NTA: Exosomes and Microvesicles: Cancer studies

Potential of Exosomes as biomarkers in Cancer

The exacerbated release of exosomes in tumor cells, as evidenced by their increased levels in blood during the late stage of a disease and their over expression of certain tumor cell biomarkers, suggests an important role of exosomes in diagnosis and biomarker studies (Simpson et al., 2009). Furthermore, recent findings that exosomes contain inactive forms of both mRNA and microRNA that can be transferred to another cell and be functional in that new environment, have initiated many microRNA profiling studies of exosomes circulating in blood. These studies highlight the potential of exosomal microRNA profiles for use as diagnostic biomarkers of disease through a non-invasive blood test (Simpson et al., 2009).

Similarly, tumor cells emit large quantities of MVs containing procoagulant, growth regulatory and oncogenic cargo (oncosomes), which can be transferred throughout the cancer cell population and to nontransformed stromal cells, endothelial cells and possibly to the inflammatory infiltrates (oncogenic field effect). These events likely impact tumor invasion, angiogenesis, metastasis, drug resistance, and cancer stem cell hierarchy. Ongoing studies explore the molecular mechanisms and mediators of MV-based intercellular communication (cancer vesiculome), with the hope of using this information as a possible source of therapeutic targets and disease biomarkers in cancer (Camussi et al., 2011). For a list of exosome protein markers that are most often identified in exosomes, see ExoCarta, an exosomal protein and RNA database (http://exocarta.ludwig.edu.au).

Exosomes have also been studied as biomarkers for Prostate Cancer (PCa). While the biomarker protein, prostate-specific antigen (PSA), has been considered the gold standard for the detection of PCa and has acceptable sensitivity, it lacks the specificity for discriminating benign prostate diseases (e.g. benign prostatic hyperplasia and infection), indolent PCa and aggressive PCa. Accordingly, screening for PSA is also associated with a high risk of over-diagnosis and over-treatment based on findings on complementary diagnostic prostate biopsies. In a recent paper, Duijvesz et al. (2010) focused on the potential role of exosomes as novel biomarkers for PCa. They showed that exosomes, being small vesicles (50–100nm) secreted by almost all tissues, represent their tissue origin. Purification of prostate- and PCa- derived exosomes allow the profiling of exosomes as a promising source of protein and RNA biomarkers for PCa.

In a further interesting development, dendritic cell (DC)-derived exosomes have been shown to allow targeted RNAi delivery to the brain after systemic injection, demonstrating the first proof-of-concept for the potential of these naturally occurring vesicles as vehicles of drug delivery with the added advantages of in vivo safety and low immunogenicity. Ultimately, exosome-mediated drug delivery promises to overcome important challenges in the field of therapeutics, for example as delivery of drugs across otherwise impermeable biological barriers, such as the blood brain barrier, and using patient-derived tissue as a source of individualized and biocompatible therapeutic drug delivery vehicles (Lakhal and Wood 2011). Indeed, NTA has already been used in such work (Montecalvo et al., 2011).

Ezrin et al. (2012) have characterized blood derived exosomes from glioblastoma patients following exogenous loading with Gliolan to determine if tumors loaded with Gliolan could shed circulating microparticles containing 5-ALA-derived fluorophores as a novel tool to endogenously label, track, and quantify tumor-derived microparticles. Microparticles were isolated by gel filtration and characterized using NTA and bicinchonic acid assay (BCA) for microparticle size/number and protein content, respectively. Endogenous fluorescence from the microparticles was also assessed using NTA in the fluorescence detection mode (λex = 405 nm and λem > 430 nm). Preliminary results suggested that microparticles (mode diameter of 50-100 nm) were present at a concentration of about 1011 particles/mL of serum (protein content = 283.5 + 47 ug/ml of sera). They claimed that this was the first evidence that a small molecule drug following oral dosage can be absorbed by tumor cells, enzymatic ally modified, and shed back into circulating microparticles within hours of dosing and that this direct measure of tumor function affords multiple therapeutic and drug development implications for this novel “liquid biopsy” procedure. Ezrin (2013) has also, using NTA data, patented a pharmaceutical composition comprising 5-aminolevulinic acid (5-ALA) to detect the level of conversion of 5-ALA to protoporphyrin IX (PPIX) associated with brain-derived microparticles in a biological sample from the subject, thereby detecting WHO grade III or grade IV brain tumors.

