Kitty Agarwal

Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Epigenetic regulation of connective tissue growth factor by MicroRNA‐214 delivery in exosomes from mouse or human hepatic stellate cells

Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up‐regulation in fibrotic or steatotic livers, or in culture‐activated or ethanol‐treated primary mouse HSC, is associated with a reciprocal down‐regulation of microRNA‐214 (miR‐214). By using protector or reporter assays to investigate the 3′‐untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis‐inducing stimuli was due to reduced expression of miR‐214, which otherwise inhibited CCN2 expression by directly binding to the CCN2 3′‐UTR. Additionally, miR‐214 was present in HSC exosomes, which were bi‐membrane vesicles, 50‐150 nm in diameter, negatively charged (−26 mV), and positive for CD9. MiR‐214 levels in exosomes but not in cell lysates were reduced by pretreatment of the cells with the exosome inhibitor, GW4869. Coculture of either quiescent HSC or miR‐214‐transfected activated HSC with CCN2 3′‐UTR luciferase reporter‐transfected recipient HSC resulted in miR‐214‐ and exosome‐dependent regulation of a wild‐type CCN2 3′‐UTR reporter but not of a mutant CCN2 3′‐UTR reporter lacking the miR‐214 binding site. Exosomes from HSC were a conduit for uptake of miR‐214 by primary mouse hepatocytes. Down‐regulation of CCN2 expression by miR‐214 also occurred in human LX‐2 HSC, consistent with a conserved miR‐214 binding site in the human CCN2 3′‐UTR. MiR‐214 in LX‐2 cells was shuttled by way of exosomes to recipient LX‐2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3′‐UTR activity or expression of CCN2 downstream targets, including alpha smooth muscle actin or collagen. Experimental fibrosis in mice was associated with reduced circulating miR‐214 levels. Conclusion: Exosomal transfer of miR‐214 is a paradigm for the regulation of CCN2‐dependent fibrogenesis and identifies fibrotic pathways as targets of intercellular regulation by exosomal miRs. (Hepatology 2014;59:1118–1129)

Breast Cancer–Specific miR Signature Unique to Extracellular Vesicles Includes “microRNA-like” tRNA Fragments

Extracellular vesicles (EV), including exosomes and shed vesicles, have been implicated in intercellular communication; however, their biomarker potential is less clear. Therefore, EVs derived from MCF7 and MCF10A cells were analyzed to identify unique miRNA (miR) profiles that distinguish their origin. One characteristic common to the miR profiles of MCF7 EVs and their parent cells is the high abundance of miR-21, let-7a, miR-100, and miR-125b, and low levels of miR-205. A second characteristic is the high abundance of “miRNA-like” tRNA fragments, which is unique to the MCF7 EVs, and is not found in comparing the cellular profiles. In addition, correlations were examined in the MCF7 cellular expression levels of these five miRs and two tRNA-derived miRNAs, miR-720 and miR-1274b, and compared with the correlations in MCF7 EV levels. Interestingly, correlations in the cellular expression of miR-125b, miR-100, and let-7a are mirrored in the EVs. In contrast, correlations in tRNA-derived miRNA levels are found only in the EVs. The findings suggest that EV miR clusters can be defined based on functional miR interactions related to correlated cellular expression levels or physical miR interactions, for example, aggregation due to comparable binding affinities to common targets. Implications: These results point to using high levels of tRNA-derived small RNA fragments in combination with known miR signatures of tumors to distinguish tumor-derived EVs in circulation from EVs derived from other cell sources. Such biomarkers would be unique to the EVs where high abundances of tRNA fragments are amplified with respect to their cellular levels. Mol Cancer Res; 13(5); 891–901. ©2015 AACR.

Sorafenib and Mek inhibition is synergistic in medullary thyroid carcinoma in vitro

Clinical trials using kinase inhibitors have demonstrated transient partial responses and disease control in patients with progressive medullary thyroid cancer (MTC). The goal of this study was to identify potential combinatorial strategies to improve on these results using sorafenib, a multikinase inhibitor with activity in MTC, as a base compound to explore signaling that might predict synergystic interactions. Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to sorafenib, everolimus, and AZD6244 alone and in combination. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and poly (ADP-ribose) polymerase (PARP) cleavage assays were performed to measure cell survival and apoptosis. Western blots were performed to confirm activity of the compounds and to determine possible mechanisms of resistance and predictors of synergy. As a solitary agent, sorafenib was the most active compound on MTT assay. Western blots confirmed that sorafenib, everolimus, and AZD6244 inhibited their anticipated targets. At concentrations below its IC(50), sorafenib-treated TT and MZ-CRC-1 cells demonstrated transient inhibition and then re-activation of Erk over 6 h. In concordance, synergistic effects were only identified using sorafenib in combination with the Mek inhibitor AZD6244 (P<0.001 for each cell line). Cells treated with everolimus demonstrated activation of Akt and Ret via TORC2 complex-dependent and TORC2 complex-independent mechanisms respectively. Everolimus was neither additive nor syngergistic in combination with sorafenib or AZD6244. In conclusion, sorafenib combined with a Mek inhibitor demonstrated synergy in MTC cells in vitro. Mechanisms of resistance to everolimus in MTC cells likely involved TORC2-dependent and TORC2-independent pathways.

Characterization of Multiple Myeloma Vesicles by Label-Free Relative Quantitation

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell‐to‐cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label‐free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label‐free spectral counting applied to determine relative protein abundances in EVs.

