Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100–170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.


Introduction
Ewing sarcoma (EwS) is the second most frequent bone and soft tissue tumor in children and young adults with high metastatic capacity. In the case of multifocal disease or early relapse, 5-year survival rates drastically decline to less than 30% [1,2]. EwS is a non-immunogenic tumor with a low mutational burden [3] and reduced expression of human leukocyte antigen (HLA) class I molecules [4]. Immunohistochemistry and transcriptome-based analysis revealed that the EwS tumor microenvironment (TME) is scarcely infiltrated with cytotoxic T cells [5,6], mature dendritic cells (DCs), and proinflammatory M1 macrophages [7][8][9]. Instead, EwS tumors are mostly populated by immature (M0) and immunosuppressive (M2) macrophages [7][8][9], whose abundance in the TME is correlated with worse outcomes [10]. However, mechanisms promoting the immunosuppressive TME in EwS are poorly understood.
One of the potential mechanisms may involve tumor-derived extracellular vesicles (EVs) which emerged in the past decade as mediators of tumor-associated inflammation and immunosuppression [11,12]. Small EVs range from 40-160 nm in size and include exosomes (originating from multivesicular endosome compartments) and heterogeneous populations of plasma membrane-derived microvesicles [13]. EVs are secreted by healthy tissue cells as well as by tumor cells, including EwS [14][15][16]. The EV cargo is protected by a lipid membrane and reflects the physiological or pathological state of the parental cell. It is mostly comprised of proteins, lipids, and RNA, which can be transferred to the recipient cell [13,17,18].
Circulating myeloid cells including monocytes can encounter and engulf tumorderived EVs in peripheral blood or after extravasation into the tumor stroma. Monocytes can differentiate into DCs specializing in antigen processing and priming of CD4 + and CD8 + T cells, thereby activating adaptive immune responses including antitumor immunity [19]. However, proper differentiation and maturation of DCs can be disrupted by inflammatory signals [20]. Immature DCs are incapable of priming cytotoxic T cell responses, and, instead, promote T cell anergy, inflammation, and tumor progression [19,21]. Cancer-associated chronic inflammation can also lead to pathological activation of myeloid cells and expansion of myeloid-derived suppressor cell (MDSC) populations in the blood of cancer patients, including EwS [22,23]. MDSCs possess potent immunosuppressive functions and are involved in cancer, autoimmune, and chronic inflammatory diseases [24]. Tumor-derived EVs may play active roles in both chronic inflammation and conversion of myeloid cells into MDSCs and other immunosuppressive phenotypes [25]. The examples include polarization of myeloid cells into pro-tumorigenic M2 macrophages by glioblastoma-derived EVs [26] and induction of the immune checkpoint molecule PD-L1 on circulating monocytes by EVs from melanoma and chronic lymphatic leukemia [27,28].
Previously, we identified and characterized EVs isolated from EwS cell lines and patients' plasma [14,29], indicating that similar mechanisms may be operating in EwS. We also established protocols for the generation of monocytic-derived DCs (moDCs) used in the priming of allo-restricted tumor antigen-specific T cells for adoptive T cell transfer [30][31][32]. In this study, we investigated if and how EwS-derived EVs may affect the phenotype and function of CD33 + and CD14 + myeloid cells isolated from the blood of healthy donors. We show that EwS EVs induce pro-inflammatory cytokine release and impair myeloid cell differentiation as well as their T cell stimulatory function, providing novel insights into the mechanisms of EwS-associated inflammation and immunosuppression.

Cytokine Single-and Multiplex Assay
Cytokines and chemokines in conditioned medium from cell cultures were analyzed using the Bio-Plex Pro Human Chemokine TNF-α Set (171BK55MR2) and Bio-Plex Human Cytokine Screening Panel, 48-plex panel (both Bio-Rad, Munich, Germany) as described previously [34]. Conditioned medium was collected, centrifuged at 2000× g for 10 min at 4 • C, and stored at −80 • C until further analysis. Samples were measured in duplicates using the Bio-Plex ® 200 system (171000201, Bio-Rad, Munich, Germany). Cytokine concentrations were determined with the Bio-Plex ® Manager Version 6.2 (Bio-Rad, Munich, Germany).