The subject of microparticles and exosomes as biomarkers has been recently reviewed by Burger et al. (2013) in which they summarize approaches for the detection of microparticles and examine novel concepts relating to the formation of microparticles and their biological effects and well as the evidence for microparticles as both biomarkers of, and contributors to, the progression of disease.

Morton et al. (2012a) have described microvesicles as indicators of cancer progression using biomarkers in a further methodology belying their more familiar role in proteomics and genomics. Balaj (2012) has carried out BEAMing qRT-PCR analysis of mutant IDH1 mRNA in tumor microvesicles in a diagnostics context and has carried out a direct comparison of glioblastoma large oncosomes and exosomes/ microvesicles reporting that two EVs populations from glioblastoma U87 and HUVEC cells (separated by differential centrifugation and sucrose gradient and compared by NTA, cryoEM, immunofluorescence (IF), qRT-PCR, western blotting and mNMR), both produce more EMs than large oncosomes suggesting that large oncosomes may better represent the content of tumor cells.

Because exosomes carry a range of membrane and cytosolic proteins comprising endosomal compartment and transport/fusion proteins, and because specific proteins indicative of cell type and functional state and are ubiquitously found on exosomes from different biological samples and referred to as common identification markers, their expression can vary across exosomes from different sources. Following capture on an ExoTEST (2013) plate by incubation with precleared plasma samples, different capture/detection antibodies as well as NTA analysis were used to define the correspondence between expression profiles and number of exosomes in each sample (Guazzi et al., 2013). They concluded in this study that, when used in a quantitative immunoassay, some commonly acknowledged exosomal proteins can act as specific markers of tumor type and stage due to variations in overall exosome number or altered protein levels.

Lunaavat et al. (2013) used NTA in their comparison of RNA profiles between microvesicles and exosomes derived from melanoma cells, while Polanco et al. (2013) undertook a proteomic study of prostate cancer cell-derived microvesicles for identification of therapeutic targets.

In order to characterize exosomes from the saliva of oral cancer (OC) patients isolated by different methods and to compare them to exosomes from the saliva of healthy individuals (HI), Zlotogorski et al. (2013) showed that exosomes isolated from saliva of cancer patients differ from those of healthy individuals. Exosomes were isolated by two methods: chemical – Exoquick® (EQ, System Biosciences, CA, USA), and physical – ultracentrifugation (UC, 120,000 g for 3 hours) and isolated exosomes were characterized by ELISA, NTA, EM and AFM. ELISA performed on saliva of OC patients using the exosomal marker CD63 presented higher concentrations compared to HI saliva by both methods, EQ and UC. These results were confirmed in the examination of OC saliva with NTA, which revealed a higher concentration of exosomes that were of a larger size compared to HI saliva. They concluded that exosomes isolated from the saliva of OC patients seem to differ from those of HI saliva in their concentration, distribution and size and that these differences should be further explored for diagnostic and therapeutic purposes. Similarly, Yoshioka et al. (2013) undertook a comparative marker analysis of extracellular vesicles in different human cancer types. To confirm the presence of EVs in the preparations, they pointed out that researchers have utilized so-called EV marker proteins, including the tetraspanin family proteins and such cytosolic proteins as heat shock 70 kDa protein 4 (HSP70) and tumor susceptibility gene 101 (TSG101). However, studies have shown that some EV markers are not always present in all EVs, which not only complicated the identification of EVs but also precluded the quantitative evaluation of EV proteins. Thus, it was strongly required to explore well-conserved EV marker proteins that were present at similar levels, regardless of their tissue or cellular origin. In their study, they compared the presence of 11 well-known EV marker proteins by immunoblotting using EVs isolated from 4 human prostate cell lines and 5 human breast cell lines, including cancer cells with different phenotypes and found that all the tested EVs were positive for CD9 and CD81, with similar abundance that was irrespective of the EV origin. In contrast, other EV marker proteins, such as TSG101, Rab-5b and CD63, were detected in an inconsistent manner, depending on the origin of the EVs. Thus, they proposed that the detection of CD9 and/or CD81 should ensure the presence of EVs.

NTA was used to determine exosome size distribution and concentration in an examination of quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells which reveal differential expression of EMT factors (Jeppesen et al., 2013). Similarly, NTA-identified urinary exosomal microRNAs in incipient diabetic nephropathy was studied by Barutta et al. (2013) in which they showed that urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication.

Having previously demonstrated that the scaffolding protein plectin is a robust biomarker for pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive malignancies, Shin et al. (2013) reported an unexpected gain of function for plectin due to mislocalization in pancreatic cancer, DLS analysis revealing that the mean size of PDAC particles was 63.53 ± 4.46 nm in diameter and that the mode size was 50.75 nm. NanoSight analysis showed similar results (57.67 ± 20.00 nm). They proposed that it is now clear that this PDAC biomarker plays a role in PDAC, and further understanding of plectin’s contribution to PDAC could enable improved therapies.

Cancer Studies in Exosomal Intracellular Communication involving NTA

Given it is now accepted that a) microvesicles (MVs) and exosomes play a pivotal role in cell-to-cell communication and that b) tumor cells have specifically been demonstrated to release such membranous structures, described as microvesicles or exosomes depending on specific characteristics, including size and composition, and that c) these cell-derived vesicles can exhibit an array of proteins, lipids, and nucleic acids derived from the originating tumor, it is now recognized that these vesicular components are critical conveyers of intercellular communication and mediate many of the pathological conditions associated with cancer development, progression, and therapeutic failures. Accordingly, the role that exosomes and microvesicles play in cancer is currently one of the most important subjects of study and most frequently reported use of NTA in the analysis of exosomes. Following earlier disclosures that tumor microvesicles contain retrotransposon elements and amplified oncogene sequences (Balaj et al., 2011) and reviews on brain tumor microvesicles and their role in intercellular communication in the nervous system (van der Vos, 2011) and a review on historical and perspectives of the cell biology of exosomes (Cicero and Raposo, 2012), a significant amount of research has been carried out recently in the role that exosomes play in cellular communication and the potential impact this has on tumor genesis and progression. NTA has, as one of the few techniques by which these small structures can be detected, sized and concentration measured, found itself at the centre of a wide range of such studies.

Given the production of microvesicles (MVs) appears to be closely linked to activation of the cell-death programme, apoptosis, but the functional attributes of MVs released from apoptotic cells have not been defined in detail, Willems et al. (2012) hypothesized that MVs produced by apoptotic tumor cells are involved in conditioning of the tumor microenvironment, a critical aspect of tumor evolution and progression using NTA to measure concentration of MVs.

Cicero and Raposo (2012) have reviewed the general area of the cell biology of exosomes from a historical perspective and Taylor and Cicek (2012) have discussed how circulating cell-derived vesicles mediate tumor progressions. In the latter report it was suggested that through the expression of components responsible for angiogenesis promotion, stromal remodelling, signaling pathway activation through growth factor/receptor transfer, chemoresistance, and genetic intercellular exchange, tumor exosomes/microvesicles could represent a central mediator of the tumor microenvironment.

Attempting to define the mechanisms by which fetuin-A mediates the adhesion of tumor cells, Watson et al. (2012) used the concentration measuring capability of NTA to demonstrate that the secretion of exosomes increases as a function of intracellular calcium ion concentration. Graner (2012) has ebulliently reviewed the role that extracellular vesicles play in cancer and emv-target cell interactions and Arigi et al. (2012) described the proteomic profiling and characterization of human endometrial cancer cell-derived extracellular microvesicles.

The secretion, composition and biological activity of tumor derived exosomes were shown to be regulated by heparinase (Thompson et al., 2012) and King et al. (2012) have demonstrated the hypoxic enhancement of exosome release by breast cancer cells. In this study, proposing that hypoxia is an important feature of solid tumors which promotes tumor progression, angiogenesis and metastasis, potentially through exosome-mediated signaling, King and his co-workers showed that exposure of three different breast cancer cell lines to moderate (1 % O2) and severe (0.1 % O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting.

As outlined earlier (Lee et al. 2011), exosomes are thought to have a significant role in cell signaling and as such exhibit a strong relationship to disease progression. Because extracellular organelle terminology is often confounding, with many preparations reported in the literature being mixtures of extracellular vesicles, there is a growing need to clarify nomenclature and to improve purification strategies in order to discriminate the biochemical and functional activities of these moieties and that NTA is a potentially useful method for exosome detection and enumeration (Mathivanan et al., 2010).

A number of studies have begun to utilize NTA for the detection and concentration measurement of exosomal sized microvesicular structures to investigate their role in intracellular communication, specifically in the study of prostasomes, which are exosome related structures released by prostate acinar epithelial cells (Ronquist et al., 2012); transcriptomics profiling of hepatic extracellular microvesicles (Falcon-Perez et al., 2012); exosomal transfer of RNA based signals between the hematopoietic system and the brain in response to inflammation (Oesterwind et al., 2012); Syndecan–syntenin–ALIX regulation of the biogenesis of exosomes (Baietti et al., 2012); and the induction of phosphatidylserine exposure and microvesicle formation in erythrocytes by an excipient in the conventional clinical formulation of paclitaxel (Vader et al., 2012). Most recently, van Balkom (2012) has described recent developments in exosome signaling in endothelial function and angiogenesis.

Shao et al. (2012) used protein typing of circulating microvesicles to allow real-time monitoring of glioblastoma therapy and employed NTA to obtain size, size distribution (log normal) and number of MVs to develop a dedicated microfluidic chip, labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system which exhibited a much higher detection sensitivity and whicy could differentiate glioblastoma multiforme (GBM) microvesicles from nontumor host cell–derived microvesicles.

It is known that one component of the adaptive stress response is that innate immunity is primed by circulating endogenous danger-associated molecular patterns (DAMPs). Extracellular heat shock protein 72 (eHsp72) is a DAMP that is upregulated intracellularly after acute stress, but its mechanism of release is unknown. In a study on the role that exosome-associated eHsp72 plays following exposure to acute stress. Beninson et al. (2012) used NTA and EM to confirm successful exosome isolation and reported that exposure to an acute stressor increased exosome expression of eHsp72, but not other stress-inducible proteins (IL-1β and IL-6). Additionally, exosomes from stressed, but not control, rats facilitated in vivo bactericidal inflammatory response (p < 0.05) and an in vitro LPS-evoked inflammatory response (p < 0.05). These data suggested that exposure to stress can alter the proteomic composition of circulating exosomes, thereby enhancing the innate immune response. Wallner (2012) has analyzed extracellular vesicle (EV) mediated signaling in an in vitro model of atherosclerotic lesions using NTA to calculate that low density lipoprotein-induced granulocyte microparticles are produced equally over the size range 100-400nm though the LDL particles might have exhibited, in part at least, a common size range.

The role played by exosomes in prostate cancer and their analysis by NTA has been the subject of many recent research projects (Kharaziha and Panaretakis, 2012). Deep et al. (2013) have shown that exosomes secreted under hypoxia enhance invasiveness in prostate cancer cells. Human PCA LNCaP cells were exposed to hypoxic (1% O2) or normoxic (20% O2) conditions. Media was collected and exosomes, secreted under hypoxic and normoxic conditions, were isolated by ultracentrifugation and precipitation (ExoQuick) methods. Hypoxic and normoxic exosomes were characterized by NTA and EM to confirm the size/structure of the exosomes. Ramteke et al. (2013) from the same Group, showed also that exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules. NTA revealed that ExoHypoxic have smaller average size as compared to ExoNormoxic. This supported other data which suggested that hypoxic exosomes are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes, thereby promoting prostate cancer aggressiveness. Chowdhury et al. (2013) reported that prostate cancer exosomes alter the fate of mesenchymal stem cell differentiation. Their exosomes were purified using a sucrose cushion and characterized by Western blot, NTA and flow cytometry of exosome-coated beads (flow cytometry being unable to detect the exosomes directly). Hosseini-Beheshti et al. (2013) showed prostate cancer derived exosomes could promote orostate cancer progression via activation of the ERK cell signaling pathway and Kim et al. (2013) reported that microvesicles shed from DIAPH3-silenced, amoeboid prostate cancer cells enhanced growth of other tumor cells and suppressed proliferation of immune cells. NTA was used to assess the degree of MV shedding from DIAPH3 knockdown DU145 cells (DIAPH3 KD) and controls. They showed that DIAPH3 KD cells secreted about 2-fold more MVs than control DU145 cells suggesting that MVs produced in mCRPC by the loss of DIAPH3 expression may condition the tumor microenvironment, by activation of cancer cells and suppression of tumor-infiltrating immune cells. Ogorevc et al. (2013) discussed the role of extracellular vesicles derived from bladder cancer cells in intercellular communication using NTA to help further investigate the mechanisms of intercellular transfer of bladder cancer cell-derived EVs to non-cancerous and cancerous cells. Finally, Karmate et al. (2013) have for the first time shown the expression of membrane receptors such as EGFR in exosomes derived from prostate cancer cell lines, LNCap xenograft serum and patient plasma/serum. Exosomes isolation was validated by TEM, expression of exosomal markers and NTA and their study also revealed that a sucrose-assisted centrifugation method was superior for exosomes isolation as compared to ExoQuick.

Melanoma cell lines have been shown to release the stress inducible protein 1 via extracellular vesicles (Dias et al., 2013) in which size and concentration of EVs were evaluated using NTA and TEM while Burdek et al. (2013) have studied the effect of human melanoma exosomes on the function of antigen-specific CD8+ T cells. In a related study, Wang et al. (2013) demonstrated a positive role for bone marrow stromal cell (BMSC)-derived exosomes in the facilitation of multiple myeloma (MM) cell survival through inhibition of the JNK pathway, using NTA to determine the size of exosomes derived from naïve BMSCs, 5T33 BMSCs and 5T33MMvt cells. Similarly, Boswell et al. (2013) have undertaken miRNA expression profiling and proteomic analysis of circulating exosomes from multiple myeloma patients using NTA for the detection and sizing of their exosomes.

Exosomes have also been implicated in lung cancer though their contribution is still largely unknown. Huang et al. (2013) evaluated the roles of nanometer sized extracellular vesicles on lung cancer progression investigating the roles of EVs in lung cancer using a malignant pleural effusion (MPE) model, in which soluble components play important roles. EVs were isolated using both ultra-centrifugation (UC) and ultra-filtration (UF) methods, and evaluated by TEM, NTA and Western blotting, as three techniques which are now commonly used in exosomal research. TEM and NTA revealed that EVs isolated using both methods were closed vesicles of nanometer size. Their results demonstrated that the UF method is ideal for isolating tumor-associated EVs from both cell culture and clinical samples and that lung cancer-associated EVs may contribute to cancer progression by triggering oncogenic signals with the IL-6 and VEGF cargos. Wong et al. (2013) showed extracellular vesicles (EVS) from activated fibroblasts promoted lung fibrotic remodelling, the EVs being characterized by EM and NTA.

Given the central role that exosomes and EV appear to play in a wide variety of disease conditions and the fact that NTA is proving to be an extremely useful tool in their characterization and enumeration, these have been many reports of such work over the last year. NTA has been used routinely in studies on their mediation of oligodendrocyte–neuron communication by neurotransmitter-triggered transfer of exosomes (Frühbeis et al., 2013); possible defects of communication between neuron and Schwann cells (Zhu et al., 2013); the over expression of a single oncogene altering the proteomic landscape of microparticles (Amorim et al., 2013); the increased abundance of active lysyl oxidase-like-2 on the surface of exosomes by endothelial cells following stimulation with collagen-I or hypoxia (de Jong et al., 2013); the potential of B-cell derived exosomes to activate naive B Cells (Gutzeit et al., 2013).

The release of humoral factors between cancer cells and the microenvironmental cells is critical for metastasis. However, the roles of secreted miRNAs in non-cell autonomous cancer progression against microenvironmental cells remain largely unknown. Kosaka et al. (2013) have recently demonstrated that the neutral sphyngomyelinase 2 regulates exosomal miRNA secretion and promotes angiogenesis within the tumor microenvironment as well as metastasis using NTA to measure their exosomes. NTA has also been used to measure EV emission by NB4 cells derived from an acute promyelocytic leukaemia (APL) patient with t(15;17), the reciprocal translocation between chromosomes 15 and 17 which is a major causative agent in APL (Fang et al., 2013)

Gastrointestinal Stromal Tumors (GIST) are the most common mesenchymal tumors of the digestive tract and several studies have shown that tumor cells produce and utilize exosomes, transporting various cargo reflective of the cells of origin, to communicate with and alter the surrounding microenvironment. In a recent study, Atay et al. (2013) demonstrated for the first time that GIST-derived exosomes, detected by NTA and flow cytometry, could induce a GIST-like phenotype in human smooth muscle cells via the transfer of mutant KIT.

Iglesias et al. (2012) have shown that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured mutant fibroblasts or proximal tubular cells from cystinosis patients and that paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA as identified and confirmed by NTA following ultracentrifugation. In work reflective of the studies carried out by the Oxford researchers described above, Alam et al. (2012) have reported that immunomodulatory molecules are secreted from the first trimester and term placenta via microvesicles. Wang et al. (2013) have subsequently shown that mesenchymal stem cell-derived exosomes interact with monocytes and mesenchymal stem cells.

Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes had not been studied. Garnier et al. (2012) have now shown that cancer cells can indeed be induced to express mesenchymal phenotype release exosome-like extracellular vesicles carrying tissue factor using NTA to measure the number of size and size distribution of these EVs. That exosome uptake depended on ERK1/2-heat shock protein 27 signaling and that lipid raft-mediated endocytosis was negatively regulated by caveolin-1 was described by Svensson et al. (2013), the presence and purity of isolated exosomes again being confirmed by TEM with size measurement being carried out by NTA. Exosome secretion from multivesicular endosomes, as quantitated by NTA, was enhanced by specialized invasive actin structures called invadopodia and has been shown to drive invasive behavior of cancer cells (Hoshino et al., 2013).

Davila et al. (2013) used NTA and DLS to analyze the size distribution of particles in conditioned medium (CM) or plasma fractions in their research into microparticle association and heterogeneity of tumor-derived tissue factor (TF) in plasma. In attempting to find out whether it was important for coagulation activation, they found that particles <0.1μm and the supernatants of both CM and plasma gained TF activity after addition of exogenous phospholipids. While TF was found in MP-free CM supernatants, it was also present in CM and plasma pellets. They concluded that tumor-derived particles <0.1μm and non-sedimentable TF are, or can, become procoagulant in the presence of phospholipids and may contribute to the procoagulant potential of circulating TF.

Arguing that the bioactivity of exosomes resides not only in their protein and RNA contents but also in their lipidic molecules, Record et al. (2013) have proposed exosomes as representing new vesicular lipid transporters involved in cell-cell communication and various path physiologies. Because exosomes can vectorize lipids such as eicosanoids, fatty acids, and cholesterol, and their lipid composition can be modified by in-vitro manipulation. Because they also contain lipid related enzymes such that they can constitute an autonomous unit of production of various bioactive lipids, the lipid content of circulating exosomes could be useful biomarkers of lipid related diseases.

Finally, Tadokoro et al. (2013) have shown that exosomes derived from hypoxic K562 leukaemia cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 hours and quantitated by NTA enhanced tube formation in endothelial cells while Haga et al. (2013) demonstrated that PDGF-BB induces apoptotic priming of cancer-associated fibroblasts in cholangiocarcinoma. EVs were isolated by differential centrifugation, verified using EM and quantitated using NTA. Their results allowed them to conclude that EV transfer from KMBC increases fibroblast-like activity and selectively alters mRNA expression and secretion of IL6 and other cytokines/chemokines by mesenchymal stem cells that can, in turn, alter KMBC proliferation. Thus, tumor cells can "educate" MSC to modulate the microenvironment and thereby facilitate tumor growth. This was claimed to be a previously undescribed and unique mechanism by which tumor cells can modulate the microenvironment and facilitate tumor growth and offered new opportunities for therapeutic intervention in cholangiocarcinoma and possibly other cancers.

Exosomal Cancer Therapeutic Potential involving NTA

Most recently, Beckler et al. (2012) have carried out a proteomic analysis of exosomes from mutant KRAS colon cancer cells to identify intercellular transfer of mutant KRAS which occur in 30-40% of colorectal cancers and NTA allowed them to enumerate the number of exosomes per μg protein.

