Exosomal PD-L1 Derived From Osteosarcoma is Associated With Pulmonary Metastasis for Patients With Osteosarcoma


 Background

Recent studies indicated that exosomal programmed death-ligand 1 (PD-L1) derived from cancers could induce immunosuppression and tumor pathogenesis. However, it is unclear how the exosome influences the osteosarcoma (OS) progression and whether PD-L1 also exists in serum exosomes (Sr-exosomes) of patients with osteosarcoma.
Methods

We examined serum exosomes from 70 patients with osteosarcoma, 9 patients with benign tumors and 22 healthy donors. Osteosarcoma-derived exosomes and exosomal-PD-L1 were functionally evaluated using in vivo and in vitro models.
Results

The characteristics of serum exosomes of OS patients and OS cell line exosomes were confirmed by several methods. Bioinformatic analysis demonstrated that Sr-exosomes isolated from OS patients may involve in the important process of immune function and cancer pathogenesis for OS patients. Meanwhile, exosomes derived from OS cell line and serum of OS patients could induce osteosarcoma migration and invision in vitro. Then, we confirmed PD-L1 existing in Sr-exosomes of patients with osteosarcoma and higher level of Sr-exosomal PD-L1 in OS patients compared to healthy donors and patients with benign tumor. Furthermore, exosomal-PD-L1 derived from osteosarcoma promoted the pulmonary metastasis, which was demonstrated in the osteosarcoma metastatic model.
Conclusion

We confirm that osteosarcoma releases the exosomes carrying PD-L1 on their surface and induces the pathogenesis of pulmonary metastasis for OS patients.


Results
The characteristics of serum exosomes of OS patients and OS cell line exosomes were con rmed by several methods. Bioinformatic analysis demonstrated that Sr-exosomes isolated from OS patients may involve in the important process of immune function and cancer pathogenesis for OS patients.
Meanwhile, exosomes derived from OS cell line and serum of OS patients could induce osteosarcoma migration and invision in vitro. Then, we con rmed PD-L1 existing in Sr-exosomes of patients with osteosarcoma and higher level of Sr-exosomal PD-L1 in OS patients compared to healthy donors and patients with benign tumor. Furthermore, exosomal-PD-L1 derived from osteosarcoma promoted the pulmonary metastasis, which was demonstrated in the osteosarcoma metastatic model.

Conclusion
We con rm that osteosarcoma releases the exosomes carrying PD-L1 on their surface and induces the pathogenesis of pulmonary metastasis for OS patients.

Background
Exosomes are secreted as the vesicles of a size range of 30 to 150 nm diameter, which are enclosed by the cell membrane. Inward budding of late endosomes develop into intracellular multivesicular endosomes and RNA, DNA, proteins and other small moleculars are encapsulated into exosomes during the formation of exosome [1,2]. Various types of cells such as immune cells, broblast and endothelial cells, release exosomes into the extracellular space and microenviroment, involving the tumor pathogenesis [3,4]. Recent studies reported that tumor cell-derived exosomes stimulated cancer and sarcoma cell growth, survival, metastasis, angiogenesis and chemotherapy resistance [5][6][7][8][9][10][11]. Meanwhile, For iodixanol density gradient centrifugation, exosomes harvested by differential centrifugation were loaded on top of a discontinuous iodixanol gradient (5%, 10%, 20% and 40%, made by diluting 60% OptiPrep aqueous iodixanol with 0.25 M sucrose in 10 mM Tris) and centrifuged at 100,000 g for 18 h at 4 °C (Beckman Coulter). The exosomes distributed at the density range between 1.13 and 1.19 g/ml, as previously demonstrated. The exosomes were further pelleted by ultracentrifugation at 100,000 g for 2 h at 4 °C [23,24].

Characterization Of Puri ed Exosomes
For transmission electron microscopy (TEM) analysis, the exosome pellet resuspending solution was loaded onto formvar/carbon-coated EM grids, xed with 2% paraformaldehyde (PFA), and stained with 1% aqueous uranyl acetate, then dried at room temperature. The samples were examined using an device TEM. For nanoparticle tracking analysis (NTA), vesicle enriched suspension with concentrations between 1 × 107/mL and 1 × 109/mL was examined using the ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405 nm laser to determine the size and quantity of particles isolated. A video of 60-sec duration was taken with a frame rate of 30 frames/sec, and particle movement was analyzed using NTA software.

