Systemically Circulating Viral and Tumor-Derived MicroRNAs in KSHV-Associated Malignancies
[['MicroRNAs ( miRNAs ) are small , non-coding RNAs that are capable of fine-tuning gene expression through translational repression and/or mRNA degradation .', 'In the past , miRNAs have emerged as important regulators in nearly every cellular process , but perhaps the largest biological consequence of miRNA dysregulation is in cancer [1] , [2] , [3] , [4] , [5] , [6] .', 'The relationship between intra-tumor miRNA signatures and cancer progression has been well established , leading to the discovery of specific miRNAs or miRNA clusters that modulate gene expression in cancer [7] , [8] , [9] .', 'We and others have shown that miRNA signatures can classify tumors into distinct classes and are predictive of disease outcome [3] , [4] , [6] , [10] , [11] .', 'In our prior study , we found that the host miRNA profile differed depending on the degree of transformation among cells , even though all samples were infected by the same virus and thus expressed similar levels of viral miRNAs [6] .', 'This suggests that host miRNA profiles impart information about viral infection above that provided by detecting the presence of the infectious agent .', 'MiRNA regulation is complex in malignancies associated with viral infection such as herpesvirus-associated cancers [2] , [6] , [12] , [13] .', 'Viral infection can trigger changes in the miRNA profile through the expression of viral genes that modulate the host miRNA repertoire .', "Some viruses such as Kaposi's sarcoma-associated herpesvirus ( KSHV ) and Epstein-Barr Virus ( EBV ) in addition encode their own miRNAs , which fine-tune host gene expression to promote latent viral persistence , immune evasion , and tumor progression [8] , [9] , [14] , [15] , [16] , [17] .", 'These viral miRNAs are often expressed within the tumor and can reveal important information regarding viral latency and disease progression [18] .', 'Furthermore , recent studies have highlighted important functions of the viral miRNAs in regulation of the viral life cycle , immune evasion and angiogenesis through validated mRNA targets [7] , [14] , [19] , [20] , [21] , [22] , [23] .', 'In KSHV-associated cancers , the KSHV miRNAs can account for as much as 20% of all mature miRNA species within a cell and are highly conserved among isolates ( Figure S1 and [12] , [14] , [17] ) .', "KSHV is the etiological agent of Kaposi's sarcoma ( KS ) , the most common AIDS-defining cancer worldwide [24] .", "KSHV is also associated with the B cell lymphoma Primary Effusion Lymphoma ( PEL ) and with the plasmablastic variant of Multicentric Castleman's Disease ( MCD ) .", 'Despite the availability of Highly Active Anti-Retroviral Therapy ( HAART ) , KS continues to occur in the US and worldwide .', 'Treatment of KS remains a challenge and stable , minimally invasive biomarkers for diagnosis are lacking [25] , [26] .', 'Therefore , the discovery of plasma miRNA biomarkers for KSHV-associated malignancies could improve diagnostics through early detection and could influence treatment through non-invasive monitoring of tumor responses .', 'MiRNA biomarkers can be sampled from blood , saliva , or other bodily fluids , offering a feasible diagnostic test even in resource-poor regions such as the “KS belt” in sub-Saharan Africa [24] , [27] .', 'Viral microRNAs are the most attractive candidate biomarker because of their specificity for KSHV .', 'However , a combination of viral microRNAs with cellular microRNA biomarkers is even more useful , as it may help differentiate among stages of KS progression or response to therapy and as it can identify cellular microRNAs that are common among KS and other cancers .', 'We previously determined the cellular and viral miRNA profile in KS tumor biopsies as well as in PEL and found that the expression of viral miRNAs varies with disease state [3] , [4] , [6] .', 'In addition to the viral miRNAs , key cellular miRNAs are involved in KSHV transformation and KS progression [8] , [9] , [28] , [29] .', 'The detection of circulating miRNAs in plasma , serum and other bodily fluids suggests their utility as minimally invasive biomarkers for cancer diagnostics [11] , [30] , [31] , [32] , [33] , [34] .', 'These circulating miRNAs are unusually stable', '( i ) due to their packaging in microvesicles or exosomes ,', '( ii ) due to their RNA folding and size and/or', '( iii ) due to their presence in Ago-containing ribonucleic acid:protein ( RNP ) complexes [32] , [34] , [35] , [36] .', 'At this point it is unclear which of these mechanisms is the most efficient .', 'Evidence suggests that all three mechanisms contribute to diagnostic utility by increasing miRNA stability .', 'There are a variety of vesicles that are secreted from cells , each with slightly varying content and surface marker composition .', 'Microvesicles can range in size from 30 nm–1000 nm and each follow different pathways of biogenesis ( reviewed in [37] , [38] , [39] ) .', 'Recent studies have additionally shown that microvesicles from tumor cells may have altered morphology , size and surface markers , including the expression of tumor antigens compared to microvesicles that are released from non-tumor cells [40] , [41] , [42] , [43] .', 'MiRNAs have been detected in microvesicles , exosomes and/or nanovesicles .', 'This study refers to these vesicles collectively as exosomes based on common surface marker expression and morphological characteristics .', 'Transfer of exosomes and their contents from tumor cells to surrounding , uninfected cells may be an important form of cellular communication and has been demonstrated in cell culture models , for instance in EBV-associated cancers [44] , [45] .', 'Additionally , exosomes may provide a means of paracrine signaling from virally infected cells to adjacent , non-permissive cells [46] .', 'This study attempts to bridge the gap between clinical samples and cell culture models .', 'To do so we compared the detailed , circulating miRNome of KS in clinical human samples and in KS mouse models [47] , [48] , [49] .', 'This confirms the presence of circulating KS and KSHV-specific miRNAs in vivo in the context of KSHV infection .', 'Multiple KSHV miRNAs and members of the miR-17-92 cluster of cellular miRNAs were detected within patient exosomes .', 'These circulating miRNA signatures may serve as a new mechanism of paracrine signaling for mediating KSHV pathogenesis and may represent a reservoir for novel biomarkers .'], ['To date , most studies on viral exosomes have used tissue culture models of infection .', 'To expand on these studies , we utilized a series of clinical samples and two novel robust mouse models of KSHV pathogenesis [47] , [48] , [49] .', 'The sample groups and number of samples included in each group are outlined in Table S1 .', 'Briefly , human plasma from healthy , KSHV-negative controls or from AIDS patients with either KS or a non-KS malignancy was used to isolate exosomes .', 'The HIV viral load and CD4+ T cell counts were similar in both KS and non-KS malignancy groups ( data not shown ) .', 'KS tumor biopsies and primary PEL pleural fluid were also included and served as positive controls for the presence of KSHV compared to control human plasma .', 'We also used two mouse models previously characterized in our lab [47] , [48] , [49]: the 801 latency locus transgenic mouse model which expresses all viral miRNAs in B cells [50]; and a xenograft model using TIVE L1 tumor cells , which maintain KSHV [48] .', 'These cells are xenografted into SCID mice , which results in robust and reproducible tumor formation [48] .', 'H&E staining revealed similar phenotypes of KS and our TIVE xenograft mouse model while both of these differed from the staining observed in PEL ( Figure S2 ) .', 'The KSHV-TIVE model [48] represents another instance of extended yet incomplete KSHV lytic transcription , as recently demonstrated in KSHV-infected lymphatic endothelial cell cultures under puromycin selection [51] and previously in KSHV-infected mouse endothelial cells [52] .', 'Similarly , a KSHV cell line model of transformed rat mesenchymal precursors yields some lytic gene expression but with minimal amounts of virions produced [53] .', 'The KSHV-TIVE endothelial cell model maintains KSHV in the absence of selection and like other long-term KSHV-infected endothelial cell cultures they remain tightly latent .', 'Neither sodium butyrate nor exogenously provided RTA/Orf50 are able to induce infectious virus production ( R . Renne , personal communication ) or complete , genome-wide lytic transcription in TIVE L1 cells [48] .', 'Subcutaneous implantation into mice can activate many viral genes , although these represent only approximately half of the genes turned on during lytic reactivation in PEL cells and are insufficient to produce infectious virions .', 'A similar , abortive lytic expression profile has been observed in KSHV-infected human TIVE-L1 cells [48] as