Tumor-derived exosomes are emerging mediators of tumorigenesis and Peinado et al. (2012) showed, by using NTA to analyze exosomes isolated from fresh plasma derived from healthy controls and melanoma subjects, that exosome production and transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process. Itoh et al. (2012) demonstrated that prostate cancer cells in vitro released microvesicles into the culture medium, which was shown by electron microscopic study and NTA for the first time, and Huang et al. (2013) recently reviewed extracellular miRNAs embedded inside circulating microvesicles as biomarkers for diagnosis and prognosis of disease, or even as therapeutic agents for targeted treatment. They summarized recent publications involving extracellular miRNA profiling studies in three representative urologic cancers, including: prostate cancer, bladder cancer, and renal cell carcinoma, focussing on the diagnostic, prognostic, and therapeutic potential of these miRNAs in biological fluids such as serum, plasma, and urine which they had concluded were present at concentrations of 0.8 × 108 to 13.4 × 108 /mL of stocked serum or plasma.

Baj-Krzyworzeka et al. (2012b) have focussed on the interactions of tumor-derived microvesicles (TMV) with human monocytes, which are precursors of tumor associated macrophages. Their work has shown that monocytes pre-exposed to TMV and restimulated with tumor cells show M2-like cytokine secretion and that TMV significantly modulate biological activity of monocytes and may affect their function during tumor progression, thus suggesting TMV mimicks the effect of tumor cells on monocytes. They postulate that TMV should be considered as a modulator of monocyte/macrophage functions in the tumor bed and in peripheral blood.

Mizrak and his co-workers reported the first use of a therapeutic mRNA/protein via microvesicles (MVs), analyzed by NTA, for treatment of cancer (Mizrak et al., 2012). They first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein–cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (Schwannomas) in an orthotopic mouse model. They subsequently demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into Schwannomas led to regression of these tumors upon systemic treatment with the prodrug 5-fluorocytosine, which is converted within tumor cells to 5-fluorouracil – an anticancer agent. Excitingly, these studies suggest that MVs can serve as novel cell-derived “liposomes” to effectively deliver therapeutic mRNA/proteins to treatment of diseases.

In their work on determining the quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells, Choi et al. (2012) used NTA to measure the diameters of 500ng/ml extracellular microvesicles in PBS while Fonsato et al. (2012) showed that the delivery of selected miRNAs by MVs (confirmed by NTA to have been successfully isolated from stem from human liver cells) may inhibit hepatoma tumor growth in SCID mice and stimulate apoptosis. Hepatocellular carcinoma metastasis and the role export mechanisms played by exosomal microRNAs in this disease were addressed by Janas et al. (2013) while Takahashi et al. (2013) showed that chemoresistance in hepatocellular cancer, a common problem, was mediated by an increase in long non-coding RNA-ROR in tumor cell exosomes. Exosomal content was verified by density gradient centrifugation, NTA and EM in malignant (HepG2, Hep3B, HepG2ST, Huh7 and PLC) or non-malignant hepatocytes.

Bruno et al. (2012) have shown that microvesicles derived from human bone marrow mesenchymal stem cells inhibit tumor growth. The 145nm (NTA-measured) microvesicles, when administered intra-tumor into established tumors generated by subcutaneous injection of these cell lines in SCID mice, significantly inhibited tumor growth. Furthermore, MVs from human mesenchymal stem cells inhibited in vitro cell growth and survival of different tumor cell lines and in vivo progression of established tumors, suggesting a future role in tumor treatment. Penfornis et al. (2013) similarly described exosome-mediated tumor stromal support of mesenchymal stem cells.

Huan et al. (2013) reported that hypoxia alters exosome release and RNA incorporation by acute myeloid leukaemia cells while Katsuda et al. (2013) worked on the potential role of osteosarcoma-derived exosomes in pre-metastatic niche formation in the lung. Wong et al. (2013) showed that therapeutic treatment of glioblastome modulated extracellular vesicles dynamics. NTA was used in all these cases to determine exosome size and concentration.

