Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular ...communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication.
We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps.
The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles.
This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Tenosynovial giant-cell tumor (TGCT) and pigmented villonodular synovitis (PVNS) are related conditions with features of both reactive inflammatory disorders and clonal neoplastic proliferations. ...Chromosomal translocations involving chromosome 1p13 have been reported in both TGCT and PVNS. We confirm that transiocations involving p1pl3 are present in a majority of cases of TGCT and PVNS and show that CSF1 is the gene at the chromosome lp13 breakpoint. In some cases of both TGCT and PVNS, CSF1 is fused to COL6A3 (2q35). The CSF1 translocations result in overexpression of CSF1. In cases of TGCT and PVNS carrying this translocation, it is present in a minority of the intratumoral cells, leading to CSF1 expression only in these cells, whereas the majority of cells express CSFIR but not CSF1, suggesting a tumor-landscaping effect with aberrant CSF1 expression in the neoplastic cells, leading to the abnormal accumulation of nonneoplastic cells that form a tumorous mass.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The cell-surface protein CD38 mediates airway smooth muscle (ASM) contractility by generating cyclic ADP-ribose, a calcium-mobilizing molecule. In human ASM cells, TNF-α augments CD38 expression ...transcriptionally by NF-κB and AP-1 activation and involving MAPK and PI3K signaling. CD38-/- mice develop attenuated airway hyperresponsiveness following allergen or cytokine challenge. The post-transcriptional regulation of CD38 expression in ASM is relatively less understood. In ASM, microRNAs (miRNAs) regulate inflammation, contractility, and hyperproliferation. The 3' Untranslated Region (3'UTR) of CD38 has multiple miRNA binding sites, including a site for miR-708. MiR-708 is known to regulate PI3K/AKT signaling and hyperproliferation of other cell types. We investigated miR-708 expression, its regulation of CD38 expression and the underlying mechanisms involved in such regulation in human ASM cells.
Growth-arrested human ASM cells from asthmatic and non-asthmatic donors were used. MiRNA and mRNA expression were measured by quantitative real-time PCR. CD38 enzymatic activity was measured by a reverse cyclase assay. Total and phosphorylated MAPKs and PI3K/AKT as well as enzymes that regulate their activation were determined by Western blot analysis of cell lysates following miRNA transfection and TNF-α stimulation. Dual luciferase reporter assays were performed to determine whether miR-708 binds directly to CD38 3'UTR to alter gene expression.
Using target prediction algorithms, we identified several miRNAs with potential CD38 3'UTR target sites and determined miR-708 as a potential candidate for regulation of CD38 expression based on its expression and regulation by TNF-α. TNF-α caused a decrease in miR-708 expression in cells from non-asthmatics while it increased its expression in cells from asthmatics. Dual luciferase reporter assays in NIH-3 T3 cells revealed regulation of expression by direct binding of miR-708 to CD38 3'UTR. In ASM cells, miR-708 decreased CD38 expression by decreasing phosphorylation of JNK MAPK and AKT. These effects were associated with increased expression of MKP-1, a MAP kinase phosphatase and PTEN, a phosphatase that terminates PI3 kinase signaling.
In human ASM cells, TNF-α-induced CD38 expression is regulated by miR-708 directly binding to 3'UTR and indirectly by regulating JNK MAPK and PI3K/AKT signaling and has the potential to control airway inflammation, ASM contractility and proliferation.
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Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. ...Inflammatory cytokines alter calcium (Ca2+) signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3) and CD38-cyclic ADP-ribose (CD38/cADPR) are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose: Macrophages are migratory cells that are frequently recruited to the site of tumors. Their presence is associated with poor
clinical outcome in a variety of epithelial malignancies. The aim ...of this study is to examine the prognostic significance
of tumor-associated macrophages in sarcomas.
Experimental Design: Global gene expression profiling data of a series of soft tissue tumors were analyzed for macrophage-associated gene expression.
Immunohistochemistry on tissue microarrays containing leiomyosarcoma cases with known clinical outcome was used to verify
the presence of macrophages and to examine the relationship between tumor-associated macrophages and clinical outcome.