well as in KSHV-infected mouse and rat endothelial cells [52] , [53] .', 'This incomplete transcription program is incompatible with virion production and in the case of KSHV-infected LEC has been termed a novel latency program [51] .', 'For this reason , we refer to the TIVE xenograft mouse model as a latent KSHV model due to the lack of virions produced .', 'Exosomes and circulating miRNAs were purified as shown in Figure 1A and detailed in methods .', 'Following purification , total RNA was isolated from each sample group and used for Taqman-based qPCR profiling of the cellular miRNA repertoire ( 754 human miRNAs ) as described [3] , [4] , [54] .', 'Agilent RNA analysis showed that exosomes expressed small RNAs but lacked both 18S and 28S ribosomal RNA ( Figure S3 ) .', 'Figure 1 shows the distribution of miRNAs in different sample subsets ( Figure 1B–E ) .', 'Each boxplot shows the expression levels for the different sample groups .', 'The expression of individual microRNAs are denoted by solid circles .', 'As demonstrated in Figure 1B , the majority of miRNAs present in control human plasma ( KSHV− ) in the supernatant fraction are susceptible to RNase , representing free , circulating miRNAs .', 'These miRNAs are likely not encapsulated in Ago-RNP complexes nor microvesicles [35] .', 'The exceptions were miRNAs miR-16 , miR-195 and miR-197 , which could be detected despite RNase treatment .', 'This RNase resistance of these particular miRNAs is consistent with prior observations [35] .', 'Levels of the C . elegans cel-mir-39 spike-in were abolished ∼16 , 000-fold after RNase treatment and were decreased when incubated with pleural fluid prior to RNA isolation ( Figure S4 ) .', 'This verifies the activity of our RNase treatment and confirms that pleural fluid , like other body fluids , has some intrinsic RNase activity [33] , [34] , [35] .', 'Therefore , the majority of RNAs that are stable in plasma and pleural fluid are likely RNase-resistant and protected within exosomes .', 'In samples enriched for exosomes derived from either control human plasma or mouse serum , we were able to readily detect both human and mouse miRNAs ( Figure 1C ) .', 'Figure 1D denotes the relative expression levels of miRNAs in cells , exosomes and the free , circulating fractions of control human plasma .', 'As expected , exosomal and other circulating miRNAs are detectable but are present at lower levels compared with intracellular miRNAs .', 'MiRNAs are readily detected in all sample types tested including tumor biopsies and exosomes from control plasma or serum and malignant effusions such as pleural fluid ( Figure 1E ) , though the miRNA yield was highest in tumor tissue .', 'Although we used human plasma and serum from mice in the majority of experiments , we also performed miRNA profiling with control mouse plasma .', 'Importantly , we did not observe significant differences in the levels of miRNAs found in plasma versus serum in this study .', 'The comparison of this small subset of miRNAs across the different variables shown in Figure 1B–E did not afford us the statistical power to identify differences among individual miRNAs expressed in these samples .', 'However , these data establish the framework for further analysis and confirms qPCR as a reliable platform for the profiling of miRNAs in a diverse group of clinical samples [54] , [55] .', 'Furthermore , we validate the presence of exosomal miRNAs in cell-free patient plasma and mouse serum .', 'Isolation of exosomes using the Exoquick method has previously been validated to yield similar electron microscopy ( EM ) structures and miRNA array populations as other techniques [56] .', 'Nonetheless , we sought to confirm the presence of exosomes in our patient samples using two independent isolation techniques .', 'Enriched exosomes from the Exoquick protocol revealed similar structures via electron microscopy compared to exosomes enriched by differential ultracentrifugation ( Figure 2A ) .', 'However , while Exoquick samples did contain exosomes ( determined by size and morphological characteristics ) , they yielded images with high background by electron microscopy due to the crowding agent present in the ExoQuick solution .', 'This background was not due to contaminating cellular debris , as high-speed centrifugation and elimination of cellular debris using a sucrose cushion failed to eliminate background in the EM images ( Figure S5 ) .', 'By comparison , differential ultracentrifugation yielded