RNA Isolation, Whole Transcriptome Sequencing, and Bioinformatics
Total RNAs from CD14 + cells were isolated using the mirVana miRNA isolation kit (AM1561), followed by removal of DNA (Turbo DNA-free Kit, AM1907) and RNA quantification (Qubit RNA high sensitivity assay kit, Q32852, all Thermo Fisher Scientific, Waltham, MA, USA). RNA 6000 Pico chips (Agilent Technologies, Santa Clara, CA, USA) were used to analyze RNA integrity. Strand-specific RNA-seq libraries were constructed using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (8098140702, Roche, Basel, Switzerland). The quality of libraries was confirmed using the Agilent High Sensitivity DNA assay, and sequenced to about 100M reads per sample on an Illumina NovaSeq at a minimum of 2 × 100 bp.

Statistical Analysis
Statistical analysis and graphs were performed with GrapPadPrism 9 (GraphPad Software, San Diego, CA, USA). Normal distribution was analyzed with the Shapiro-Wilk test. Differences between conditions were calculated by paired or unpaired two-tailed student's t-test or one-way ANOVA with subsequent Turkey adjustment regarding mean and standard deviation (SD). Nonparametric values were analyzed with Mann-Whitney U test. p values ≤ 0.05 were considered statistically significant (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

EwS EVs Induce Pro-Inflammatory Responses in Myeloid Cells
We sought to determine if EwS EVs may play a role in the observed accumulation of immunosuppressive M2 macrophages [7,8,10] and CD14 + HLA-DR low/neg monocytes [23] in the tumor microenvironment and blood circulation of EwS patients. For EV isolation, the conditioned medium from A4573, A673, TC32, and TC71 EwS cell lines was subjected to ultrafiltration and differential centrifugation. In parallel, we purified EVs from MRC5 fibroblasts and from the blood plasma of healthy donors, to be used as negative controls. As assessed by nanoparticle tracking analysis (NTA), the median size of the EV preparations from EwS and MRC5 cells and healthy donors' plasma, ranged from 100 to 170 nm ( Figure 1A). As shown by immunoblotting and flow cytometry, these EV preparations were positive for classical exosome markers CD63, CD81, and TSG101 and negative for the frequent EV contaminant calnexin ( Figure 1B,C and Supplementary Figure S1A-C), consistent with characteristics of small EVs [13]. In addition, we identified other common EV constituents, including the protein chaperons Hsp90 and Hsp70, and the cytoskeletal proteins tubulin and actin ( Figure 1B and Supplementary Figure S1A). We also noticed some variability in the protein composition between independently isolated EV preparations and between EVs from different EwS cell lines ( Figure 1B and Supplementary Figure S1A,B), which could be due to the protein loss during EV isolation or the variable expression of these proteins in the respective parental cells, or both. The subsequent testing of EV activities was thus performed based on the NTA-quantified number of particles, to ensure standardized comparisons between the experiments [38]. We also examined independently isolated EV preparations, including those with higher and lower levels of CD81, CD63, or TSG101, to identify common properties of EwS EVs.
To assess the potential pro-inflammatory effects of EwS EVs on blood-circulating myeloid cells, we representatively used CD33 + myeloid cells and CD14 + monocytes isolated from peripheral blood of healthy donors. We first tested independently isolated EV preparations for their ability to induce secretion of tumor necrosis factor (TNF), one of the major mediators of pro-inflammatory response [39]. Analysis of the conditioned medium by cytokine singleplex assay showed a significant elevation of TNF released by CD14 + cells after the 6-h treatment with any of three different EV preparations from A4573, A673, TC32, and TC71 cells, but not with EVs from MRC5 cells or healthy donor plasma, or PBS control ( Figure 1D).
We next performed multiplex cytokine profiling of the conditioned medium after the 24-h treatment of CD33 + and CD14 + cells with EVs from A4573, A673, and TC32 EwS cell lines. In addition to TNF, we identified the upregulation of the pro-inflammatory cytokines and chemokines IL-1α, IL-1β, IL-6, IL-8, CCL2/MCP1, CCL3/MIP-1α, and CCL4/MIP-1β, whose levels were induced by 10-1,000-fold compared to PBS control (Figure 2A,B). Similar effects were observed in both CD33 + and CD14 + cells, with A4573 EVs eliciting the strongest response. In contrast to healthy donor plasma EVs, A4573 EV-induced release of IL-6, IL-8, and TNF by CD33 + cells was strongly dose-dependent ( Figure 2C). These cytokines were undetectable in pre-conditioned medium with added spike-in A4573 EVs ( Figure 2C), excluding a possibility of cross-contamination from the growth medium or EVs themselves. Likewise, A673 EVs (but not PBS control or MRC5 EVs) induced persistent and dose-dependent secretion of IL-6 and IL-8, and the effect was evident 18 h after a short Cells 2021, 10, 2081 6 of 17 6-h exposure to A673 EVs ( Figure 2D). Despite some variations between preparations from different cell lines (e.g., A673 EVs were less efficient in inducing TNF release compared to EVs from TC32 and TC71 cells; compare Figure 2D,E), overall pro-inflammatory activities of EV preparations from A673, TC32, and TC71 cells were comparable, indicating that induction of pro-inflammatory responses is a common property of EwS EVs. However, because A4573 EVs exhibited unexplainably high induction of a large number of proinflammatory cytokines and chemokines (Figure 2A,B), they were considered an outlier and were excluded for the subsequent analysis. To investigate whether EwS EVs may induce pro-inflammatory responses during the 5-day differentiation towards moDCs, CD33 + myeloid cells were differentiated with IL-4 and GM-CSF in the presence of EVs from TC32 cells or MRC5 fibroblasts. By day 3, levels of IL-6 and TNF were increased by 480-and 16-fold, respectively, in the conditioned medium from TC32 EV-treated CD33 + cells compared to PBS control ( Figure 2F), while only IL-6 reached statistical significance (p ≤ 0.05, unpaired two-tailed t-test). Moreover, increased IL-6 and TNF secretion by TC32 EV-treated immature DCs persisted even after the medium change and was detected by day 5, in contrast to MRC5 EVs which exhibited much weaker effects ( Figure 2F). Together, these results demonstrate a strong EwS EV-driven induction of pro-inflammatory responses in CD33 + and CD14 + myeloid cells.

EwS EVs Impair DC Differentiation and Maturation of CD33 + Myeloid Cells and CD14 + Monocytes
To examine if EwS EVs may preferentially affect myeloid cells at early stages of differentiation or later on, during their maturation towards moDCs, TC32, A673, or TC71 EVs were added to freshly isolated CD33 + or CD14 + cells during differentiation (days 0 and 3) or maturation (day 5), alongside with the appropriate cytokine cocktails ( Figure 3A,E). Flow cytometry analysis by day 7 showed that CD14 + cells ( Figure 3B, p ≤ 0.05, one-way ANOVA with multiple comparison Turkey test) and CD33 + cells ( Figure 3D, p ≤ 0.05, paired two-tailed t-test) failed to upregulate co-stimulatory molecules CD80, CD86 and HLA-DR after being exposed to TC32, A673 or TC71 EVs at day 0, in contrast to MRC5 EVs which had no effect ( Figure 3B,D and Supplementary Figure S2A-C). Compared to control CD14 + cells, downregulation of CD80, CD86, and HLA-DR was significant with all three independently isolated TC32 EV preparations (p ≤ 0.05, unpaired two-tailed t-test), with preparation #3 exhibiting the strongest effect ( Figure 3C). Although there might be manifold reasons for the stronger activity of this preparation, compared with preparation #1 (the weakest one), it exhibited enrichment with Hsp90 and Hsp70, while having similar levels of TSG101 and reduced levels of CD81, tubulin, and actin (Supplementary Figure S1A,B). Adding TC32 EVs at later time points during differentiation (day 3) and maturation (day 5) did not significantly interfere with the expression of maturation markers ( Figure 3E, Supplementary Figure S2A-C). Since independently isolated EV preparations including those from three distinct EwS cell lines exhibited similar activities, these results indicate that EwS EVs impair the maturation of myeloid cells, especially when they are exposed to EwS EVs at very early stages of differentiation.

EwS EVs Rewire Gene Expression Programs Associated with Inflammatory Responses and Maturation of Myeloid Cells
To identify gene expression programs affected by EwS EVs in myeloid cells, we performed a whole transcriptome analysis of CD14 + cells differentiated in the absence or presence of EVs from TC32 or TC71 EwS cells ( Figure 4A). We found that both TC32 and TC71 EVs induced similar gene expression patterns, with 412 transcripts commonly detected in both EwS EV-treated compared to control CD14 + cells ( Figure 4B and Supplementary Table S1). In contrast, only 228 and 282 were shared between control and either TC32 or TC71 EV-treated cells, respectively ( Figure 4B). CIBERSORT analysis of the immune cell gene expression programs revealed activation of monocyte and macrophageassociated signatures in both EwS EV-treated cells, while "activated DC" signatures were downregulated ( Figure 4C), supporting a potential link between EwS EVs and the reported prevalence of these subtypes in the TME of EwS [7][8][9]. Differential gene expression analysis of the canonical genes associated with M1 and M2 macrophages or DCs (Supplementary  Table S2) further confirmed a coordinated switch of gene expression programs in both TC32 and TC71 EV-treated compared to control cells. Concordant with cytokine profiling, genes encoding pro-inflammatory cytokines and chemokines, such as IL1B, IL6, CXCL8/IL8, TNF, CCL3, and CCL4) were upregulated ( Figure 4D, "M1" panel), alongside with immunosuppressive cytokines (IL10 and TGFB1), and extracellular matrix-degrading enzymes ADAM8 and MMP14 ( Figure 4D, "M2" panel). Also, in line with our flow cytometry data, in both TC32 and TC71 EV-treated cells we observed downregulation of CD86 and HLA-DRA and B1, although CD80 and several other markers of maturation (CD40, CD209/DCSIGN, and LAMP3) were upregulated ( Figure 4D, "DC" panel).