Exosome Protein Quanti cation And Western Blot Analysis
The concentration of exosomal total protein was quanti ed by the bicinchoninic acid (BCA) assay (Thermo Fisher Inc.) using bovine serum albumin (BSA) as standard. The concentration of exosomal total protein in the serum was analyzed between six healthy donors and six patients with osteosarcoma.
The pellets were also dissolved in 200 µL RIPA buffer for protein assay. The samples were individually homogenized in 5 mM Tris-HCI (4 mM EDTA, pH 7.4, containing 1 M pepstatin, 100 M leupeptin, 100 M phenylmethyl sulfonyl uoride, and 10 g/ml aprotinin) and cleared by centrifugation at 14,000 g for 10 min at 4 °C. Approximately 100 ug of protein were run on a discontinuous SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in TBS Comparision of RNA quanti cation in Sr-exosomes between patients with OS and healthy donors Total RNA was isolated from serum exosomes of patients with osteosarcoma and healthy donors using TRIzol Reagent (Invitrogen) and the concentration of exosomal RNA in the serum was analyzed between six healthy donors and six patients with osteosarcoma.
Quantitative Pcr (qpcr) Total RNA was isolated from 143B-WT and 143B-Rab27a-KD using TRIzol Reagent (Invitrogen), and reverse transcribed into rst-strand complementary DNA (cDNA) with random primer with RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scienti c). The samples were then analysed in an Applied Biosystems QuantStudio 3 Real-Time PCR system. GAPDH was used as an internal control.

Immunohistochemistry Analysis
Para n sections were incubated with the corresponding antibodies and stained with nonimmune serum in PBS instead of the primary antibody as the negative control. Based on the average percentage of positive cells calculated from at least 10 representative elds, positive staining was de ned as a positive cell percentage ≥ 10%. Staining intensity was classi ed as follows: 0, no staining or staining in < 10% of tumor cells; 1+, weak to moderate staining in 10 to 20% of tumor cells; 2+, strong staining in 10 to 20% of tumor cells or weak staining in 20 to 50% of tumor cells; 3+, moderate to strong staining in 20 to 50% of tumor cells or staining in 50% of tumor cells. The immunostaining assessment was conducted by two independent pathologists without any previous knowledge of the clinical characteristics and outcomes [26]. The primary antibodies were as follows: (1) Mouse anti-PD-L1 (sc-293425, Santa Cruz, CA, USA), (2) Rabbit monoclonal anti-E-cadherin (ab194982), (3) Rabbit polyclonal anti-N-cadherin (ab18203), (4) Rabbit monoclonal anti-vimentin (ab92547).

Immunogold Labeling
Fixed specimens at an optimal concentration were placed onto a 400 mesh carbon/formvar coated grids and allowed to absorb to the formvar for a minimum of 1 minute. The grids were placed into a blocking buffer for a block/permeabilization step for 1 hour. Without rinsing, the grids were immediately placed into the primary antibody at the appropriate dilution overnight at 4 °C (Mouse anti-PD-L1, sc-293425, Santa Cruz, CA, USA). As controls, some of the grids were not exposed to the primary antibody. The following day, all the grids were rinsed with PBS then oated on drops of the appropriate secondary antibody attached with 10 nm gold particles (AURION, Hat eld, PA) for two hours at room temperature. Grids were rinsed with PBS and were placed in 2.5% Glutaraldehyde in 0.1M Phosphate buffer for 15 min.
After rinsing in PBS and distilled water the grids were allowed to dry and stained for contrast using uranyl acetate. The samples were viewed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR) and images were taken with an AMT CCD Camera (Advanced Microscopy Techniques, Danvers, MA).
Bioinformatics analysis of Sr-exosomes from 20 OS patients and 6 healthy donors Exosomal RNA was extracted using RNeasy® Mini kit (Qiagen, cat. No. 217004) according to the manufacturer's instructions. RNA quality was assessed by using UV 260/280. The pellets were also dissolved in 1 ml Trizol LS reagent (Invitrogen) for RNA isolation. Then, we used NGS to get sequencing data of mRNA in different Sr-exosomes from 20 patients with osteosarcoma and 6 healthy donors. These sequencing data were analyzed by bioinformatics method. Differentially expressed genes were identi ed based on RVM t test and false discovery rate (FDR) analysis. Differentially expressed genes with at least 1.2-fold change in either direction with P < 0.05 were considered to be up or downregulated. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, signi cantly changed pathways were identi ed. GO analysis was used to organize differentially expressed genes into hierarchical categories.