exosomes of similar size and morphology with minimal background ( Figure 2A ) .', 'Patient pleural fluid and BCBL1 cell supernatant yielded exosomes that appeared similar by EM .', 'We therefore pursued the Exoquick method for further study , as these samples required much less sample input , a key benefit when working with clinical samples and mouse models .', 'To further establish the purity of our exosomes , we performed Western blots for previously established exosomal markers including the tetraspanin CD9 , Hsp90 alpha/beta and flotillin [57] , [58] .', 'We first analyzed the expression of flotillin , which is enriched in exosomes [58] , [59] , [60] .', 'Flotillin was expressed in all human and mouse exosome samples ( Figure 2B , C ) but was not present in the supernatant fractions containing freely circulating miRNAs ( Figure 2D , S ) .', 'Hsp90 alpha and beta , which are also highly enriched in exosomes , were detected in PEL cells and pleural fluid-derived exosomes but , as expected , were absent in the supernatant fraction ( Figure 2E–G ) .', 'Finally , we assessed the expression of the tetraspanin CD9 , another exosomal marker .', 'The KS exosomal subgroup ( KS-E ) expressed detectable levels of CD9 whereas the supernatant fraction ( S ) did not express the exosome marker ( Figure 2H ) .', 'As a negative control for exosomes , we used the exosome-depleted supernatant fraction from BJAB cells ( - ) .', 'The mouse exosome samples isolated from serum of control and transgenic ( Tg ) mice also showed robust expression of the CD9 exosome marker , indicating that these samples are enriched for exosomes ( Figure 2H ) .', 'The increased expression observed in the mouse samples most likely reflects the ratio of input used to the total fluid volume present in human and mouse .', 'A mouse has a total blood volume of 1 . 5 mls of which we use 250 ul ( ∼17% ) whereas human blood volume is approximately ∼5 L of which we used 250 ul ( 0 . 005% ) .', 'We also tested the presence of exosomal markers in samples purified by ultracentrifugation and received similar results , validating the use of the Exoquick method for our study ( Figure S6 ) .', 'Furthermore , the ExoQuick method yields more exosomes than other methods tested and uses approximately 100-fold less starting material .', 'Increased expression of KSHV miRNAs correlates with disease state and tumor progression in endothelial cells [3] , [6] .', 'EBV viral miRNAs have been detected in exosomes isolated from cultured lymphoma cell lines , NPC patients and xenografted mice [44] , [45] , but thus far it has not been shown that KSHV miRNAs are also loaded into exosomes .', 'To address this question , we examined patient-derived exosomes for the presence of KSHV-encoded miRNAs .', 'Figure 2I shows the qPCR products separated by size on a Caliper nanofluidics platform .', 'Exosomes derived from serum of three independent KSHV-positive TIVE L1 xenograft tumor mice and PEL fluid contained KSHV miR-K2 ( Figure 2I ) .', 'Total RNA from KSHV-positive latently-infected BCP-1 PEL cells were used as a positive control .', 'KSHV miRs K12-4-5p , K12-4-3p , K12-5 , K12-6-5p , K12-10a and K12-11 were also detected in pleural fluid and xenograft tumor mice ( data not shown ) .', 'KSHV miRNAs were undetectable in the KSHV-negative BJAB cell line ( Figure 2I ) .', 'This shows that systemically circulating exosomes contain appreciable levels of mature KSHV miRNAs and therefore exosomes containing KSHV miRNAs can travel from the subcutaneous tumor graft into the bloodstream and are stable enough to circulate systemically .', 'Since we harvested the blood at day 10–15 after tumor cell injection , this result is likely to reflect steady-state levels of exosomal miRNAs .', 'Notably , the L1 TIVE xenograft model does not generate infectious virus [48] .', 'Hence , exosome encapsulated KSHV miRNAs show promise as a highly sensitive marker for latent KSHV tumor cells .', 'In order to study exosome-associated viral miRNAs in more detail , we used the BCBL1 PEL cell line to assess KSHV miRNA expression .', 'We found that 14 out of 14 KSHV microRNAs tested were expressed at detectable levels in exosomes from latent BCBL1 cells ( Figure S7 ) .', 'Methods for purifying virions and exosomes can lead to co-precipitating of both exosomes and virions , therefore making it difficult to physically separate them for analysis .', 'To distinguish the source of these viral miRNAs as exosomal or virion-associated , we purified exosomes using three different techniques .', 'In addition to using the ExoQuick method of purification , we also utilized differential ultracentrifugation and a new bead affinity purification technique that positively selects for CD63+ exosomes , an exosomal marker not present on KSHV virions .', 'Expression of KSHV microRNAs was then assessed following enrichment of exosomes using either method ( Figure 3 , Figure S7 ) .', 'In addition , we passed the samples through a 0 . 2 µm filter prior to exosome isolation but after the removal of cellular debris .', 'Although KSHV virions are approximately 180 nm in size , they tend to aggregate , a phenomenon well-recognized in earlier studies studying infectivity of cell-free virus [61] , [62] , [63] .', 'This aggregation of virions makes it difficult to clear even a 0 . 2 µm filter .', 'This was experimentally confirmed by filtering concentrated KSHV stocks , which resulted in a decrease in titer of approximately 4 logs ( data not shown ) .', 'Exosomes , however , which range in size from 30–100 nm , can easily pass through a 0 . 2 µm filter , as is evident from EM imaging of filtered patient-derived exosomes ( Figure 2A ) .', 'Consistent with this , the expression levels of KSHV miRNAs were only slightly affected by filtering ( Figure 3A , Figure S7 ) .', 'By contrast , filtering of exosome samples resulted in a dramatic decrease in viral load ( Figure 3B ) .', 'We observed similar expression patterns of viral miRNAs in exosomes isolated and filtered following both ExoQuick and ultracentrifugation methods .', 'This was confirmed by Caliper gel electrophoresis , which showed the presence of KSHV miRNA products in both the exosome and filtered exosome fractions ( Figure S7 ) .', 'A lower shifted band corresponding to primer dimers was detected in the no template control reactions .', 'Note also that the Caliper images represent non-quantitative accumulation of product after 55 cycles , whereas quantification was based on the exponential phase of the PCR reaction .', 'Using the CD63+ exosome isolation method , we consistently observed expression of KSHV miRNAs regardless of filtration ( Figure 3A ) .', 'The levels of viral miRNAs were not significantly different in exosome preparations from latent or lytically induced PEL cells ( Figure 3A ) .', 'If these miRNAs were predominantly present within virions , we would expect a robust increase in viral miRNA levels concomitantly with increased virion production following reactivation as we observed for KSHV load ( Figure 3B ) .', 'Furthermore , RNase treatment of samples slightly decreased viral miRNA levels in the CD63− supernatant but did not affect KSHV miRNA expression in CD63+ fractions , suggesting that these miRNAs are primarily protected within exosomes ( data not shown ) .', 'Analysis of exosomes isolated by CD63 affinity capture confirmed the presence of CD9 , another well established exosomal marker ( Figure 3F ) .', 'CD9 levels were unaffected by filtering samples and RNase treatment ( Figure 3F ) .', 'Taken together , this demonstrates that the KSHV miRNAs are predominantly contained within exosomes released from latently-infected tumor cells .', 'Having determined that viral miRNAs were present in exosomes , we next sought to analyze the distribution of KSHV DNA among our samples and biochemical fractions .', 'There are two mechanisms that lead to KSHV viral DNA being detectable in body fluids:', '( i ) virions [64] ,', '( ii ) tumor cell-released free viral DNA , as has been demonstrated for EBV [65] , [66] , [67] , [68] .', 'To eliminate the contribution of cell-free viral DNA , we treated all samples with DNase prior to DNA isolation .', 'We evaluated BCBL1-derived exosomes purified using different techniques for the presence of KSHV DNA ( Figure 3B , C ) .', 'Exosome-enriched samples were passed through a 0 . 2 µm filter , which led to a drastic decrease in KSHV load using both purified virus stock and exosomes ( Figure 3B , data not shown ) .', 'Although the viral load increased following reactivation , filtering of exosomes from lytic BCBL1 cells abolished viral load to approximately the limit of detection .', 'DNase treatment of samples , which effectively eliminated freely circulating tumor-associated DNA , further decreased KSHV load in filtered fractions ( data not shown ) .', 'We also compared the presence of viral DNA in exosome and supernatant fractions of samples enriched by CD63 bead affinity purification or differential centrifugation ( Figure 3C ) .', 'Viral DNA was detected in the exosome-depleted supernatant fraction ( CD63− ) after bead affinity purification but was undetectable in the CD63+ exosome fraction .', 'Conversely , viral DNA was enriched in the exosome pellet following differential ultracentrifugation , as both virions and exosomes sediment at similar densities during centrifugation .', 'This establishes CD63-based affinity capture as an efficient way to separate exosomes and virions .', 'Since we detected KSHV miRNAs , but not KSHV DNA in the CD63-affinity purified exosomes , this suggests that the primary source of the viral miRNAs we observe is exosomes rather than virions .', 'We also evaluated viral load in exosomes purified using the ExoQuick method .', 'The advantage of the ExoQuick method compared to CD63 capture is greater efficiency ( using only 250 µl as input ) , which is essential when profiling large numbers of clinical samples .', 'We found KSHV DNA in both exosomal and free supernatant fractions of plasma and pleural fluid ( Figure 3D , E ) .', 'The highest viral load was found in exosomes derived from PEL pleural fluid .', 'No viral DNA was detected in our negative control samples or in exosomes purified from non-KS , HIV+ patients ( Figure 3D , 3E lanes CHP and AMT respectively ) .', 'Both ExoQuick and ultracentrifugation methods yielded KSHV DNA in the KS patient plasma and PEL fluid exosome fraction ( Figure 3 ) .', 'Thus , neither differential centrifugation not ExoQuick can with certainty be used to separate exosomes from virion particles .', 'However , CD63+ exosomes contain little KSHV DNA , especially following filtering of exosome samples .', 'This novel method confirms that the majority of the signal detected in the viral load assay was due to free DNA and KSHV virion DNA .', 'To further address the possibility that virions may also be present in our exosome fraction and may contribute to our results , we looked for KSHV virions and viral proteins in our exosome-enriched samples .', 'We did not detect any virions by EM following ExoQuick or ultracentrifugation isolation of exosomes ( Figure 2A ) .', 'We analyzed more than 20 grids for the presence of virions in our exosome-enriched samples .', 'Quantitative analysis of exosome-enriched pellets by differential centrifugation revealed 2 , 319 exosomes and no virions on three sample grids .', 'The supernatant fraction was also imaged by EM for the presence of exosomes and only 13 exosomes were detected , validating ultracentrifugation as an efficient method for exosome isolation .', 'We also compared the number of exosomes from latent and lytic BCBL1 cell supernatants by EM .', 'Both latent and lytic samples had similar numbers of exosomes detected on representative grids , averaging 135 and 126 , respectively ( while the DNase resistant viral load differed by >10-fold ) .', 'This suggests that the presence of exosomes in our samples may be static and independent of virus production .', 'Finally , we probed exosome-enriched samples for KSHV structural proteins .', 'KSHV K8 . 1 was readily detected in BCBL1 PEL cells following lytic reactivation ( Figure S8 ) .', 'However , PF-derived exosomes , which contained the highest viral load of our samples , did not express K8 . 1 .', 'These data confirm that our exosome-enriched samples do not contain appreciable levels of KSHV virions ( Figures S7 , S8 ) .', 'Although KSHV protein and DNA are not found in exosomes , we find systemically circulating KSHV miRNAs in exosomes derived from patients , tissue culture models and mouse models of KS ( Figure 2I , Figures S7 , S8 ) .', 'This establishes exosome-associated viral miRNAs as new biomarkers for KSHV-associated cancers .', 'It also suggests that detecting viral miRNAs may offer greater sensitivity of diagnosing viral infection than viral load measurements .', 'To obtain a more complete picture , we profiled the host miRNA repertoire in each of our sample groups using both exosomal and exosome-depleted supernatant preparations .', 'The C . elegans cel-mir-39 spike-in was used as an internal normalizing control .', 'Unsupervised clustering analysis revealed two distinct clusters , which are shown as projected onto the first three principal components ( Figure 4A ) .', 'Unsupervised clustering groups samples and the different miRNAs based on similar expression levels .', 'The result is typically shown as a heatmap .', 'Principal component analysis is used to reduce the complexity of the data further