CD14 + Monocytes Differentiated in the Presence of EwS EVs Suppress T Cell Activation
Sustained release of pro-inflammatory cytokines and type I/III IFNs by tumor and immune cells eventually lead to immunosuppression and tolerance, affecting both innate and adaptive immunity [40,41]. Given a persistent release of pro-inflammatory cytokines by EwS EV-treated myeloid cells and their impaired maturation, we next examined a potential impact of these cells on T cell activation, using IFNγ release and T cell proliferation as readouts. Specifically, CD14 + cells differentiated in the absence or presence of EVs from TC32 EwS cells or MRC5 fibroblasts were co-cultured with allogeneic CD14-depleted PBMCs and analyzed by cytokine profiling and T cell proliferation assays ( Figure 5A). Compared to PBS control, we observed a significant increase in IL-6 and IL-10 levels and reduction of IFNγ in the conditioned medium from co-cultures with CD14 + cells differentiated in the presence of TC32 EVs ( Figure 5B; p ≤ 0.05, Mann-Whitney U test). The effect was specific to TC32 EVs and was not observed with MRC5 EV-pretreated CD14 + cells. In contrast, TNF was unselectively induced by both TC32 EV-and MRC5 EV-pretreated CD14 + cells ( Figure 5B), in line with unspecific TNF release from MRC5 EV-treated CD33 + cells ( Figure 2F).
We next assessed the effect of EV-pretreated CD14 + cells on the proliferation of T cells, by co-culturing them with allogeneic CD14-depleted PBMCs. We found that compared to control, CD14 + cells differentiated in the presence of TC32 or A673 EVs (but not with MRC5 EVs) significantly reduced the proliferation of both CD4 + and CD8 + T cells ( Figure 5C; p ≤ 0.001 and p ≤ 0.01, one-way ANOVA with multiple comparison Turkey test), with CD4 + T cells being stronger affected. In line with reduced proliferation, flow cytometry analysis of CD8 + T cells co-cultured with A673 EwS EV-treated CD14 + cells revealed a significant reduction of the activation marker CD69 compared to untreated control cells (p ≤ 0.05, one-way ANOVA with multiple comparison Turkey test), while CD25 expression was not affected (Supplementary Figure S3A,B). Although the detailed mechanisms remain to be studied, these results indicate that CD14 + cells differentiated in the presence of EwS

EwS EVs Rewire Gene Expression Programs Associated with Inflammatory Responses and Maturation of Myeloid Cells
To identify gene expression programs affected by EwS EVs in myeloid cells, we performed a whole transcriptome analysis of CD14 + cells differentiated in the absence or presence of EVs from TC32 or TC71 EwS cells ( Figure 4A). We found that both TC32 and TC71 EVs induced similar gene expression patterns, with 412 transcripts commonly detected in both EwS EV-treated compared to control CD14 + cells ( Figure 4B and Supplementary Table S1). In contrast, only 228 and 282 were shared between control and either TC32 or TC71  design (B-D). CD14 + or CD33 + myeloid cells purified from healthy donors were differentiated in the presence of GM-CSF and IL-4 for 5 days and matured with the IL-1β, IL-6, PGE 2 , and TNF cocktail for additional 2 days to moDCs. The indicated EVs (3 × 10 9 /mL) or PBS control (Ctrl) were added at day 0 of differentiation. (B-E) Flow cytometry analysis of the indicated maturation markers at day 7. (B) Geometric mean fluorescence intensity (gMFI) of CD80, CD86, and HLA-DR normalized to IT of CD14 + monocytes treated with EVs from MRC5, A673, TC32, or TC71 cells, or PBS. Fold change to Ctrl is shown. (C) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD14 + myeloid cells treated with three independent EV preparations from TC32 cells or PBS. (D) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD33 + myeloid cells treated with MRC5 EVs, TC32 EVs, or PBS. (E) TC32 EVs were added to CD14 + or CD33 + myeloid cells during differentiation (day 0 and 3) or maturation (day 5). Representative pseudocolor plots of CD80, CD83, CD86, and HLA-DR compared to the respective IT antibodies are shown. The results were obtained from one (C) or three (B,D,E) independent donors with three (B-E) independent EV preparations. Data are presented as mean ± SD. One-way ANOVA with multiple comparison Turkey test (B), and unpaired (C) and paired (D) two-tailed t-tests were used to calculate p values. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001.