Detection of extracellular RNAs in cancer and viral infection via tethered cationic lipoplex nanoparticles containing molecular beacons.

Noninvasive early detection methods have the potential to reduce mortality rates of both cancer and infectious diseases. Here, we present a novel assay by which tethered cationic lipoplex nanoparticles containing molecular beacons (MBs) can capture cancer cell-derived exosomes or viruses and identify encapsulated RNAs in a single step. A series of ultracentrifugation and Exoquick isolation kit were first used to isolate exosomes from the cell culture medium and human serum, respectively. Cationic lipoplex nanoparticles linked onto the surface of a thin glass plate capture negatively charged viruses or cell-secreted exosomes by electrostatic interactions to form larger nanoscale complexes. Lipoplex/virus or lipoplex/exosome fusion leads to the mixing of viral/exosomal RNAs and MBs within the lipoplexes. After the target RNAs specially bind to the MBs, exosomes enriched in target RNAs are readily identified by the fluorescence signals of MBs. The in situ detection of target extracellular RNAs without diluting the samples leads to high detection sensitivity not achievable by existing methods, e.g., quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Here we demonstrate this concept using lentivirus and serum from lung cancer patients.

Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers.

UNLABELLED Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patients serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers. BIOLOGICAL SIGNIFICANCE Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.

Analysis of exosome release as a cellular response to MAPK pathway inhibition.

Exosome size distributions and numbers of exosomes released per cell are measured by asymmetric flow-field flow fractionation/multi-angle light scattering (A4F/MALS) for three thyroid cancer cell lines as a function of a treatment that inhibits MAPK signaling pathways in the cells. We show that these cell lines release exosomes with well-defined morphological features and size distributions that reflect a common biological process for their formation and release into the extracellular environment. We find that those cell lines with constitutive activation of the MAPK signaling pathway display MEK-dependent exosome release characterized by increased numbers of exosomes released per cell. Analysis of the measured exosome size distributions based on a generalized extreme value distribution model for exosome formation in intracellular multivesicular bodies highlights the importance of this experimental observable for delineating different mechanisms of vesicle formation and predicting how changes in exosome release can be modified by pathway inhibitors in a cell context-dependent manner.

Abstract P3-09-04: Exosome-Specific microRNA Signatures in Combination with Characteristic Surface Markers on the Circulating Exosomes Themselves Provide New Insights into the EMT

Background: Recent discoveries have established that cancer tumors exhibit distinct microRNA (miR) expression profiles compared to normal tissues. Detection of miRs in the peripheral blood of cancer patients is possible due to their high stability. This stability is attributed to encapsulation of the miRs inside microvesicles (MVs) where they are protected from endogenous RNase activity in circulation. The tumor cell-secreted MVs of primary interest are circulating exosomes, a subpopulation of MVs distinguished by their relatively small size: 40 to 100 nm in diameter. The isolation of circulating tumor-derived exosomes from the other cell-secreted MVs in peripheral blood is the critical barrier to successful development of a robust assay for cancer-specific miR signatures. We describe the development of such an assay to: (1) capture/isolate circulating exosomes based on characteristic surface markers that correlate with different cell types, and (2) characterize exosome-specific miR signatures based on these surface markers. Materials and Methods: We apply sequential ultracentrifugation to isolate cell-secreted MVs, followed by asymmetric flow field-flow fractionation to selectively separate exosomes based on their intrinsically small size and characteristic surface markers. Isolated exosomes are then screened for surface markers by selectively capturing them on microarrays printed with antibodies against a library of known cell-surface markers for breast cancer. For the assessment of exosome-specific miR signatures, we have devised an antibody microarray assay that also enables the in situ characterization of miR profiles of the captured exosomes locally by qRT-PCR analysis confined to subarrays of printed spots in 50-µl microwells of a custom-designed, multi-well microarray. Assay development has been carried out using two human cancer cell lines (MCF 7 and MDA MB 231) and a non-malignant cell line (MCF 10a) representing pre-malignant cells and cancer of the breast at different epithelial and mesenchymal states. Results: We have used light scattering and cryo-transmission electron microscopy to characterize the size, size distribution, and morphology of exosomes as a sub-population of the secreted MVs from each of the three cell lines. We have also screened for surface markers by selective capturing the exosome sub-populations derived from these cell lines on antibody microarrays, and correlated these surface markers with the miR content of MVs using qRT-PCR analysis. Discussion: Our results demonstrate that correlating the miR signatures of circulating exosomes with characteristic surface markers on these exosomes leads to robust distinctions between the three cell lines that could not be achieved using either analysis or characterization alone. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-04.

Dynamic Scaling of Exosome Sizes

A model is proposed for characterizing exosome size distributions based on dynamic scaling of domain growth on the limiting membrane of multivesicular bodies in the established exosome biogenesis pathway. The scaling exponent in this model captures the asymmetry of exosome size distributions, which are notably right-skewed to larger vesicles, independent of the minimum detectable vesicle size. Analyses of exosome size distributions obtained by cryogenic transmission electron microscopy imaging and nanoparticle tracking show, respectively, that the scaling exponent is sensitive to the state of the cell source for exosomes in cell culture supernatants and can distinguish exosome size distributions in serum samples taken from cancer patients relative to those from healthy donors. Finally, we comment on mechanistic differences between our dynamic scaling model and random fragmentation models used to describe size distributions of synthetic vesicles.