Results: Gene expression profiling revealed high-level expression of several macrophage-associated genes such as CD163 and CD68 in a subset of leiomyosarcomas, indicating the presence of variable numbers of tumor-infiltrating macrophages. This was confirmed
by CD68 and CD163 immunostaining of a tissue microarray containing 149 primary leiomyosarcomas. Kaplan-Meier survival analysis
showed that high density of tumor-infiltrating macrophages as identified by CD163 or CD68 staining is associated with a significantly
worse disease-specific survival in nongynecologic leiomyosarcomas, whereas leiomyosarcomas arising from the gynecologic tract
showed no significant association between macrophage infiltration and survival. The presence of tumor necrosis did not correlate
significantly with outcome.
Conclusions: An increased density of CD163- or CD68-positive tumor-infiltrating macrophages is associated with poor outcome in nongynecologic
leiomyosarcomas. This may help the clinical management of patients with leiomyosarcomas.
Intercellular communication is vital to the ecosystem of cancer cell organization and invasion. Identification of key cellular cargo and their varied modes of transport are important considerations ...in understanding the basic mechanisms of cancer cell growth. Gap junctions, exosomes, and apoptotic bodies play key roles as physical modalities in mediating intercellular transport. Tunneling nanotubes (TNTs)-narrow actin-based cytoplasmic extensions-are unique structures that facilitate direct, long distance cell-to-cell transport of cargo, including microRNAs, mitochondria, and a variety of other sub cellular components. The transport of cargo via TNTs occurs between malignant and stromal cells and can lead to changes in gene regulation that propagate the cancer phenotype. More notably, the transfer of these varied molecules almost invariably plays a critical role in the communication between cancer cells themselves in an effort to resist death by chemotherapy and promote the growth and metastases of the primary oncogenic cell. The more traditional definition of "Systems Biology" is the computational and mathematical modeling of complex biological systems. The concept, however, is now used more widely in biology for a variety of contexts, including interdisciplinary fields of study that focus on complex interactions within biological systems and how these interactions give rise to the function and behavior of such systems. In fact, it is imperative to understand and reconstruct components in their native context rather than examining them separately. The long-term objective of evaluating cancer ecosystems in their proper context is to better diagnose, classify, and more accurately predict the outcome of cancer treatment. Communication is essential for the advancement and evolution of the tumor ecosystem. This interplay results in cancer progression. As key mediators of intercellular communication within the tumor ecosystem, TNTs are the central topic of this article.
Tunneling nanotubes are actin-based cytoplasmic extensions that function as intercellular channels in a wide variety of cell types.There is a renewed and keen interest in the examination of modes of ...intercellular communication in cells of all types, especially in the field of cancer biology. Tunneling nanotubes -which in the literature have also been referred to as "membrane nanotubes," "'intercellular' or 'epithelial' bridges," or "cytoplasmic extensions" - are under active investigation for their role in facilitating direct intercellular communication. These structures have not, until recently, been scrutinized as a unique and previously unrecognized form of direct cell-to-cell transmission of cellular cargo in the context of human cancer. Our recent study of tunneling nanotubes in human malignant pleural mesothelioma and lung adenocarcinomas demonstrated efficient transfer of cellular contents, including proteins, Golgi vesicles, and mitochondria, between cells derived from several well-established cancer cell lines. Further, we provided effective demonstration that such nanotubes can form between primary malignant cells from human patients. For the first time, we also demonstrated the in vivo relevance of these structures in humans, having effectively imaged nanotubes in intact solid tumors from patients. Here we provide further analysis and discussion on our findings, and offer a prospective 'road map' for studying tunneling nanotubes in the context of human cancer. We hope that further understanding of the mechanisms, methods of transfer, and particularly the role of nanotubes in tumor-stromal cross-talk will lead to identification of new selective targets for cancer therapeutics.
Tumor metastases pose the greatest threat to a patient's survival, and thus, understanding the biology of disseminated cancer cells is critical for developing effective therapies.
Microarrays and ...immunohistochemistry were used to analyze primary breast tumors, regional (lymph node) metastases, and distant metastases in order to identify biological features associated with distant metastases.
When compared with each other, primary tumors and regional metastases showed statistically indistinguishable gene expression patterns. Supervised analyses comparing patients with distant metastases versus primary tumors or regional metastases showed that the distant metastases were distinct and distinguished by the lack of expression of fibroblast/mesenchymal genes, and by the high expression of a 13-gene profile (that is, the 'vascular endothelial growth factor (VEGF) profile') that included VEGF, ANGPTL4, ADM and the monocarboxylic acid transporter SLC16A3. At least 8 out of 13 of these genes contained HIF1alpha binding sites, many are known to be HIF1alpha-regulated, and expression of the VEGF profile correlated with HIF1alpha IHC positivity. The VEGF profile also showed prognostic significance on tests of sets of patients with breast and lung cancer and glioblastomas, and was an independent predictor of outcomes in primary breast cancers when tested in models that contained other prognostic gene expression profiles and clinical variables.
These data identify a compact in vivo hypoxia signature that tends to be present in distant metastasis samples, and which portends a poor outcome in multiple tumor types.This signature suggests that the response to hypoxia includes the ability to promote new blood and lymphatic vessel formation, and that the dual targeting of multiple cell types and pathways will be needed to prevent metastatic spread.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRNA) are 20 approximately 25 nucleotide non-coding RNAs that inhibit the translation of targeted mRNA, and they have been implicated in the development of human malignancies. High grade ...serous ovarian carcinomas, the most common and lethal subtype of ovarian cancer, can occur sporadically or in the setting of BRCA1/2 syndromes. Little is known regarding the miRNA expression profiles of high grade serous carcinoma in relation to BRCA1/2 status, and compared to normal tubal epithelium, the putative tissue of origin for high grade serous carcinomas.
Global miRNA expression profiling was performed on a series of 33 high grade serous carcinomas, characterized with respect to BRCA1/2 status (mutation, epigenetic silencing with loss of expression or normal), and with clinical follow-up, together with 2 low grade serous carcinomas, 2 serous borderline tumors, and 3 normal fallopian tube samples, using miRNA microarrays (328 human miRNA). Unsupervised hierarchical clustering based on miRNA expression profiles showed no clear separation between the groups of carcinomas with different BRCA1/2 status. There were relatively few miRNAs that were differentially expressed between the genotypic subgroups. Comparison of 33 high grade serous carcinomas to 3 normal fallopian tube samples identified several dysregulated miRNAs (false discovery rate <5%), including miR-422b and miR-34c. Quantitative RT-PCR analysis performed on selected miRNAs confirmed the pattern of differential expression shown by microarray analysis. Prognostically, lower level miR-422b and miR-34c in high grade serous carcinomas were both associated with decreased disease-specific survival by Kaplan-Meier analysis (p<0.05).
High grade serous ovarian carcinomas with and without BRCA1/2 abnormalities demonstrate very similar miRNA expression profiles. High grade serous carcinomas as a group exhibit significant miRNA dysregulation in comparison to tubal epithelium and the levels of miR-34c and miR-422b appear to be prognostically important.
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Fatty liver induced by alcohol abuse is a major worldwide health hazard leading to morbidity and mortality. Previous studies indicate antifatty liver properties of garlic. This study investigated the ...molecular mechanisms of garlic oil (GO) or diallyl disulfide (DADS) imparted hepatoprotection against alcohol induced fatty liver in C57BL/6 mice using microarray-based global gene expression analysis. Alcohol liquid diet resulted in severe fatty liver with increased levels of serum aspartate aminotransferease and alanine aminotransferease as well as triglycerides and decreased levels of liver glutathione and antioxidant enzymes. The major canonical pathways implicated by alcohol treatment are the metabolisms of xenobiotics by cytochrome P450, glutathione, and arachidonic acid. Treatment with DADS or GO normalized the serum aminotransferease levels and liver antioxidant enzymes and reduced the contents of triglycerides and cholesterol. The canonical pathways involved in the amelioration of liver include arachidonic acid metabolism, altered T cell and B cell signaling, tryptophan metabolism, antigen presentation pathway for DADS, metabolism of xenobiotics, mitotic roles of Polo-like kinase, fatty acid metabolism, LPS/IL-1 mediated inhibition of RXR function, and C21-steroid hormone metabolism for GO.
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