OR 1.56 95%CI 0.83-2.95), (SNP-gender interaction P = 0.048). We also identified a SNP-SNP interaction between T-cell activation related genes GATA3 and CD81 (interaction P = 0.0003). The fact that bladder cancer incidence is 3-4 times higher in males suggests the involvement of hormone levels. This biologic process-based analysis suggests candidate susceptibility markers and supports the theory that disrupted hormone regulation plays a role in bladder carcinogenesis.

BACKGROUND: CD81 is a transmembrane protein that serves as a putative receptor for hepatitis C virus. In addition, CD81 has been suggested to be involved in a broad range of other cellular functions. Its putative implication in tumorigenesis has so far, however, remained largely unexplored. To assess the candidacy of CD81 as a tumor suppressor in gastric cancer development, we investigated its expression and function in a series of primary gastric tumors and gastric tumor-derived cell lines.
METHODS: The expression and concomitant methylation status of the CD81 gene and its effect on tumor development and cellular signaling were evaluated.
RESULTS: CD81 mRNA levels were found to be low in 16 of 40 (40 %) primary tumors and 9 of 14 (64.2 %) cell lines, and these low expression levels were found to correlate with the stage and grade of the tumors. Genomic alterations of CD81 were not encountered, whereas its expression could be re-activated in low expressing cells upon 5-aza-dC treatment. Bisulfite DNA sequencing analysis of 10 CpG sites within the 5' proximal region of the CD81 gene promoter revealed that the observed transcriptional silencing was tightly associated with aberrant hypermethylation. Subsequent restoration of CD81 expression induced a G1 cell cycle arrest and apoptosis, whereas siRNA-mediated CD81 down-regulation promoted cell proliferation and attenuated cellular responses to various apoptotic stress stimuli. Also the colony-forming ability of the tumor cells could be inhibited and enhanced through CD81 up- and down-regulation, respectively. CD81 was found to inhibit p38 (but not ERK, JNK and AKT) phosphorylation and its growth suppressive effect could be abolished through p38 up- and down-regulation.
CONCLUSION: From our data we conclude that epigenetic inactivation of CD81 is a common feature of gastric tumors and that this inactivation may render growth and survival advantages to the tumor cells, at least partially through p38 signaling.

UNLABELLED: The polymorphisms in the interleukin (IL)-28B (interferon-lambda [IFN]-λ3) gene are strongly associated with the efficacy of hepatitis C virus (HCV) clearance. Dendritic cells (DCs) sense HCV and produce IFNs, thereby playing some cooperative roles with HCV-infected hepatocytes in the induction of interferon-stimulated genes (ISGs). Blood dendritic cell antigen 3 (BDCA3)(+) DCs were discovered as a producer of IFN-λ upon Toll-like receptor 3 (TLR3) stimulation. We thus aimed to clarify the roles of BDCA3(+) DCs in anti-HCV innate immunity. Seventy healthy subjects and 20 patients with liver tumors were enrolled. BDCA3(+) DCs, in comparison with plasmacytoid DCs and myeloid DCs, were stimulated with TLR agonists, cell-cultured HCV (HCVcc), or Huh7.5.1 cells transfected with HCV/JFH-1. BDCA3(+) DCs were treated with anti-CD81 antibody, inhibitors of endosome acidification, TIR-domain-containing adapter-inducing interferon-β (TRIF)-specific inhibitor, or ultraviolet-irradiated HCVcc. The amounts of IL-29/IFN-λ1, IL-28A/IFN-λ2, and IL-28B were quantified by subtype-specific enzyme-linked immunosorbent assay (ELISA). The frequency of BDCA3(+) DCs in peripheral blood mononuclear cell (PBMC) was extremely low but higher in the liver. BDCA3(+) DCs recovered from PBMC or the liver released large amounts of IFN-λs, when stimulated with HCVcc or HCV-transfected Huh7.5.1. BDCA3(+) DCs were able to induce ISGs in the coexisting JFH-1-positive Huh7.5.1 cells. The treatments of BDCA3(+) DCs with anti-CD81 antibody, cloroquine, or bafilomycin A1 reduced HCVcc-induced IL-28B release, whereas BDCA3(+) DCs comparably produced IL-28B upon replication-defective HCVcc. The TRIF-specific inhibitor reduced IL-28B release from HCVcc-stimulated BDCA3(+) DCs. In response to HCVcc or JFH-1-Huh7.5.1, BDCA3(+) DCs in healthy subjects with IL-28B major (rs8099917, TT) released more IL-28B than those with IL-28B minor genotype (TG).
CONCLUSION: Human BDCA3(+) DCs, having a tendency to accumulate in the liver, recognize HCV in a CD81-, endosome-, and TRIF-dependent manner and produce substantial amounts of IL-28B/IFN-λ3, the ability of which is superior in subjects with IL-28B major genotype.

Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.

BACKGROUND: Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome.
METHODS: The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis.
RESULTS: The overwhelming majority (97.3%) of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p < 0.05), while CASP8 with older (p < 0.01). MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p < 0.05), while methylation of CASP8 was more frequent in patients who survived shorter than 36 months (p < 0.05). Cox regression analysis showed patient age, treatment, MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p < 0.05). MGMT and GATA6 were independent predictors for patient survival in younger patients' group, while there were no significant associations observed in older patients' group when adjusted for therapy.
CONCLUSIONS: High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome.