Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression

This article is featured in Highlights of This Issue, p. 3477

Osteosarcoma is a very aggressive bone tumor, which mainly affects children and adolescents. Lung metastases are present in approximately 20% of osteosarcoma patients at diagnosis and represent the main cause of death. However, undetectable micrometastases seem to be present in at least 80% of patients at initial diagnosis, and they are mostly resistant to the aggressive chemotherapy regimen used for osteosarcoma (1). As a consequence, the 5-year survival rate in the presence of metastatic disease does not exceed 20% (2). The rarity and heterogeneity of osteosarcoma, together with the chaotic genomic rearrangements and exceptionally frequent chromotripsis (3), are major obstacles in the search for molecular therapeutic targets. Indeed, no improvements in osteosarcoma survival have been achieved in the last 30 years (2). Recent studies pointed to a defining role for the local and systemic environment in osteosarcoma initiation and progression (4, 5). Osteosarcoma develops during the adolescent growth spurt at sites of rapid bone growth, and preferentially affects male individuals that are taller for their age (6, 7). Intercepting the environmental factors sustaining osteosarcoma may halt or even reverse malignant progression, thereby providing novel therapeutic options.

The tumor microenvironment takes part in virtually every aspect of cancer development and progression (8). Mesenchymal stem cells (MSC) are established contributors to malignant dissemination in multiple cancer types, including breast cancer, brain tumors, colon cancer, and osteosarcoma (9–12). MSCs are adult stem cells that home to sites of inflammation, where in response to environmental cues they can differentiate into cancer-supporting cells. Cancer-associated MSCs provide essential factors for malignant progression (13). MSC-derived CCL5 promotes metastasis formation in breast and prostate cancer (9, 14), IL6 released by MSCs supports tumor growth and angiogenesis in colorectal cancer (15), and MSC-derived stromal-derived growth factor-1 (SDF-1) favors epithelial–mesenchymal transition (EMT) and metastasis in prostate cancer (16). However, how tumor cells influence MSC behavior to favor metastatic progression is not understood.

Tumor cells are prolific producers of extracellular vesicles (EV), including a significant proportion of vesicles of endosomal origin called exosomes (17). Exosomes are 40 to 100 nm vesicles carrying a bioactive cargo of the cell of origin, including proteins, lipids, and regulatory RNAs (18). In addition, tumor cells shed membrane vesicles from their surface that are difficult to distinguish from exosomes based on protein content (19). Therefore, we will refer to the heterogeneous population of vesicles released by cancer cells using the term EVs. Once released, EVs can be taken up by surrounding cells or carried to distant sites via the blood or lymph circulation and influence target cell behavior (18, 20, 21). Cancer EVs can transport functional RNAs that promote angiogenesis (22), oncoproteins involved in premetastatic niche formation (20), and heat shock proteins (HSP) that can suppress antitumor immune responses (23). Circulating tumor EVs can be detected in cancer patients and have remarkable diagnostic and prognostic potential (24) and may predict response to treatment (25).

We provide evidence that osteosarcoma-produced EVs trigger a prometastatic inflammatory loop by altering the physiology of MSCs. We reveal that EVs from metastatic osteosarcoma cells carry a membrane-associated form of TGFβ that educates human MSCs to produce IL6 in vitro. When injected in a preclinical mouse model, “tumor-educated” MSCs (TEMSC) promote osteosarcoma growth and lung metastasis formation. Importantly, coadministration of a therapeutic IL6 receptor (IL6R) antibody abolishes the cancer-promoting effects of TEMSCs. Our study reveals IL6 and TGFβ as rational targets for therapeutic intervention in osteosarcoma patients.

Tissue microarrays from paraffin-embedded tumor tissue were previously constructed (26). All specimens were handled according to the ethical guidelines described in Code for Proper Secondary Use of Human Tissue in The Netherlands of the Dutch Federation of Medical Scientific Societies. Immunohistochemistry was performed as described previously (27). The phospho-STAT3(Tyr705)(D3A7) antibody (Cell Signaling Technology) was used at a 1:400 dilution. Lung carcinoma tissue was used as a positive control for titration. Cores from 103 tumor tissues were scored by staining intensity and percentage of positive cells (average scores from 3 cores/tumor were calculated), and the percentage of pSTAT3-positive tumors was calculated (cutoff value, 5% positive cells/tumor tissue). The analysis was performed by two operators independently.

Osteosarcoma tissues for RNA-seq analysis were collected from 18 patients in Vietnam who had histologically confirmed osteosarcoma and were allocated for surgery. Tumor and normal bone samples were collected from the removed bone immediately after the operation. Samples were stored at −80°C until RNA extraction. Protocols were approved from the ethics committee on biomedical research of the Hue University hospital. All the participants or representative of patients signed the informed consent.