without loss of statistical power .', 'It combines the multiple measurements of each sample ( or each miRNA ) to such that the data can be represented in three dimensions ( the principal component axes ) .', 'Individual analysis of the human and mouse profiling samples ( Figures 4B , C ) illustrates the more divergent clusters representing miRNAs elevated in tumor versus control samples in the mouse model .', 'We expected to see more defined clusters in our mouse models since the xenografts represent biological replicates with limited variability compared with human clinical samples .', 'The miRNA profile in the human samples alone clearly separated samples into KSHV-associated and control groups .', 'When we further narrowed the miRNAs to known oncomiRs and tumor suppressor miRNAs , the classification improved ( Figure 4D ) .', 'A list of these ∼150 oncomirs and tumor suppressor miRNAs is shown in Table S2 .', 'As a negative control for our analysis we clustered an unrelated sample .', 'We performed unsupervised clustering of miRNAs in HEK293 cells following infection with West Nile Virus ( WNV ) ( Chugh and Dittmer , unpublished data ) .', 'This comparison yielded very different clusters of miRNAs compared with the KSHV exosome data as noted by further predicted target analysis ( Table 1 ) .', 'This establishes a unique oncomir signature of KS- and PEL-associated exosomes .', 'We further examined the expression of individual oncomirs and tumor suppressor miRNAs in the mouse exosome subset by heatmap analysis ( Figure 4E ) .', 'Oncomirs were defined as host miRNAs readily studied for their role in tumorigenesis and related cancer signaling pathways while tumor suppressor miRNAs have been demonstrated to functionally inhibit these processes ( Table S2 ) .', 'We identified this subset of oncogenic miRNAs because', '( a ) we previously extensively validated these assays [3] , [4] , [55] and', '( b ) they represent miRNAs with experimentally verified expression and function .', 'The most distinct expression pattern was the apparent separation between TIVE xenograft and control mouse serum ( Figure 4E , Panel', 'i ) .', 'The majority of oncomiRs in this cluster were increased in exosomes from KS xenograft tumor models and were only minimally or not detectable in the control mice ( ctrl versus xeno ) .', 'We next compared miRNA expression profiles of control and xenograft mice to our latency locus transgenic mouse model ( Figure 4E , Panel', 'ii ) .', 'In this novel model , only the KSHV latent genes and miRNAs are expressed in B cells [50] .', 'However , none of the viral structural genes are present .', 'We found that exosomes derived from the transgenic model differed from that of control mice and shared some oncogenic miRNA expression with the xenograft mice .', 'As this transgenic mouse model phenotype represents B cell hyperplasia , these highly expressed miRNAs may be reflective of change in the miRNome regulated by the KSHV latency locus prior to tumor formation ( Figure 4E ) .', 'We further compared the exosomal miRNA profile of transgenic mice to that of PEL-associated exosomes from primary pleural fluid ( Figure 4E , panels iii , iv ) .', 'This yielded similarities in induced exosomal oncogenic miRNA expression between the 801 transgenic mouse model and PEL patient fluid ( Panel iii , Cluster 1 ) .', 'Interestingly , we also identified a subset of microRNAs that was solely induced in the KSHV latency locus transgenic mouse model ( Figure 4E , Panel iv , Cluster 2 ) .', 'Figure S9 further compares the exosomal miRNA profile in an independent set of transgenic and control mice and indicates elevated levels of oncogenic miRNA expression in the transgenic mouse model .', 'Analysis of miRNA profiles in both Clusters 1 and 2 also revealed a subset of oncogenic miRNAs that were exclusively expressed in exosomes , suggesting that these miRNAs may be preferentially incorporated from the tumor site into exosomes for intercellular communication ( Figure 4E , Panels iii , iv , Figure S10 ) .', 'Taken together , we find that the most elevated oncomiR levels in exosomes were observed in the TIVE xenograft tumor group , as these mice were bearing large , well-vascularized tumors , which facilitates expression and release of miRNAs .', 'This demonstrates for the first time that exosomal miRNAs , including KSHV miRNAs , can be detected in mouse models of KS .', 'Our human clinical samples of AIDS-KS recapitulated the trends in oncogenic miRNA expression