Microarray Analysis Of Exosomes Isolated From 143b And Uos
Exosomal RNA of 143B and U 2 OS was extracted using Affymetrix Pico Kit and puri ed with an RNeasy mini kit (Qiagen GmbH, Hilden, Germany). cDNA was synthesized with One-Cycle Target Labeling and Control Reagents, and cRNA was created using a GeneChip IVT Labeling kit (both from Affymetrix; Thermo Fisher Scienti c, Inc.). cRNA was fragmented, and then hybridized to an Affymetrix Human Clariom D Array (Affymetrix; Thermo Fisher Scienti c, Inc). GeneChips were washed and stained in the Affymetrix Fluidics Station 450. All arrays were scanned using the Affymetrix GeneChip Command Console, which was installed in the GeneChip Scanner 3000 7G. The data were analysed with the Robust Multichip Analysis (RMA) algorithm using the Affymetrix default analysis settings and global scaling as a normalization method. Values presented are log2 RMA signal intensities. Differentially expressed mRNA were identi ed using the Student's t-test for comparison of 143B (high metastatica ability) and U 2 OS (low metastatica ability) [18]. Genes were considered differentially regulated between the two groups when there was > 2-fold difference in expression with P < 0.05. Differentially expressed mRNA with at least a 2-fold change in either the positive or negative direction were respectively considered up-or downregulated.

Exosome labeling and osteosarcoma cell uptake in vitro
To examine whether Sr-exosomes derived from the patient with osteosarcoma can be taken up by osteosarcoma cell lines, 143B and U 2 OS, PKH26 (Sigma-Aldrich, PKH26GL) was used to label exosomes.
The exosomes or PBS were stained with PKH26 dye in 400 µl of Diluent C for 4 min at room temperature. Then an equal volume of 1% BSA were used to stop the labeling reaction, after which they were washed with PBS and ultracentrifuged again. The labeled exosomes or PBS were respectively incubated with 143B and U 2 OS cells with complete medium for four hours at 37 °C in an atmosphere of 5% CO2. Then the cells were washed three times in PBS to eliminate the in uence of serum exosomes. The cell nuclei was counterstained with DAPI for 8 min and cell membrane were counterstained with CFSE (Sigma-Aldrich) for 5 min. The uptake of the labeled exosomes by 143B and U 2 OS cells was assessed using an inverted confocal microscope.

Cell Counting Kit 8 Assay And Transwell Assay
For the assessment of serum exosomes of patients with OS on 143B and U 2 OS cell proliferation, cells were seeded at a density of 2000 cells per well in 96-well plates. The cells were treated with different exosome concentration of 10, 20, 30, 40 ug/ml for 24, 48 and 72 hours. Cell proliferation was analyzed using Cell Counting Kit 8 (Dojindo, Kumamoto, Japan) according to the manufacturer's protocol.
For the evaluation of serum exosomes of patients with OS and healthy donors on 143B and U 2 OS migration and invision, cells (6 × 10 4 ) were respectively seeded into the non-coated upper chamber of transwell plates (8 mm pore size; Corning) for a migration assay and into matrigel coated upper chamber (BD Bioscience, 354234) for an invasion assay with 50 ug/ml exosomes. For the evaluation of exosomes derived from osteosarcoma on 143B and U 2 OS migration and invision, the same procedure as above mentioned was performed with 10 ug/ml and 50 ug/ml. After culturing for 24 h, cells were xed with methanol and stained with a 0.1% crystal violet solution. Migrated cell populations were evaluated in ve elds per well under a microscope.

Wound-healing Assay
Con uent 143B cell monolayers were scratched with a sterile 100-µl pipette tip and treated with or without GW4869 at a dose of 1ug/ml. Cell migration was monitored for 24 and 48 hours under a microscope. The widths of the 'wound' (scratched areas) were measured and the proportion of wound healing was calculated by the following formula: 100%-(width after 24 h/width at the beginning) × 100%.
ELISA analysis of Sr-exosomal PD-L1 expression for patients with OS For detection of PD-L1 on exosomes, the serum from patients was analyzed by ELISA. Exosomes were isolated as above mentioned and then resuspended in 100 ul PBS. 50 ul suspense was analyzed as the schematic diagram shown in Figure-4N. Equilibrate all materials and prepared reagents to room temperature prior to use. All standards, controls and samples were analyzed in duplicate. The protocol was followed as the instruction (ab214565, Human PD-L1 SimpleStep ELISA® Kit). Calculate the average absorbance value for the blank control (zero) standards. Subtract the average blank control standard absorbance value from all other absorbance values. Create a standard curve by plotting the average blank control subtracted absorbance value for each standard concentration (y-axis) against the target protein concentration (x-axis) of the standard. Use graphing software to draw the best smooth curve through these points to construct the standard curve. Determine the concentration of the target protein in the sample by interpolating the blank control subtracted absorbance values against the standard curve. Multiply the resulting value by the appropriate sample dilution factor, if used, to obtain the concentration of target protein in the sample. Highest standard should be further diluted and reanalyzed. Similarly, samples which measure at an absorbance values less than that of the lowest standard should be retested in a less dilute form.

Pulmonary Metastatic Model Study
All protocols were approved by the animal care committee of the Peking University Health Science Center. We have minimized the number of animals used and their suffering.
Experiment NO 1 in vivo, to evaluate the effect of 143B-derived exosomes and inhibitor of exosome secretion GW4869 on pulmonary metastasis, 143B cells were injected into tail vein of six-week-old female BALB/c nude mice (Vitalriver, Beijing, China) in a volume of 10 µL (50,000 cells) as the primary injection. Administration of PBS, GW4869 (at a dose of 2.5 mg/kg, from day 3 to day 21, once three days, eight times in total. Sigma-Aldrich, cat. D1692) and 143B-derived exosome (at a dose of 10ug per mouse, from day 3 to day 21 once three days, eight times in total) as secondary injection were performed after primary injection. Mice were euthanized at four weeks after the secondary injection. Experiment NO 2 in vivo, the model and treatment were followed as the owchart indicated in Figure-6H. The mice were sacri ced at four weeks after secondary injection. The lung specimen were stored at -80℃ for experiments.

Haematoxylin And Eosin Staining
Mice were sacri ced at four weeks (Experiment NO 1 and 2 in vivo) after secondary injection and the lung tissue were dissected. Osteosarcoma specimen of patients were stored at -80℃ after resection. The tissue was xed in 10% formalin at room temperature overnight and consequentially dehydrated in 70% ethanol. Samples were mounted in para n blocks and sectioned at 5 mm of thickness. Slides were baked at 60℃ for 15 minutes, then treated with Xylene Substitute (Citrisolv) (Fisher chemical cat. x3p-1GAL) then rehydrated and stained with Hematoxylin, followed with washes with running water. An incubation with Acid Alcohol (Sigma, cat. A3429) was performed, followed with washes with running water. The samples were then stained with eosin (Thermo Scienti c) then dehydrated with ethanol followed by Xylene Substitute (Citrisolv). Samples were mounted with Permount (Fisher Scienti c, cat. SP15-500). Slides were submitted for pathologic evaluation. Pathologist was blinded to the sample.
The mouse weight was recored for analysis and lungs were collected after H&E staining. Metastasis was measured by visually counting the number of metastatic nodules, maximum diameter per metastatic nodule and cross-section area per metastatic nodule in the entire mouse lung section for each mouse, using Nikon NIS-Elements software (Nikon Corporation Instruments, Tokyo, Japan).

Results
Con rming the characteristics of serum exosomes (Sr-Exo) of OS patients and OS cell line exosomes The plan for analysis of patients with OS and benign tumor and healthy donors was shown in the owchart (Figure-1A). The exosome fraction was characterized by transmission electron microscopy (TEM) and nanoparticle tracking system for morphology, concentration and size, as well as by western blot analysis for exosome associated biomarkers. As nanoparticle tracking analysis was shown in Figure This study was designed to progress the global mRNA screening included the serum samples from 20 osteosarcoma patients and 6 healthy volunteers. The clinical characteristics of 20 patients with OS was shown in Table-1. Our research provided a new insight into RNA sequence in exosomes and may make a little contribution to the understanding of exosomal mRNA composition in patients with OS. We found parts of differential expression of RNAs, signi cant KEGG pathways and GO enrichment in exosomes between patients with OS and healthy donors.
Volcano plots of the differentially expressed genes in the Sr-exosome of six healthy donors and twenty patients with OS, log2 fold change > 1 with statistical signi cance (P < 0.05) detected by RNA-seq using NGS. Signi cantly upregulated and downregulated genes were colored in red and green, respectively. X axis: log2 fold change of gene expression. Y axis: statistical signi cance of the differential expression in the scale of − log10 (p value) (Figure-1F).
Bioinformatic analysis revealed 248 mRNAs as exclusively signi cant expression in the Sr-exosomes derived from OS patient than that from the healthy donors (Table-S2). Heatmap of differential mRNA expression showed 75 Sr-exosomal mRNAs were identi ed to be highly expressed in OS patients compared to healthy donors and 173 Sr-exosomal mRNAs were low expression in patients with OS compared with healthy donors ( Figure.1I). KEGG pathways analysis revealed that which was enriched in mRNA. The number of according mRNAs was labeled for each pathway. It revealed that several mRNAs in Sr-exosome derived from OS patietns were enriched in the cancer-associated and immune-associated pathways. The cancer-associated pathways included FOXO signaling, Jak-STAT signaling, MAPK signaling, PI3K-Akt signaling, Ras signaling and TNF signaling pathways. Meanwhile, the cancerassociated and immune-associated pathways contained antigen processing and presentation, B cell receptor signaling, chemokine signaling, Fc-gammaR-mediated cytotoxicity and T cell receptor signaling pathways ( Figure. Table.S3&4). These results illustrated that Sr-exosomal mRNAs derived from OS patietns may participate in the osteosarcoma pathogenesis, especially the immune process in the microenviroment.
We also found the different expressed mRNAs in the Sr-exosome between OS patietns and healthy donors, likely FLOT2, is related to the vesicle genesis. Venn diagram showed FLOT2 as the co-expression genes between Sr-exosome DEG (different expression genes, OS patients vs healthy donors) and gene of associating with exosome biogenesis (Figure-1J). A list of genes identi ed from the literature with known function in exosome biogenesis [27]. Reviewing the recent literature, we have listed genes with known functions in vesicular tra cking and secretion, like the ESCRT components TSG101, MVB12, VPS36 and PDCD6IP (ALIX), and of typical extracellular vesicle markers (e.g., FLOT1, FLOT2 and the tetraspanins CD9, CD63 and CD81) was assembled (Table. S1). GO analysis demonstrated that Sr-exosomes of OS patients involved in the process of immune function during osteosarcoma pathogenesis, which was pointed by arrowhead shown in Figure-1K.

Exosomes derived from OS cell line and patient serum induces osteosarcoma migration and invision
We rst tested if exosomes derived from OS cell line and patient serum were internalized to the recipient 143B and U 2 OS cells. 143B and U 2 OS cells respectively labeled with CFSE (green) were treated with red PKH26-labeled exosomes isolated from 143B cell line and patient serum. Immuno uorescent microscopy analysis revealed the presence of PKH26 labeled exosomes from 143B cells or patient serum exclusively in the cytoplasm of 143B and U 2 OS cells, especially around the perinuclear region ( Figure-2D). Thus, the recipient cells were inundated with the exogenous exosomes. These results indicated that osteosarcoma cells can internalize exosomes derived from osteosarcoma cell. Furthermore, the upregulation of E-cadherin and downregulation of N-cadherin and vimentin was observed in the metastatic lesion treated by GW4869 and the inverse condition occurred in 143B exosomes treated group (Figure-3L). It illustrated exosomes derived from OS could stimulate the tumor to disseminate to the lung and GW4869 can be used to inhibit the secretion of osteosarcoma cells and other supporting cells in the microenviroment. Thus it can be a new targeted therapy to reduce the pulmonary metastasis in the future.

