Circulating miRNAs, especially those within exosomes, have emerged as novel biomarkers 
. Their main advantage is stability and ease of detection as all miRNAs can be profiled with a common platform. We previously established and validated such a miRNA profiling platform 
. Bodily fluids such as plasma can be obtained using minimally invasive techniques and lend themselves to repeat sampling, for instance to follow therapy. In the case of PEL, periodic (in extreme cases every few days) draining of pleural cavities is medically indicated.
Although the exosomal miRNA profile of malignancies associated with EBV have been previously reported 
, this is the first study to examine the circulating miRNA profile of KSHV-associated cancers. This is also one of a few studies to compare patient tumors to xenograft mouse models 
. We extend previous findings on exosomal miRNAs, which were largely based on cell culture models. KSHV-encoded miRNAs were detectable in systemically circulating exosomes ( and ), including in xenograft mouse models of KS. This suggests that viral miRNAs can have effects far from the site of the infected cell. Furthermore, viral microRNAs could potentially serve as highly specific biomarkers of KSHV-associated malignancies, particularly if the lesions are internal and comprised of mostly latently infected cells. We found similar levels of viral miRNAs in exosomes derived from latently infected PEL cells compared to PEL cells undergoing lytic reactivation (). Most KS tumor cells and most PEL are latently infected and even if lytic gene expression is observed in a subset of cells, virions are seldom produced 
A significant complication of characterizing exosomal miRNAs in virally associated diseases is that miRNAs may be incorporated into virions. Previous studies have shown that viral RNAs can be detected within herpesvirus virions, including KSHV and EBV 
. Recently, Lin et al. demonstrated the presence of viral, as well as cellular miRNAs in purified KSHV virions 
. Exosomes are difficult to physically separate from virions due to their similar sedimentation velocities, buoyant densities, biogenesis and heterogeneous nature of exosomes 
. Others have circumvented this issue using cell culture models that are incapable of virus production, such as HCV subgenomic replicon (SGR) cells 
. Analogous to this model, we employed several latent models of KSHV infection, including the latently infected TIVE xenograft mice, the latency locus transgenic mice and the BCBL1 latent PEL cell line 
. We believe that the majority of miRNAs we detect here are exosomal, rather than virion-associated. To support this interpretation, we offer three lines of evidence.
First, we were able to detect all viral miRNAs in latent BCBL1 exosomes and filtering samples led to decreased viral load but did not significantly affect levels of KSHV miRNAs (, Figure S7
). We detected similar amounts of KSHV miRNAs in exosomes isolated from latent PEL supernatant as in exosomes from supernatant of induced PEL (). In the same samples, we observed a greater than 10-fold increase in viral DNA. This suggests that KSHV miRNAs are released into exosomes from latently infected PEL, analogous to exosomal EBV miRNAs which are released from latently infected cells 
. Note, that we are able to detect KSHV miRNAs in exosomes from 250 µl of latently infected cell supernatant, whereas at least 500 mls were previously used to enrich for virion-associated miRNAs 
. We could also detect KSHV miRNAs in the bloodstream of mice, which carry KSHV latently-infected TIVE-E1/L1 xenografts. These cells do not generate infectious virions 
and (R. Renne, personal communication).
Second, we were able to isolate exosomes by CD63-mediated affinity purification (). Herpesvirus virions and exosomes co-purify in almost all centrifugation schemas designed to enrich for exosomal fractions (i.e. differential ultracentrifugation, sucrose gradients, ExoQuick solution). By contrast, anti-CD63 Dynabeads positively select exosomes which carry CD63 as one of their surface markers 
while CD63(−) virions are eliminated. This resulted in an enrichment of KSHV miRNAs and concomitant depletion of viral DNA (), demonstrating that indeed viral miRNAs are present in exosomes. We were also unable to detect any contaminating virions in our samples enriched for exosomes by electron microscopy and structural viral proteins were absent in our exosome-enriched samples ( and Figure S8
Thirdly, KSHV miRNAs could be detected in exosomes isolated from the serum of our xenograft mouse model. These xenograft mice harbor latently infected cells, which do not generate infectious virus. This suggests that viral miRNAs are constantly released and circulate systemically in exosomes in mice (and patients) who harbor KSHV latently infected cells. Taken together, these data suggest that KSHV latently infected cells can release viral miRNAs and further demonstrates that exosomes are the source of these circulating miRNAs.
Human oncogenic miRNAs were easily detected in tumor-derived exosomes isolated from patient plasma and pleural fluid (). Further analysis confirmed increased levels of the well-studied miR-17-92 cluster miRNAs. Our data also show potentially important similarities and differences in the miRNA profile from AIDS patients with KS compared to patients with other non-viral AIDS-associated malignancies (). This subset of exosomal miRNAs could reflect differences between the varying progression of different malignancies in AIDS patients or similarities among AIDS-associated cancers and merits further study. Exosomal miRNAs are readily detected in pleural fluid samples, representing an alternate sample source with potentially higher correlation to disease state for patients with malignant effusions. Since pleural fluid is more proximal to the tumor site than plasma, which circulates throughout the body, we reason that the circulating miRNome from malignant effusions may be more reflective of the tumor itself. However, further studies comparing the miRNA signatures of pleural fluid-derived exosomes from PEL and other non-KSHV-associated malignancies such as lung cancer are necessary to reveal diagnostic biomarkers unique to PEL.
We also demonstrated that human and viral miRNAs are present in circulating exosomes in xenografted mice (,). We used the TIVE L1 
xenograft model, which has been shown to be predictive of anti-KS therapies 
. The KSHV miRNAs that we consistently detected in these mouse models could only stem from the human graft. Due to the high conservation of cellular miRNAs within the oncogenic clusters, the cross-species detection of miRNAs using the human assays makes it difficult to distinguish miRNAs of human versus mouse origin in these models (Table S3
, ABI product information, miRBase). In some cases, the mature miRNAs share 100% sequence homology across the entire length, not just the seed region (miRBase, 
) and in many cases the targets have co-evolved as well 
. We observed greater levels of miRNAs in the mouse exosomes compared to human exosomes, which may be due to the fixed 250 µl sample size with respect to the overall amount of blood circulating within a human (approximately 5 L) or mouse (approximately 0.0015 L).
Specific host miRNA markers of tumorigenesis also emerged in our mouse models. We showed previously that host miRNAs are distinct for different stages of KS tumor progression 
. Therefore, tumorigenic miRNAs combined with viral miRNAs would offer a very specific biomarker signature and may also identify biomarkers for other related cancers. Therefore, we analyzed expression of the oncogenic miR-17-92 and 106b/25 clusters and found that they were significantly enriched in exosomes from TIVE tumor-bearing mice compared with controls (). Several of the oncogenic miRNAs expressed in exosomes were previously found at highly expressed levels in the TIVE cell line independently (R. Renne, personal communication). The mir-17-92 cluster was previously shown to be upregulated in KS tumor biopsies 
. This is the first demonstration that the miR-17-92 cluster miRNAs are incorporated into exosomes from KSHV-associated malignancies. These oncogenic miRNAs have also been detected in exosomes derived from leukemia cells and those derived from breast milk 
, suggesting that their function is at least in part to mediate paracrine phenotypes. Viral and cellular miRNAs originating from the tumor enter the mouse circulatory system and are readily detected in serum. Since our mouse model exosome signatures recapitulate the clinical KS signatures, this supports the validity of xenograft mice as a reliable model system for KS.
We also observed a subset of miRNAs that were highly induced in exosomes and were virtually undetectable in the free, circulating miRNA fraction (). The miRNAs detected exclusively in the exosome fractions are either known to be oncogenic or shown to be upregulated by KSHV infection 
. This suggests that certain miRNAs are preferentially incorporated into exosomes and that many proliferative and tumor-associated miRNAs fall into this class. Recently, Palma et al 
found that selectively exported miRNAs from malignantly transformed cells may be incorporated into customized exosomal particles distinct from the microvesicles that originate from untransformed cells. It is conceivable that these have different systemic stability and thus become enriched in a blood sample. This may be the case with KSHV-induced miRNAs as well since we found miRNAs originating from our transgene model also to be enriched in this fraction.
Exosomes serve as a means of intercellular communication with surrounding cells and the contents of exosomes can be shared between cells through the mechanism of exosomal transfer 
. Exosomes can deliver functional miRNAs to recipient cells and consequently downregulate expression of target genes 
. Leukemia cell-derived exosomes have recently been shown to affect endothelial cell function through microRNA transfer 
. Moreover, tumor-derived microRNAs were recently reported to play a functional role through binding to Toll-like receptors, thereby inducing an inflammatory response and influencing tumor growth and metastasis 
. This in vivo
relevance was further demonstrated by inhibiting tumor-secreted miRNAs, which altered tumor formation in mice 
. Dendritic cell-derived exosomes can be used to prime the immune response as cancer immunotherapy to suppress tumor burden 
. Exosomes have also been recently tested in clinical trials to reduce tumor size 
. Collectively, these studies further demonstrate the in vivo
relevance of exosomes and their potential as mediators of disease phenotypes.
In this study, we find stable, systemic KSHV miRNAs and oncomiRs. GO pathway analysis of predicted targets of the oncogenic miRNAs expressed in exosomes revealed a variety of pathways targeted by KSHV during pathogenesis (). Since several of these pathways shared a role in cell migration, we further tested the effects of patient-derived exosomes on migration of hTERT-HUVECs. Treatment of cells with exosomes from pleural fluid led to earlier, enhanced migration of endothelial cells, giving these patient-derived exosomes a functional biological role (). Therefore, it is possible that miRNAs specifically expressed within exosomes play a role in disease progression and mediate paracrine effects, which are a hallmark of KSHV tumorigenesis.