observed in our mouse models ( Figure 4F , Figure S11 ) .', 'Cluster 1 represents a subset of oncogenic miRNAs that are most highly expressed in exosomes derived from PEL pleural fluid ( Figure 4F ) .', 'Several miRNAs in this cluster were also elevated in KS patient-derived exosomes .', 'This pattern of miRNA expression may reflect a signature of KSHV-associated malignancies .', 'A subset of miRNAs within this cluster could also represent miRNAs overexpressed in KS and other cancers since we observed oncogenic miRNA induction in other AIDS malignancies as well as KS-associated exosomes ( Figure 4F ) .', 'Cluster 2 shows another subset of miRNAs with elevated expression in exosomes from KS or AIDS malignancy patients .', 'This cluster also includes several miRNAs that seem to be preferentially expressed within exosomes compared to the supernatant fraction .', 'We noticed little difference in the miRNA profile from control plasma exosomes versus RNase-treated control plasma exosomes , indicating that exosomes are indeed resistant to RNase treatment [35] ( Figure 4F , lanes CHP exo and RNase-CHP exo ) .', 'We also compared the miRNA profile in 
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['MicroRNAs ( miRNAs ) are stable , small non-coding RNAs that modulate many downstream target genes .', 'Recently , circulating miRNAs have been detected in various body fluids and within exosomes , prompting their evaluation as candidate biomarkers of diseases , especially cancer .', "Kaposi's sarcoma ( KS ) is the most common AIDS-associated cancer and remains prevalent despite Highly Active Anti-Retroviral Therapy ( HAART ) .", 'KS is caused by KS-associated herpesvirus ( KSHV ) , a gamma herpesvirus also associated with Primary Effusion Lymphoma ( PEL ) .', 'We sought to determine the host and viral circulating miRNAs in plasma , pleural fluid or serum from patients with the KSHV-associated malignancies KS and PEL and from two mouse models of KS .', 'Both KSHV-encoded miRNAs and host miRNAs , including members of the miR-17–92 cluster , were detectable within patient exosomes and circulating miRNA profiles from KSHV mouse models .', 'Further characterization revealed a subset of miRNAs that seemed to be preferentially incorporated into exosomes .', 'Gene ontology analysis of signature exosomal miRNA targets revealed several signaling pathways that are known to be important in KSHV pathogenesis .', 'Functional analysis of endothelial cells exposed to patient-derived exosomes demonstrated enhanced cell migration and IL-6 secretion .', 'This suggests that exosomes derived from KSHV-associated malignancies are functional and contain a distinct subset of miRNAs .', 'These could represent candidate biomarkers of disease and may contribute to the paracrine phenotypes that are a characteristic of KS .']
['Circulating microRNAs ( miRNAs ) , such as those found in exosomes , have emerged as diagnostic tools and hold promise as minimally invasive , stable biomarkers .', 'Transfer of tumor-derived exosomal miRNAs to surrounding cells may be an important form of cellular communication .', "Kaposi's sarcoma-associated herpesvirus ( KSHV ) is the etiological agent of Kaposi's sarcoma ( KS ) , the most common AIDS-defining cancer worldwide .", 'Here , we survey systemically circulating miRNAs and reveal potential biomarkers for KS and Primary Effusion Lymphoma ( PEL ) .', 'This expands previous tissue culture studies by profiling clinical samples and by using two new mouse models of KSHV tumorigenesis .', 'Profiling of circulating miRNAs revealed that oncogenic and viral miRNAs were present in exosomes from KS patient plasma , pleural effusions and mouse models of KS .', 'Analysis of human oncogenic miRNAs , including the well-known miR-17-92 cluster , revealed that several miRNAs were preferentially incorporated into exosomes in our KS mouse model .', 'Gene ontology analysis of upregulated miRNAs showed that the majority of pathways affected were known targets of KSHV signaling pathways .', 'Transfer of these oncogenic exosomes to immortalized hTERT-HUVEC cells enhanced cell migration and IL-6 secretion .', 'These circulating miRNAs and KS derived exosomes may therefore be part of the paracrine signaling mechanism that mediates KSHV pathogenesis .']
['animal models of infection', 'medicine', 'infectious diseases', 'viral persistence and latency', 'virology', 'viruses and cancer', 'host-pathogen interaction', 'biology', 'microbiology', 'viral disease diagnosis']