Pd-l1 Is Located At Surface Of Osteosarcoma Exosomes
Although PD-L1 is well known to be upregulated in many tumor tissues, such as melanoma, breast cancer and lung carcinama, few studies have been focused on osteosarcoma, especially circulatory PD-L1 delivered by exosomes. We rst revealed the level of serum exosome-delivered PD-L1 for OS patients and con rmed the existence of PD-L1 at the surface of exosomes, which derived from osteosarcoma cell and serum of patients with OS.
As shown in Figure Higher level of Sr-exosomal PD-L1 in OS patients compared to healthy donors and patients with benign tumor In order to reveal whether several parameters of exosomes can distinguish OS patients from healthy donors, we performed the following analysis. We compared the particle concentration of Sr-exosomes between OS patietns and healthy donors and the result showed there was no signi cant difference between them by using the nanoparticle tracking system (Figure-4J, K). We found there was no or only marginal difference in the concentration of serum exosomes or the total protein or mRNA level in these exosomes ( Figure-4L, M).
The analysis of density gradient centrifugation experiment con rmed that PD-L1 existied in Sr-exosomes derived from OS patients (Figure-4I). Western blot analysis showed Sr-exosomal PD-L1 expression of OS patients was signi cantly higher than that of healthy donors and patients with benign tumors (P < 0.05) (Figure-4F, G). By immunogold labeling for Sr-exosomal PD-L1, we detected PD-L1 at the surface of exosome derived from osteosarcoma cells and serum of healthy donors and patietns with osteosarcoma and benign tumor, whereas PD-L1 positive exosomes from serum of healthy donors and patients with benign tumor were fewer than that from patients with osteosarcoma ( Figure-4D, H). Next, we evaluated whether Sr-exosomal PD-L1 from patients with OS was higher than that of healthy donors and patients with benign tumor. Schematic of ELISA to measure PD-L1 concentration at the surface of exosomes isolated from healthy donors and patients with benign tumor and osteosarcoma was shown in Figure-4N. We found that the level of Sr-exosomal PD-L1 from 50 OS patients was signi cantly higher than levels noted from healthy donors and patients with benign tumor (P < 0.05; Figure-4O). Receiver operating characteristic curve (ROC) indicated that Sr-exosomal PD-L1 showed an AUC of 0.695 (95% CI: 0.577-0.814) (P = 0.006, Figure-4P). The result of ROC indicated a strong correlation between Sr-exosomal PD-L1 and osteosarcoma in ELISA analysis These results encouraged us to perform further analysis to potentially reveal on the mechanism of Sr-exosome, which was derived from OS, especially PD-L1 contained in Sr-exosomeas, for osteosarcoma pathogenesis and metastasis.
Change of Sr-exosomal PD-L1 for OS patients between pre-treatment and post-surgery In light of the highly distinguishing patients with osteosarcoma from healthy donors and patients with benign tumor, we next evaluated whether Sr-exosomal PD-L1 expression could identify patients with pulmonary metastasis from ones without metastasis at diagnosis. The radiological images of four OS patietns with and without pulmonary metastasis were shown in Figure Western blot analysis also revealed Sr-exosomal PD-L1 expression was signi cantly lower, whereas some patients didn't show obvious decrease of PD-L1 expression (eg, Patient 1) (Figure-5I). The disease was stable in the lung assessment for Patient 2 and 3 during the follow-up. The radiological images detected the pulmonary metastasis after neo-chemotherapy for Patient 1 (Figure-5H). It illustrated that the change of Sr-exosomal PD-L1 expression in OS patients was related to the disease progression during the followup.
In order to reveal the in uence of Sr-exosomes isolated from OS patietns on the PD-L1 expression of osteosarcoma cells, we performed the western blot analysis. The clinical information of three OS patietns, whose Sr-exosomes were used as was shown in Figure- Based on this study, we set 143B as a higher invasive cell line and U 2 OS as a lower one. We found 5792 mRNA upregulated and 983 mRNA downregelated of 143B compared to U 2 OS (Figure-6B).
Establishment of co-expression network among Sr-exosomal different expressed mRNA between 20 OS patietns and 6 healthy donors Based on the bioinformatic mRNA DEG data of serum exosomes between 20 OS patietns and 6 healthy donors, we established the co-expression network among the different expression genes, which had PD-L1 as the core gene (Figure-6A). From the co-expression network, we found the some genes, likely ALPL and C5AR1 which were associated with tumor invasion and metastasis shown in Venn diagram, to be close to core gene PD-L1 (Figure-6A