Serum samples used in this study were prepared and stored by CRO-Biobank (CRO National Cancer Institute, Aviano, Italy). The CRO-Biobank project has been approved by the CRO Institutional Ethics Committee and all participants provided written informed consent. Briefly, blood samples were collected in Serum Z tubes (Monovette, Sarstedt), placed on ice, and centrifuged at 2,608 × g for 10 minutes at room temperature. Aliquots of serum were then stored at −80°C.

All clinical samples used in this study were used in compliance with the Declaration of Helsinki.

Human adipose tissue samples were obtained from the department of Plastic Surgery of the Tergooi Hospital (Hilversum, the Netherlands) after institutional Ethical committee approval and written informed consent. MSCs were isolated as previously described (28). GFP-positive adipose-derived MSCs were obtained from the Department of Medical and Surgical Sciences for Children and Adults (University of Modena and Reggio Emilia, Italy; ref. 29). MSCs were expanded in alpha-MEM (Lonza), supplemented with 5% platelet lysate and 10 U/mL heparin (Leo Pharma). MG63, HOS, and 143B osteosarcoma cells were cultured in IMDM supplemented with 10% FBS. Primary human fibroblasts were a kind gift from J.M. Middeldorp (VU University Medical Center, Amsterdam, the Netherlands) and were cultured in DMEM 10% FBS. Primary osteosarcoma cells were kindly provided by V.W. van Beusechem (VU University Medical Center, Amsterdam, the Netherlands) and cultured in EMEM 10% FBS. All media were supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco).

The endolysosomal compartment of osteosarcoma cell lines was characterized by confocal laser scanning microscopy and transmission electron microscopy (TEM) as previously described (28).

For cell-cycle analysis, MSCs were exposed to osteosarcoma or control EVs for 48 hours (two EV treatments) and incubated overnight with nocodazole (250 ng/mL) to arrest them in G₂–M. The following day cells were collected, washed, and resuspended in PBS containing 0.6% NP-40, 50 mg/mL RNaseA and 50 mg/mL propidium iodide for 10 minutes, and analyzed using a FACSCalibur flow cytometer (BD Biosciences). For the osteogenic differentiation assay MSCs were cultured in the presence of 0.1 mol/L ascorbic acid and 10⁻⁸ mol/L dexamethasone. At cell confluence, 10 mmol/L β-glycerophosphate was added to the cultures, and after one week mineral deposition was assessed by Alizarin red staining.

To evaluate whether IL6 induction in MSCs was dependent on the EV RNA, MSCs were seeded in 12-well plates at a density of 70,000 cells per well. The day after cells were transfected with EV-RNA or 20 ng poly(I:C) (Sigma-Aldrich; positive control) using Lipofectamine 2000 (Life Technologies), or treated with matching amounts of osteosarcoma EVs. Forty-eight hours after transfection, MSCs were lysed in TRIzol (Life Technologies) for IL6 expression analysis.

To assess whether IL6 induction was dependent on TGFβ signaling, MSCs were preincubated for 30 minutes with the activin receptor-like kinase (ALK) receptor inhibitor SB-431542 (Sigma-Aldrich) at a concentration of 10 μmol/L and then exposed to osteosarcoma EVs or 5 ng/mL soluble human TGFβ (ProSpec). A second EV/TGFβ treatment was performed after 24 hours. At 24 and 48 hours cells were lysed in TRizol for mRNA expression analysis. The MSC conditioned medium was harvested to perform IL6 ELISA.

EVs were isolated from the culture supernatant using differential centrifugation (28). EV preparations and corresponding cells of origin were characterized by Western blot analysis for enriched EV proteins as CD63 and CD81 and by TEM, and the EV RNA profile was analyzed using the Bioanalyzer (Agilent) as previously described (28).

Vesicle internalization after staining with PKH67 (Sigma-Aldrich) was assessed by fluorescence microscopy and FACS analysis. To inhibit EV internalization, cells were pre-incubated for 30 minutes with 50 μmol/L dynasore (Sigma-Aldrich).

EVs from serum were purified by size-exclusion chromatography (SEC) as previously described (30). Fractions 9 and 10 were considered as EV-enriched fractions, and subjected to TGFβ protein quantification by ELISA.

Quantification of a panel of inflammatory cytokines was performed using the BD Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit (BD Biosciences) following the manufacturer's instructions.

IL6 and TGFβ protein concentration were assessed using the Human IL-6 and Human TGF-beta 1 DuoSet ELISA (R&D Systems) respectively. Quantification of soluble IL6R was carried out using the Human IL-6 R alpha Quantikine ELISA Kit (R&D Systems), according to the manufacturer's instructions.

Total RNA was isolated using TRizol Reagent (Life Technologies). Exosome preparations were pre-treated with RNase A (Sigma-Aldrich) at a final concentration of 400 ng/μL at 37°C for 1 hour to degrade unprotected RNAs. IL6 mRNA expression was analyzed using SYBR Green PCR master mix in a LightCycler 480 Real-time PCR System (Roche) with the following primer sets:

IL6-F: 5′-AGTGAGGAACAAGCCAGAGC-3′; IL6-R: 5′-CATTTGTGGTTGGGTCAGG-3′.

The mRNA expression of TLR3, TLR7, TLR8, and TLR9 was analyzed using the Universal ProbeLibrary system (Roche Applied Science). Probes and primers were selected using the web-based ProbeFinder software. Results were normalized to GAPDH.

Animal experiments were performed in accordance with the Dutch law on animal experimentation with the approval of the Committee on animal experimentation of the VU University medical center (Amsterdam, the Netherlands).

For orthotopic tumor xenografting, a single-cell suspension of exponentially growing luciferase-positive 143B cells was injected into the tibia of nude mice. Briefly, 6-weeks old athymic nude-Foxn1^nu mice (Harlan; n = 6/treatment arm) received buprenofine s.c. (0.05 mg/kg) and were anesthetized with isoflurane (2%–3% in oxygen). After anesthesia, the knee was flexed beyond 90°, a skin incision was made to expose the tibia and a pinhole was made using a 0.8 mm drill. A volume of 1 μL cell suspension (approximately 2 × 10⁵ cells) was injected into the hole using a 25-gauge needle. The hole was closed with tissue glue to prevent backflow, and the skin was closed with sutures. The anti-IL6R antibody tocilizumab (100 μg/mouse, i.p.) was administered at day 1 and every other day until the experimental endpoint. Control animals were treated with PBS. One million GFP-positive MSCs educated or not educated with osteosarcoma EVs were injected intravenously (100 μL) at day 2. Tumor growth was monitored by bioluminescence imaging (BLI). Briefly, 150 μL of D-luciferin (0.03 g/L, Gold Biotechnology) were injected intraperitoneally, and 10 minutes after administration mice were anesthetized with isoflurane and positioned in the IVIS camera. The bioluminescence signal was determined with the IVIS Lumina CCD camera. Mice were monitored daily for discomfort and weight loss. When the first animal presented moderate to severe symptoms of discomfort (weight loss of >15% or tumor diameter >15 mm), all animals were sacrificed. The duration of the experiments was approximately 3 weeks. The bioluminescence signal of the ex vivo tissues was measured with the IVIS, and tissues were formalin-fixed or cryopreserved for histological analysis. Two animal experiments with 6 mice per experimental group were performed. In Fig. 2, results from both experiments were pooled. Figure 5 shows the results of the second experiment only.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) mouse tissue (lung) slides was performed according to standard protocols. Briefly, heat-mediated antigen retrieval was performed using citrate buffer. Slides were incubated with the Vimentin antibody (V9; Santa Cruz Biotechnology) diluted 1:150, and counterstained with hematoxylin.

To assess the presence of GFP-positive MSCs in tumor and bone marrow tissues, mouse tibias were decalcified in EDTA pH 7.2–7.4. Antigen retrieval was performed using citrate buffer. Tissue slides were stained with a rabbit polyclonal anti-GFP antibody (Abcam, ab290) in a 1:900 dilution, and counterstained with DAPI.

For the pSTAT3 staining of FFPE osteosarcoma xenograft slides, antigen retrieval was performed with a TRIS-EDTA buffer pH 9, and slides were incubated with the phospho-STAT3(Tyr705; D3A7; 1:100) and with the goat-anti–rabbit-Alexa 546 (Life Technologies; 1:200) antibodies and counterstained with DAPI. Images were acquired with an LSM700 confocal laser scanning microscope equipped with an LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective (Zeiss). Positivity was determined using the LSM Image Browser (version 4.2.0.121, Zeiss).

Bone samples (40–50 mg) were grinded into powder with nitrogen in a mortar and lysed using TRizol. Total RNA was extracted with the RNeasy Fibrous Tissue Mini Kit (Qiagen) according to the manufacturer's protocol. The quality of total RNA was assessed with Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit (Agilent Technologies). Fifty ng of total RNA were amplified by applying Ovation RNA-Seq System V2 (NuGen). The resulting cDNAs were pooled in equal amounts and the DNA fragment library was prepared with SOLiD System chemistry (Life Technologies). Sequencing was performed using SOLiD 5500W platform and DNA sequencing chemistry (Life Technologies). Raw reads (75 bp) were color-space mapped to the human genome hg19 reference using the Maxmapper algorithm implemented in the Lifescope software (Life Technologies). Mapping to multiple locations was permitted. The quality threshold was set to 10, giving the mapping confidence was more than 90. Reads with score less than 10 were filtered out. Average mapping quality was 30. Analysis of the RNA content and gene-based annotation was done within whole transcriptome workflow. For statistical analysis DeSeq2 package for R was used (31).

Statistical analysis was performed using the IBM SPSS statistics software. Data were expressed as means ± standard deviation or standard error of the mean (SEM). Two-tailed t test and one-way ANOVA were applied to assess the effects of independent variables on quantitative results. The post-hoc Fisher's Least Significant Difference (LSD) test was applied to highlight the differences between individual groups. P values ≤ 0.05 were considered statistically significant.

To study the EV population released by osteosarcoma cells, we used two non-metastatic (MG63 and HOS) and one metastatic (143B) osteosarcoma cell lines. We first analyzed the cellular endosomal compartment by immunofluorescent staining for CD63. We found high punctate expression of CD63, localized both in acidic and nonacidic vesicles throughout the cell body as determined by lysotracker costaining (Fig. 1A, top). By transmission electron microscopy (TEM) we revealed the presence of multiple 500 nm late endosomes with internal vesicular structures of 40 to 100 nm (Fig. 1A, bottom), resembling multivesicular bodies (MVB).

We then isolated osteosarcoma EVs using differential centrifugation. The purity of the preparations was confirmed by TEM (Fig. 1B) and Western blot analysis for the exosomal proteins CD63 and CD81 (Fig. 1C). TEM analysis of cell compartments and purified vesicles suggests that osteosarcoma cells release a greater amount of EVs compared to their normal counterparts (bone marrow-MSCs) we previously analyzed (28).

To investigate whether osteosarcoma EVs can interact with MSCs, we labeled purified vesicle preparations with a fluorescent lipid dye (PKH67) before incubation with MSCs. After 24 hours, we observed EV internalization by fluorescence microscopy and FACS analysis (Fig. 1D; Supplementary Fig. S1). To identify non-malignant vesicles that could be used as controls for following assays we evaluated the ability of MSCs to internalize human fibroblast (hF) and MSC EVs. We found that the uptake efficiencies of osteosarcoma EVs (96.9%–99.2% positive cells) and control EVs (93.3%–99.2% positive cells) were highly similar (Supplementary Fig. S1). These observations suggest that besides phagocytic cells, as dendritic cells and macrophages (32, 33), human primary MSCs efficiently capture and internalize EVs from various cell types, including tumor (osteosarcoma) cells.

To investigate whether osteosarcoma EVs alter the physiology of MSCs such that they promote tumor progression, we developed a bioluminescent orthotopic xenograft mouse model of osteosarcoma. Human primary GFP-positive MSCs were expanded and exposed for 48 hours to EVs purified from metastatic 143B cells. Metastatic luciferase-positive 143B cells were inoculated in the tibia of nude mice, and after 2 days the osteosarcoma-bearing mice were subjected to a single systemic administration of “tumor-educated” MSCs (TEMSC, Fig. 2A). Mice receiving non-educated MSCs or no MSCs were used as control groups. Tumor growth was monitored by bioluminescence imaging (BLI). As early as day 10 after inoculation we observed increased tumor growth in mice that received TEMSCs compared with the control groups. The difference in tumor volume became increasingly prominent at the following time points (Fig. 2B and C).

The dynamics of the tumor microenvironment seem to have features of a wound-healing process (34), including the recruitment of MSCs (35). We searched for the presence of GFP-positive MSCs in decalcified mouse tibias 4 days after systemic injection, and found GFP-positive cells within the tumor mass and in the adjacent bone marrow tissue of mice receiving TEMSCs and control MSCs (Fig. 2D).

Next, we investigated whether TEMSCs promote osteosarcoma metastatic progression. Ex vivo BLI of lungs, liver, and spleen (Fig. 2E) revealed that metastatic dissemination exclusively occurred in the lungs, the most common metastatic sites in osteosarcoma patients. The number of lung metastases across the experimental groups was determined by BLI and naked eye evaluation. Strikingly, administration of TEMSCs significantly increased the number of metastases compared with the control groups (Fig. 2F). The presence of metastases in the lungs was confirmed by human vimentin staining (Fig. 2G). No GFP-positive MSCs were detected in the lung tissue at the experimental endpoint.

Collectively these observations demonstrate that osteosarcoma EVs prompt MSCs to acquire a protumorigenic and prometastatic phenotype in vivo.

Because MSCs have specialized immunomodulatory functions (36), we wondered whether osteosarcoma EVs affect cytokine production by MSCs. We used a multiplexed bead-based assay to profile the cytokine production of MSCs upon treatment with EVs. Interestingly, we observed that education of human primary MSCs with EVs from the metastatic 143B cells increased the production of IL6 and IL8 when compared with control EVs (Fig. 3A; Supplementary Fig. S2A). We noticed that osteosarcoma cell lines and primary osteosarcoma cells release IL8, but do not produce detectable levels of IL6 (Fig. 3B; Supplementary Fig. S2B and S2C). Therefore, we postulated that MSCs may act as tumor-supporting stroma cells by supplying exogenous IL6 in vivo. We confirmed that 143B EVs educate MSCs to produce IL6 both at the mRNA (Supplementary Fig. S2D) and protein level (Fig. 3C). The responsiveness of target cells to IL6 depends either on the expression of the surface IL6 receptor (IL6R) or on the availability of a soluble form of the IL6R (sIL6R). Although the expression of the surface IL6R is limited to few cell types in vivo, we found that both osteosarcoma cells and MSCs produce sIL6R (Fig. 3D). The production of sIL6R by MSCs was however not influenced by treatment with osteosarcoma EVs (Supplementary Fig. S2E). Taken together, these findings demonstrate that osteosarcoma cells release EVs inducing IL6 production in MSCs, and can respond to MSC-derived IL6 in a cell-autonomous fashion.

Because IL6 is implicated in proliferation and stemness maintenance of MSCs (37), we investigated the effects of osteosarcoma EVs on cell-cycle progression and osteogenic differentiation of these cells. We cultured MSCs in the presence of osteosarcoma EVs for 48 hours and then treated cells with nocodazole overnight to prevent mitosis. FACS analysis showed that 143B EVs determined greater accumulation of cells in the G₂–M phase compared with control EVs or untreated condition (set at 0; Fig. 3E and F), suggesting that osteosarcoma EVs accelerate the transition from G₁ to G₂–M. To study whether EVs affect the differentiation of MSCs we cultured early passage MSCs in osteogenic conditions and evaluated the formation of mineral nodules with Alizarin red staining (Supplementary Fig. S2F). No differences were observed in response to osteosarcoma EV treatment, suggesting that osteosarcoma EVs promote cell-cycle progression, but do not affect the osteogenic differentiation ability of MSCs.

Osteosarcoma EVs induce IL6 release and cell-cycle progression in MSCs, but the mechanism underlying these effects is unclear. One possibility is that osteosarcoma EVs transfer inflammatory small RNAs that are recognized by intracellular sensors within the endosomal compartments of the MSCs (32, 38, 39). We extracted RNA from osteosarcoma EVs and analyzed their small RNA profile using the Bioanalyzer. The small RNA profile showed characteristic exosomal RNA peaks between 20 and 70 nt (Fig. 4A). RNA-seq analysis revealed high abundance of polymerase III transcripts (data not shown), which can induce inflammatory responses in recipient cells (32, 39). We then determined the expression of endosomal Toll-like receptors (TLR) in MSCs (Fig. 4B), and transfected cells with the RNA isolated from osteosarcoma EVs (EV-RNA). Although MSCs strongly responded to the TLR3 agonist poly(I:C), no effect on IL6 expression was observed in response to isolated EV-RNA (Fig. 4C, left). However, a single treatment with matching amounts of intact tumor EVs prompted a 2-fold increase in IL6 mRNA expression (Fig. 4C, right). These observations suggest that EV components other than RNAs induce IL6 production in recipient MSCs.

One emerging concept is “direct signaling” of EVs where factors located at the surface of EVs can change the physiology of target cells (40, 41). To investigate this possibility, we blocked EV endocytosis in MSCs using dynasore and subsequently incubated MSCs with PKH67-labeled 143B EVs for 24 hours. Although dynasore treatment decreased 143B EV internalization by more than 60%, EV-mediated induction of IL6 MSCs was not affected (Fig. 4D), suggesting that this is not fully dependent on EV internalization by MSCs.

It has been recently shown that several growth factors, such as TNFα, FGF, and TGFβ, can be detected in association with EVs (42–44). TGFβ is a pleiotropic cytokine highly expressed by high-grade osteosarcoma (45) and presumably functions as an autocrine growth factor for osteosarcoma (46). Apart from the well-established actions of soluble TGFβ, a vesicle-associated form of TGFβ has been implicated in the stimulation of cytokine production and cancer progression (44). We quantified TGFβ in osteosarcoma EVs by ELISA and found high protein levels in osteosarcoma EVs compared to non-malignant fibroblast control vesicles (143B EVs: 593.9 ± 29.5 pg/mL; hF EVs: 146.1 ± 15.5 pg/mL; Fig. 4E). Importantly, blocking TGFβ signaling in MSCs by means of a TGFβ type I receptor (ALK) inhibitor strongly decreased EV-mediated IL6 induction in MSCs (Fig. 4F; Supplementary Fig. S3A and S3B). Addition of recombinant soluble TGFβ (sTGFβ) to MSC cultures did not reproduce IL6 induction in the MSCs (Fig. 4F; Supplementary Fig. S3A and S3B). These observations implicate the EV-associated form of TGFβ in the prometastatic inflammatory loop established by osteosarcoma EVs.