Discussion
In this study, we show that EwS EVs induce sustained pro-inflammatory signaling and impair myeloid cell maturation, affecting CD33 + myeloid cells and CD14 + monocytes. Consistent with their semi-mature phenotypes, these cells interfered with the activation of CD4 + and CD8 + T cells ( Figure 6). We thus propose that EwS EVs may pathologically activate blood-circulating and tumor-infiltrating myeloid cells, thereby contributing to systemic inflammation and immunosuppression, and influencing both innate and adaptive immunity.
Conditioned medium was assessed by cytokine multiplex assay. (C) Allogeneic CD14-depleted PBMCs were labeled with eFluor450 dye, stimulated with coated OKT3, IL2, and anti-CD28 antibodies, and co-cultured for 4 days with CD14 + monocytes differentiated in the presence of the indicated EVs. Representative flow cytometry results (top) and their quantification (bottom) of CD4 + and CD8 + T cell proliferation (representative of three independent donors). Results were obtained with two (B) or three (C) independent EV preparations. Data are presented as mean ± SD. Mann-Whitney U test (B) and one-way ANOVA with multiple comparison Turkey test (C) were used to calculate p values. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Discussion
In this study, we show that EwS EVs induce sustained pro-inflammatory signaling and impair myeloid cell maturation, affecting CD33 + myeloid cells and CD14 + monocytes. Consistent with their semi-mature phenotypes, these cells interfered with the activation of CD4 + and CD8 + T cells ( Figure 6). We thus propose that EwS EVs may pathologically activate blood-circulating and tumor-infiltrating myeloid cells, thereby contributing to systemic inflammation and immunosuppression, and influencing both innate and adaptive immunity. These conclusions are based on several lines of evidence. First, EwS EVs (but not those from healthy donor plasma or fibroblast cells) induce prolonged cytokine and chemokine release and reprogram myeloid cells at the transcriptomic level, activating pro-inflammatory and type I/III IFN response gene expression programs. In particular, EwS EVs induced the release of IL-6, IL-8, and TNF by myeloid cells, consistent with similar findings in other cancers [27,28,42,43]. Despite the change of growth medium, the release of IL-6 persisted during DC differentiation and in co-cultures of EwS EV-pretreated CD14 + cells with allogeneic CD14-depleted PBMCs. Furthermore, RNA-seq analysis and co-culture experiments also showed an increase of IL-10, an immunosuppressive cytokine whose production can be triggered in myeloid and Th2 cells by IL-6 [44]. Second, as demonstrated by RNA-seq and flow cytometry, CD14 + cells differentiated in the presence of EwS EVs exhibited a semi-mature immunosuppressive phenotype, including reduced expression of co-stimulatory molecules associated with DC maturation (CD74, CD86, and HLA-DR), and with MDSC phenotypes (TRIB1, JAK2, STAT3, CXCR4, IDO1, ARG2) [45][46][47]. By transcriptomic analysis, we also observed upregulation of immune checkpoint molecules CD47 and CD274/PD-L1, which requires further investigation in terms of their impact on immunosuppressive activities of CD14 + monocytes. Similar effects were reported with EVs from hematological and other solid malignancies [27,28,43,48,49], indicative of common mechanisms operating in different types of cancer. Third, in accordance These conclusions are based on several lines of evidence. First, EwS EVs (but not those from healthy donor plasma or fibroblast cells) induce prolonged cytokine and chemokine release and reprogram myeloid cells at the transcriptomic level, activating pro-inflammatory and type I/III IFN response gene expression programs. In particular, EwS EVs induced the release of IL-6, IL-8, and TNF by myeloid cells, consistent with similar findings in other cancers [27,28,42,43]. Despite the change of growth medium, the release of IL-6 persisted during DC differentiation and in co-cultures of EwS EV-pretreated CD14 + cells with allogeneic CD14-depleted PBMCs. Furthermore, RNA-seq analysis and co-culture experiments also showed an increase of IL-10, an immunosuppressive cytokine whose production can be triggered in myeloid and Th2 cells by IL-6 [44]. Second, as demonstrated by RNA-seq and flow cytometry, CD14 + cells differentiated in the presence of EwS EVs exhibited a semi-mature immunosuppressive phenotype, including reduced expression of co-stimulatory molecules associated with DC maturation (CD74, CD86, and HLA-DR), and with MDSC phenotypes (TRIB1, JAK2, STAT3, CXCR4, IDO1, ARG2) [45][46][47]. By transcriptomic analysis, we also observed upregulation of immune checkpoint molecules CD47 and CD274/PD-L1, which requires further investigation in terms of their impact on immunosuppressive activities of CD14 + monocytes. Similar effects were reported with EVs from hematological and other solid malignancies [27,28,43,48,49], indicative of common mechanisms operating in different types of cancer. Third, in accordance with their immunosuppressive phenotype, the CD14 + cells differentiated in the presence of EwS EVs diminished activation of CD4 + and CD8 + T cells, by reducing their proliferation and IFNγ release, with a stronger effect on CD4 + cells.