Exosomal Pd-l1 Facilitates Osteosarcoma Pulmonary Metastasis
To investigate the effect of osteosarcoma-derived exosomal PD-L1 on the pulmonary metastasis in vivo, we performed different 143B metastastic models in nude mice as the owchart and the respective treatments (Figure-7H). Importantly, we revealed that knockdown of Rab27A, which mediated the release of exosomes, blocked exosomal PD-L1 secretion (Figure-7G). We injected 143B-WT and 143B-Rab27a-KD into tail vein as the primary injection to acquire Control, 143B-WT and 143B-Rab27a-KD metastastic model. Then, injection of PBS, 143B-WT-Exo and 143B-PD-L1-KD-Exo as the secondary injection to reveal the effect of different exosomes on the pulmonary metastasis (Figure-7H).
Based on the analysis of metastastic models, we can evaluate the effect of osteosarcoma-derived exosome on the pulmonary metastasis among A, B and C groups. Likewise, the assessment of exosomal PD-L1 on the in uence of lung metastasis can be achieved among C, D and E groups. Secretion of exosomes derived from osteosarcoma was decreased in Group C due to knockdown of Rab27a (Figure-

Discussion
Our study is aiming to illuminate how the exosome in uence the osteosarcoma progression and whether PD-L1 also exists in serum exosome of patients with osteosarcoma. Exosomes act as messengers in most cancers, which eventually leads to angiogenesis, immune suppression and tumor metastasis [23,24]. Several studies reported that exosomes isolated from osteosarcoma were associated with the tumor pathogenesis and illustrated many mechanisms of metastatic progression in their studies [28][29][30][31].
In the present study, bioinformatic analysis demonstrated that Sr-exosomes isolated from OS patients may involve important process of immune function and cancer pathogenesis. Furthermore, it showed 248 mRNAs as exclusively signi cant expression in the Sr-exosomes derived from OS patient compared to that from healthy donors and several mRNAs were enriched in cancer and immune associated pathways, which can be drawn the conclusion from KEGG analysis. We also found the different expressed mRNAs in the Sr-exosome between OS patietns and healthy donors, likely FLOT2. FLOT2is considered as the exosome marker and related to the vesicle biogenesis [33]. These results illustrated that Sr-exosomal mRNAs derived from OS patietns may participate in the osteosarcoma pathogenesis, especially the immune process in the microenviroment. Interestingly, PD-L1 in Sr-exosomes of OS patients was signi cantly higher than that of patients with benign and healthy donors. This result con rmed the exosomes and exoxomal PD-L1 may be related to the pathogenesis of osteosarcoma from the clinical perspective. Afterwards, based on the bioinformatic mRNA DEG data of serum exosome between 20 OS patietns and 6 healthy donors, we established the co-expression network among the different expression genes, which had PD-L1 as the core gene. From the co-expression network, we found the some genes, likely ALPL and C5AR1 which were associated with osteosarcoma invasion and metastasis shown in Venn diagram, to be close to core gene PD-L1. It reveal that several genes in exosomes derived from OS cells may be related to exosomal PD-L1. Furthermore, exosomal PD-L1 played an important role in osteosarcoma pathogenesis, with KEGG and GO analysis which was shown in Figure In the present study, we found Sr-exosomes isolated from OS patients could induce the migration and invasion of osteosarcoma in vitro. We reckoned that Sr-exosomes derived from patients with OS included other origins such as supporting cells It has been reported that osteosarcomas may communicate with their environment by secretion of soluble proteins and generating exosomes [34][35][36][37][38]. Studies of tumor-derived exosomes may reveal possible new important targets for cancer therapy, as well as perhaps yield tumor antigens that are diagnostic or prognostic for tumor aggressiveness [24,25]. However, speci c isolation of exosomes from the serum of cancer patients remains a challenge due to the lack of speci c markers that can be used to identify and distinguish cancer exosomes from exosomes produced by other cells. In the literature, Melo reported a cell surface proteoglycan, glypican-1 (GPC1), speci cally enriched on cancer cell-derived exosomes, with high sensitivity and speci city, to detect early stages of pancreas cancer [33]. However, lack of good biomarker for sarcoma lead to us to nd out a better approach to screen the osteosarcoma prognosis.
Our results provided clear visual evidence of PD-L1 existence at the surface of exosome produced by osteosarcoma cells using electron transmission microscopy for immunogold labeling. Western blot analysis also showed that these exosomes expressed typical exosomal biomarkers, which represented components of the endosomal sorting complex required for transport (ESCRT). Furthermore, we can use Sr-exosomal PD-L1 to differentiate OS patietns from patients with benign tumor and healthy people, with ROC analysis revealing an AUC of 0.695. This nding illustrated that Sr-exosomal PD-L1 can be used as the important biomarker to monitor the progression of OS patients.