T-cell tolerance of allergic cutaneous contact sensitivity induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. In order to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles, Bryniarski et al. (2013) induced T-cell tolerance by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Using NTA, tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8+ suppressor T cells that were not regulatory T cells. Nonsuppressive nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150 thus showing, for the first time, that T-cell regulation through systemic transit of exosome-like nanovesicles could deliver a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains. In a related study, Bryniarski et al. (2013) showed antigen-specific, antibody-coated, exosome-like nanovesicles could deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity thereby highlighting a possible link to inflammatory skin diseases.

TNF-related apoptosis-inducing ligand (TRAIL) is a protein functioning as a ligand that induces the process of cell death and has been shown to kill in vitro a wide variety of tumor cells with minimal effects on normal cells but has so far shown limited efficacy in vivo. In contrast, recent reports have shown that significant apoptosis can be observed both in vitro and in vivo when TRAIL is expressed on the cell membrane (mTRAIL). By innovatively delivering the bioactive proapoptotic TRAIL through its expression by extracellular vesicles (EVs), Buttiglieri et al. (2013) have demonstrated potent in vivo anti-tumor activity of extracellular vesicles isolated from genetically engineered primary mesenchymal stromal cells, thus paving the way to the use of EVs for therapeutic purposes. NTA revealed that EVs had a variable size, up to approximately 400 nm in diameter, with a predominant peak at 273 nm. Similarly, Huber et al. (2013) employed mTRAIL-armed exosomes as a novel and effective anti-tumor therapy which represented a more efficient tool for delivering death signals to the tumor, as compared to soluble TRAIL. The poorly immunogenic erythroblastoid cell line K562 was stably transduced with a lentiviral vector containing membrane-bound TRAIL. Subsequently, TRAIL-transduced K562 cells secreted significant amounts of highly mTRAIL-positive exosomes which induced relevant apoptosis in SUDHL4 (80%) and KMS11 (40%) haematological cancers and melanoma cell lines. Exosomes were isolated by differential centrifugations. TRAIL expression and exosomal nature were assessed by flow cytometry, ELISA, electron microscopy, and NTA.

Tian et al. (2013) described another example of a targeted drug delivery vehicle with low immunogenicity and toxicity for cancer therapy, namely a doxorubicin delivery platform using engineered natural membrane vesicle exosomes. Purified exosomes from mouse immature dendritic cells were loaded with doxorubicin via electroporation, with an encapsulation efficiency of up to 20%. The αv integrin-specific iRGD peptide targeted exosomes showed highly efficient targeting and doxorubicin delivery to αv integrin-positive breast cancer cells in vitro as demonstrated by NTA, confocal imaging and flow cytometry. The intravenously injected targeted exosomes delivered doxorubicin specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity.

Bretz et al. (2013), in showing that body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via TLR signaling, suggested that exosomes (confirmed by NTA as 100-300nm in diameter) triggered TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. They concluded that this process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases.

Finally, the role that exosomes may play in the fight against infection has become of interest recently and NTA has been used routinely in visualizing and quantifying these structures. Szabo et al. (2013) have shown that Exoquick-purified and NTA-analyzed exosomes in Hepatitis C virus (HCV) infection mediate CD81-independent transmission and are rich in Ago2-miR122-HSP90 complexes and, as such, can significantly suppress exosomal transmission of HCV infection, suggesting their use when treatment failure occurs with anti-HCV immune therapies. Similarly working with HCV and with HIV, Gupta et al. (2013) demonstrated that activated monocytye-derived exosomes mediate miRNA transfer to neural cells with implications for neurodysfunction in HIV/HCV coinfection. Hu et al. (2013) reported that release of luminal exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum and contributes to TLR4-mediated epithelial shuttling of antimicrobial peptides (cathelicidin-37 and beta-defensin 2) in antimicrobial defence, thereby revealing a new arm of mucosal immunity relevant to antimicrobial defence. A time-dependent apical exosome release was detected in infected H69 monolayers by NTA and other techniques. Finally, Twu et al. (2013) showed that Trichomonas vaginalis, a common sexually transmitted parasite that colonizes the human urogenital tract, produces exosomes (sized by NTA) that deliver cargo to host cells and mediate host:parasite interactions. They suggested that exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells and that these studies are the first to reveal a potential role for exosomes in promoting parasite:parasite communication and host cell colonization. The T.vaginalis microvesicles had physical and biochemical properties similar to mammalian exosomes.

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