Although potential mechanisms remain to be studied, our data indicate that induction of immunosuppressive myeloid cells by EwS EVs may involve both protein and RNA components. Among proteins that we identified in EwS EVs, Hsp70 and Hsp90 have been implicated in the induction of pro-inflammatory responses by myeloid cells in various cancers [27,42,[50][51][52]. However, based on our RNA-seq data, the major pathways commonly activated by Hsp90 and Hsp70, such as TLR2-MyD88 and TLR4, were reduced in the EwS EV-treated CD14 + cells compared to control. Instead, we observed the upregulation of type I/III IFN stimulated genes, including the major IFN type I/III receptors.
IFNAR1 and IFNAR2, first-line antiviral defense proteins IFITM1-3, and the cytosolic pattern-recognition receptors DDX58/RIG-1 and IFIH1/MDA5. Both RIG-1 and MDA5 are capable of recognizing pathogenic non-self RNAs and are essential components of the innate immunity activated in response to viral infections [53]. Their upregulation in both TC32 and TC71 EV-treated CD14 + monocytes may thus be indicative of the presence of pathogenic viral-like RNAs in EwS EVs. This possibility is supported by our recent findings showing that EwS EVs purified from patients' plasma and TC32, TC71, and A4573 EwS cell lines are enriched with the endogenous retroviral and retroelement-derived RNAs, including HERV-K, LINE-1, SINE/ALU, 7SL/SRP, and HSAT2 RNAs [29], each of which is immunogenic and capable of inducing RIG-1 and MDA5 [54,55]. This phenomenon may therefore underlie the observed pro-inflammatory response and acquisition of immunosuppressive properties by CD14 + and CD33 + myeloid cells. In turn, the production of pro-inflammatory and immunosuppressive cytokines by these cells, including IL-6, IL-8, TNF, and IL-10, may further alter the differentiation of myeloid cells.
Overall, our study indicates that EVs released by EwS tumors may compromise both arms of the immune system, by pathologically activating myeloid cells, skewing their differentiation, and inducing immunosuppressive activity. As shown here, EwS EV-induced semi-mature moDCs can impair activation of T cells, similar to the immunosuppressive activity of MDSCs reported in other cancers and chronic infections [22,24,56]. EwS EVs may thus contribute to the observed scarce infiltration of EwS tumors with T cells, M1 macrophages, and mature DCs, as well as the prevalence of pro-tumorigenic immunosuppressive M2 macrophages and resting DCs in the EwS TME [8,10]. Furthermore, the reported expansion of abnormally differentiated immunosuppressive CD14 + HLA-DR low/neg myeloid cells [23] and CD34 + HLA-DR + IDO + fibrocytes [57] in the blood of patients with EwS support a possibility that conversion of immature myeloid cells into immunosuppressive phenotypes may occur in patients' blood, affecting early stages of myeloid cell differentiation. As such, our study uncovers a pivotal role of EwS-and, potentially, other tumor-derived EVs in immunosuppression and cancer-associated inflammation, warranting further investigation into the mechanisms of their dissemination and targeting myeloid cells in blood circulation and the local TME.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by IRB and informed consent from the DRK-Blutspendedienst Baden-Wuerttemberg-Hessen, Ulm, Germany.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.