We then asked whether an anti-IL6R antibody (tocilizumab) could reverse the effects of TEMSCs on tumor progression. Mice bearing bioluminescent osteosarcoma xenografts were injected with TEMSCs 2 days after tumor cell orthotopic inoculation. Tocilizumab (100 μg/mouse) was administered intraperitoneally one day after tumor cell inoculation and every other day until the end of the experiment. Mice receiving noneducated MSCs and no MSCs (not shown) were used as control groups. We found that tocilizumab reduced tumor growth as early as day 10 after inoculation with osteosarcoma cells (Fig. 5A and B). Of note, mice receiving tocilizumab treatment displayed BLI signals that overlapped with the control group receiving noneducated MSCs.

It is well-established that the prooncogenic effects of IL6 are mediated by STAT3, which links inflammation to cancer (47). We observed that TEMSCs induce an increase in nuclear pSTAT3 in tumor tissues, which was prevented by the concurrent administration of tocilizumab (Fig. 5C and D, Supplementary Fig. S3C). Most importantly, the administration of tocilizumab reverted the prometastatic effects of TEMSCs in vivo (Fig. 5E and F). Collectively, these data show that tumor EVs activate a prometastatic IL6/STAT3 signaling axis in osteosarcoma by engaging MSCs (Fig. 5G). However, we cannot rule out a possible contribution of mouse IL6 to cancer progression in our model, as the anti-IL6 receptor antibody would also prevent the potential cross-reaction between mouse IL6 and human IL6R.

To confirm the role of IL6/STAT3 signaling in primary osteosarcoma tissues, we first analyzed the IL6 mRNA expression in 84 pre-treatment high-grade osteosarcoma diagnostic biopsies using a publicly available dataset (48) in the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). In accordance with our in vitro data, IL6 mRNA levels in osteosarcoma biopsies were low compared to osteoblasts, MSCs and bone tissue (Fig. 6A). However, the immunohistochemical analysis of osteosarcoma tissue microarrays (TMA) revealed pSTAT3 nuclear staining in 65% of biopsies (n = 103, cutoff value: 5% positive cells/tumor tissue; Fig. 6B), suggesting that STAT3 activation in tumor tissues is most likely determined by an exogenous source of IL6.

To confirm these data, we performed RNA-seq analysis of osteosarcoma tissues and surrounding bone of 18 osteosarcoma patients (Supplementary Table S1). Again, IL6 expression was relatively low in tumors and does not significantly differ from that of normal bone (mean rpm: 11.71 ± 3.4 vs. 9.6 ± 2.9; Supplementary Fig. S4A). Curiously, TGFβ mRNA was also not differentially expressed between normal and tumor tissues (Supplementary Fig. S4B), whereas multiple TGFβ-induced genes were strongly upregulated (Fig. 6C and D; Supplementary Fig. S4C and S4D; Supplementary Table S2). Among these, COL11A1 and TGFβI were the top two upregulated genes in the analysis (log2 FC 1.51, P = 1.06E−14 and log2 FC 1.40, P = 1.35E−11, respectively). Gene set enrichment analysis (GSEA) of the top upregulated genes in osteosarcoma tumors (log₂ FC > 1, P < 0.0001) showed an overlap with 4 extracellular matrix genes early induced by TGFβ in fibroblasts (49). These include three collagen genes identified as definite TGFβ/SMAD3 targets (COL3A1, COL6A1, COL6A3), and MMP14 (Fig. 6D). In support of the role of TGFβ in osteosarcoma, elevated TGFβ mRNA in high-grade osteosarcoma biopsies is associated with a decrease in metastasis-free survival of osteosarcoma patients (Supplementary Fig. S4E).

Finally, we examined whether osteosarcoma patients have increased levels of EV-bound TGFβ in circulation. To this end, we purified EVs from patient serum (Supplementary Table S3) using size-exclusion chromatography and quantified TGFβ levels by ELISA. Our analysis revealed that serum levels of EV-associated TGFβ are significantly higher in osteosarcoma patients compared to healthy control individuals (277.5 ± 35.3 vs. 119.3 ± 39.1 pg/mL; Fig. 6E). To evaluate how well TGFβ levels discriminate between osteosarcoma patients and healthy individuals, we used an ROC curve and obtained an AUC score of 0.88 (Fig. 6F). These data suggest that these TGFβ-carrying vesicles, arguably of tumor origin, might act on stromal and tumor cells to sustain cancer progression.

Tumor-secreted EVs promote metastasis formation in various mouse models. Using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we show that tumor cells instruct MSCs to activate the oncogenic IL6/STAT3 signaling axis, which is consistent with MSC receptiveness to local stimuli (9–12, 16). We demonstrate that the osteosarcoma-secreted EVs carry functional TGFβ molecules that interact with MSCs and alter their behavior to promote tumor growth and metastasis formation. Our observations suggest that blocking IL6 and TGFβ signaling might represent a valid therapeutic strategy for osteosarcoma.