Several studies revealed the presence of PD-L1 in exosomes isolated from blood samples of patients with cancer, and the level of PD-L1 correlates with pathological features of these patients [23,24,39,40]. Chen et al. reported metastatic melanomas released exosomes, delivering PD-L1 on their surface. And furthermore, stimulation with interferon-γ increased the amount of PD-L1 on these vesicles, which suppressed the function of CD8 T cells and facilitated tumour growth. It also showed that in patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varied during the course of anti-PD-1 therapy. The number of tumour-in ltrating CD8 T lymphocytes (TILs) decreased signi cantly after the injection of exosomes. B16-F10 exosomes also decreased the proportion of proliferating PD-1 + CD8 T cells in both spleen and lymph nodes, suggesting that exosomal PD-L1 suppresses anti-tumour immunity systemically[24]. Troyer et al. reported the negative effect of OS exosomes on CD4 + and CD8 + T cell proliferation was much greater than that of osteoblast derived exosomes [41]. In the present study, we showed that in the metastatic model, mice injected with143B-Rab27a-KD, which failed to secrete more exosomes, had abundant metastatic lesions in the lung compared to ones received injection of wild type 143B osteosarcoma cell. It also illustrated that exosomes derived from osteosarcoma facilitated the progression of pulmonary metastasis. Afterwards, we found that injection of exosomes derived from parental 143B-PD-L1-KD cells was obviously inhibited compared to that receiving injection of exosomes derived from 143B-WT in the metastatic model. These results showed that the exosomal-PD-L1 can stimulate the pulmonary metastasis in the osteosarcoma metastatic model.
Personalized medicine strategies are widely advocated for the treatment of osteosarcoma patients with pulmonary progression [42][43][44][45][46]. However, we are currently faced with rigorous problem due to lack of excellent therapy which can control the tumor development [47][48][49][50]. Detecting a good personalized medicine strategy is promising for osteosarcoma patients. Liu et al. reported A485, a p300/CBP inhibitor abrogated immuno process and decreased the secretion of exosomal PD-L1 by blocking the transcription of CD274, which combined with the anti-PD-L1 antibody to reactivate T cells function for tumor attack [25]. Thus, suppression of exosomal PD-L1 may be a promising personalized therapy. It has been reported that GW4869 could decrease the secretion of tumor cell exosome, which inhibit the cell growth [51]. Although we can not observe the signi cantly inhibiting effect of GW4869 on the osteosarcoma cell growth in vitro in the present study, inhibition of exosome secretion by GW4869 could suppress the pulmonary metastasis in vivo study. It may be related to the effect of inhibiting the exosome secretion of supporting cells (i.g. endothelial and broblast cells, which support metastatic osteosarcoma cell in the lung metastatic lesion) in the microenviroment for OS patients.
There were several limitations in the present study. It has been reported that one of the mechanisms of tumor metastatic progression is the exosome associated with immune suppression [12,52,53]. However, it was not evaluated in this study how exosomes derived from osteosarcoma interact with the immune cells. We found there was no signi cant difference of Sr-exosomal PD-L1 level in patients with metastasis compared to those without metastasis. This phenomenon may be related to the small samples and patients with the pulmonary metastasis being at the early stage, which didn't have enough tumor burden of metastatic lesions. Thus, in the future study, we need to collect more samples to reveal the objective results.

Conclusion
In conclusion, to our knowledge, we con rm for the rst time that osteosarcoma releases extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface and induces the osteosarcoma pathogenesis. Abbrevisions OS, Osteosarcoma; Sr-exosome or Sr-Exo, Exosome derived from serum; PD-L1, Programmed death-ligand 1; TEM, Transmission electron microscopy; EV, Extracellular vesicle.

Declarations Ethical Approval and Consent to participate
This study was reviewed and approved by the Ethics Committee of Peking University People's Hospital and all the participants signed an informed consent form.

Consent for publication
All the patients involved in our study obtained written consent for publication.

Availability of data and materials
The datasets used and/or analyzed during the current study are availablewithin the manuscript and its supplementary information les.

Competing interests
The authors declare that they have no competing interests