Tumor-derived EVs can contribute to malignant progression by aiding in the formation of the premetastatic niche (20, 50, 51). This process may involve the education of bone marrow–derived and/or local specialized cells that shape a favorable environment at the metastatic site for malignant cells to seed and grow (20, 50, 51). Moreover, others have shown that stromal cell EVs also participate in tumorigenesis by providing a favorable environment (52). We propose a third protumorigenic EV-mediated mechanism by which tumor cell-secreted EVs act on defined subset of stromal cells directly at the primary tumor site establishing a local proinflammatory loop. We show that short-time ex vivo conditioning of MSCs by tumor EVs is sufficient to increase metastasis formation in vivo. The MSC contribution to metastasis formation is largely dependent on increased IL6 expression, leading to the activation of the STAT3 oncogene in the primary bone tumor.

In various cancer types, chronic or even short activation of the IL6/STAT3 axis is a key event in cancer development and progression (53). IL6/STAT3 signaling supports cancer cell proliferation, metastasis formation, tumor immunosuppression, and cancer stem cell self-renewal (47). In addition, overexpression of IL6 and its receptor (IL6R) is observed frequently in multiple cancer types (54). Osteosarcoma patients seem to have high IL6 serum levels compared with control individuals (55, 56), and sustain activated intra-tumor STAT3 signaling as we demonstrate in this study (Fig. 6B). Surprisingly, osteosarcoma tumor cells in vivo, primary osteosarcoma cells cultured in vitro as well as most osteosarcoma cell-lines that we studied express low to virtually undetectable levels of IL6 (Figs. 3B and 6A; and Supplementary Fig. S4A). These observations, combined with the activated STAT3 signaling observed in osteosarcoma tumors, suggests that an exogenous source of IL6 must be involved, strengthening the notion that tumors progress with the support of the microenvironment (8).

The complexity and heterogeneity of EVs complicates the identification of biomolecules that modify the physiology of recipient cells. We and others previously showed that both tumor EV-protected small RNAs and virus-derived small RNAs can induce inflammatory responses in target cells by triggering intracellular RNA sensors (32, 38, 39). Although we found that MSCs express functional endosomal TLRs, they are unresponsive to isolated tumor EV-RNA suggesting that IL6 induction in response to osteosarcoma EVs is mediated by alternative mechanisms. Multiple EV-associated proteins including proto-oncogenes and HSPs have been implicated in the intercellular communication networks that support cancer progression (20, 23). Depending on their localization EV-associated proteins might not require internalization by target cells to signal. However, until now, conclusions on EV function and tropism have been mostly drawn based on vesicle uptake by recipient cells (20, 21, 33, 50–52). We demonstrate that osteosarcoma EVs alter MSC physiology independently of internalization, by carrying membrane-associated TGFβ to the surface of MSCs, where TGFβ can interact with its receptor (Fig. 4D–F).

TGFβ is a key molecule in many metastasis models (57), and has a central role in the communication between cancer and stromal cells leading to disease progression (58). In osteosarcoma TGFβ has been previously implicated as an autocrine growth factor. Indeed, TGFβ mRNA expression in osteosarcoma tissues associates with high-grade tumors (45) and negatively correlates with metastasis-free survival (R2: Genomics Analysis and Visualization Platform, Kuijjer dataset). We show that in osteosarcoma patients metastasis-associated TGFβ-induced genes are overexpressed in the tumor tissue compared with the surrounding normal bone (Supplementary Table S2). Intriguingly, we could not detect differential expression of TGFβ, at least at the mRNA level (Supplementary Fig. S4B). This suggests that TGFβ, secreted in a latent form, is activated within the tumor mass more so than in the surrounding normal bone tissue. An alternative explanation is that the levels of EV-bound TGFβ, rather than the total amount of TGFβ protein, ultimately determines downstream target gene expression. In fact, we demonstrate that malignant osteosarcoma EVs carry high levels of membrane-bound TGFβ compared with EVs secreted by non-transformed cells (Fig. 4E), which corresponds with their ability to educate MSCs to produce IL6 (Fig. 3C). Importantly, soluble TGFβ did not reproduce the effects of the EV-associated form on the MSCs (Fig. 4F; Supplementary Fig. S3A and S3B), a finding supported by recent independent studies (44, 59). Thus, a growth factor in association with EVs has distinct signaling properties than its soluble form (40, 41). We propose that the conformation acquired by TGFβ on the EV surface, the combination with other EV-associated factors, or the presence of costimulatory signals on tumor EVs (59), might enhance or alter the TGFβ signaling properties.

To demonstrate the clinical significance of vesicle-associated TGFβ in osteosarcoma, we quantified the levels of EV-bound TGFβ in human serum. We found that osteosarcoma patients have much higher levels of EV-associated TGFβ compared with healthy control individuals. Arguably, multiple cell types, including immune cells, might be exposed to the high levels of local or systemic EV-bound TGFβ in osteosarcoma patients. Although the use of a xenograft mouse model allows to study the interactions between cell types of human origin in vivo, it limits the possibility to investigate the contribution of immune components such as tumor-infiltrating T cells to cancer progression. Further studies using syngeneic mouse models need to be performed to obtain a more complete picture of tumor–stromal cell interactions in osteosarcoma, and to evaluate the potential role of EV-associated TGFβ and IL6 in tumor immune escape.

Currently, adolescent osteosarcoma patients receive one of the most aggressive treatment regimens, whereas prognosis in the presence of metastases remains discouraging (2). This is the first study addressing the role of EV-mediated tumor–stroma communication in osteosarcoma. We describe the establishment of a prometastatic inflammatory loop initiated by osteosarcoma EVs that can be disrupted to inhibit osteosarcoma progression. This is relevant because IL6 and TGFβ inhibitors are novel attractive targets for anti-cancer therapy (60, 61). In particular, the anti-IL6R antibody used in this study (tocilizumab), already approved for the treatment of rheumatic diseases, has been evaluated with encouraging results in a phase I trial for recurrent ovarian cancer (NCT01637532), and will be tested for the treatment of pancreatic cancer (NCT02767557) and Chronic Lymphocytic Leukemia (NCT02336048). Osteosarcoma is a rare tumor of childhood and adolescence, which complicates large clinical studies stressing the need for preclinical models. Although it is unlikely that IL6-blocking antibodies, used as single therapeutic agents, may result in patient response, combination with current chemotherapy treatment may improve osteosarcoma survival and allow to lower the dosage of chemotherapeutic drugs, reducing toxicity. Moreover, our findings suggest that combination of IL6-blocking agents with TGFβ inhibitors might halt osteosarcoma progression while reducing resistance.

D.M. Pegtel is an employee of and holds ownership interest (including patents) in Exbiome BV and is a consultant/advisory board member for Takeda Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S.R. Baglio, T. Lagerweij, X.D. Ho, T.D. de Gruijl, K. Maasalu, N. Baldini, D.M. Pegtel

Development of methodology: S.R. Baglio, L. Roncuzzi, N. Zini, A. Martson, D.M. Pegtel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.R. Baglio, T. Lagerweij, M.P. Lanzón, X.D. Ho, N. Léveillé, S.A. Melo, A.-M. Cleton-Jansen, E.S. Jordanova, M. Dominici, R. Bonafede, S. Cervo, A. Steffan, V. Canzonieri, A. Martson, K. Maasalu, S. Köks, T. Wurdinger

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.R. Baglio, T. Lagerweij, M.P. Lanzón, X.D. Ho, E.S. Jordanova, A. Martson, K. Maasalu, N. Baldini, D.M. Pegtel

Writing, review, and/or revision of the manuscript: S.R. Baglio, T. Lagerweij, M.P. Lanzón, E.S. Jordanova, L. Roncuzzi, M. Dominici, T.D. de Gruijl, K. Maasalu, T. Wurdinger, N. Baldini, D.M. Pegtel

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Lagerweij, M.P. Lanzón, M. Greco, M.A.J. van Eijndhoven, G. Grisendi, R. Bonafede, S.M. Lougheed, S. Cervo, V. Canzonieri, A. Martson, K. Maasalu, S. Köks

Study supervision: D.M. Pegtel

Other (funding): N. Baldini

The authors thank H.W.M. Niessen (VU University Medical Center, Amsterdam, the Netherlands) for the provision of human MSCs; J.M. Middeldorp (VU University Medical Center, Amsterdam, the Netherlands) for the provision of human fibroblasts; V.W. van Beusechem (VU University Medical Center, Amsterdam, the Netherlands) for providing primary osteosarcoma cells; A. Avan and E. Giovannetti (VU University Medical Center, Amsterdam, the Netherlands) for their support in the in vivo experimentation; G. Bonuccelli (Istituto Ortopedico Rizzoli, Bologna, Italy) for her assistance in tissue staining; J. Letterio (UH Cleveland Medical Center, Cleveland, OH) for constructive discussion.

S.R. Baglio was supported by a fellowship by Associazione Italiana per la Ricerca sul Cancro (AIRC) co-funded by the European Union, by the Veronesi Foundation and by the L'Oréal UNESCO “For Women in Science.” This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 660200 (to S.R. Baglio). In addition, this work was supported by grants from the Dutch Cancer Society (KWF, grant No VU2012-5510; to D.M. Pegtel), and the Italian Association for Cancer Research (AIRC, grant No 15608; to N. Baldini).

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