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The expansion of MDSCs induced by exosomal PD-L1 promotes the progression of gastric cancer

Journal of Translational Medicine volume 22, Article number: 821 (2024) Cite this article

Abstract

Background

Myeloid-derived suppressor cells (MDSCs) are the major factor in gastric cancer (GC) immune evasion. Nevertheless, the molecular process underlying the expansion of MDSCs induced by tumor-derived exosomes (TDEs) remains elusive.

Methods

The levels of exosomal and soluble PD-L1 in ninety GC patients were examined via enzyme-linked immunosorbent assay (ELISA) to determine their prognostic value. To investigate the correlation between exosomal PD-L1 and MDSCs, the percentage of MDSCs in the peripheral blood of 57 GC patients was assessed via flow cytometry. Through ultracentrifugation, the exosomes were separated from the GC cell supernatant and detected via Western blotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The function of exosomal PD-L1 in MDSCs was evaluated via immunofluorescence, Western blotting and flow cytometry in a GC cell-derived xenograft (CDX) model.

Results

The overall survival (OS) of GC patients in the high exosomal PD-L1 group was significantly lower than that of patients in the low exosomal PD-L1 group (P = 0.0042); however, there was no significant correlation between soluble PD-L1 and OS in GC patients (P = 0.0501). Furthermore, we found that the expression of exosomal PD-L1 was positively correlated with the proportions of polymorphonuclear MDSCs (PMN-MDSCs, r = 0.4944, P < 0.001) and monocytic MDSCs (M-MDSCs, r = 0.3663, P = 0.005) in GC patients, indicating that exosomal PD-L1 might induce immune suppression by promoting the aggregation of MDSCs. In addition, we found that exosomal PD-L1 might stimulate MDSC proliferation by triggering the IL-6/STAT3 signaling pathway in vitro. The CDX model confirmed that exosomal PD-L1 could stimulate tumor development and MDSC amplification.

Conclusions

Exosomal PD-L1 has the potential to become a prognostic and diagnostic biomarker for GC patients. Mechanistically, MDSCs can be activated by exosomal PD-L1 through IL-6/STAT3 signaling and provide a new strategy against GC through the use of exosomal PD-L1 as a treatment target.

Introduction

Gastric cancer (GC) ranks fourth in the world in terms of cancer-related deaths and is the fifth most common type of cancer worldwide [1]. The 5-year relative survival rate for GC patients remains remarkably low (20–30%) [2]. Surgery followed by adjuvant chemotherapy or chemoradiotherapy is the standard-of-care treatment for half of GC patients who are eligible for surgery [3, 4]. Glutamic acid and its derivatives glutamine, antifungal drugs, thalidomide and its derivatives, and nanocarrier drugs have good anticancer effects on some cancer cells [5,6,7,8,9]. In the preclinical model, combined blocking of PD-L1 and IL-6 receptor ( IL6R ) can synergistically regress the established large tumors and significantly improve the anti-tumor CD8 + cytotoxic T lymphocyte ( CTL ) response compared with the use of anti-PD-L1 alone, and IL-6-STAT3 signaling inhibited the classical cytotoxic differentiation of CTLs in vitro [10]. The activation of IL-6/STAT3 signaling can also lead to GC cells growth and migration [11, 12].

The tumor microenvironment (TME) is a highly structured ecosystem that contains cancer cells, immune cells, cancer-associated fibroblasts (CAFs), endothelial cells (ECs), pericytes, and other cell types. It is associated with maintaining proliferation signaling, resisting cell death, inducing angiogenesis, activating invasion and metastasis, triggering proinflammatory cytokines, and preventing immune damage in cancer [13,14,15]. The TME of GC is a complex system that can inhibit the immune response and promote tumor progression, which is largely affected by the matrix components. Tumor cells can mobilize these matrix components to inhibit multiple steps of T cell activation, leading to immunosuppression [16]. MDSC is the main immunosuppressive cell in TME. Elevated PD-L1 expression in the gastric epithelium can increase the number of MDSCs infiltrating tumors, and the levels of chemokines and cytokines are related to the migration, differentiation, recruitment and immune activity of MDSCs [17].

Exosomes are structures with an average size of 100 nm that can transport proteins, lipids, and nucleic acids, which are able to modulate components of the tumor microenvironment and influence the proliferation and migration rates of cancer cells [18]. Notably, exosomes are present in different body fluids, such as blood circulation, and can be employed as biomarkers for the diagnosis of diabetic patients [19, 20]. Tumor-derived exosomes (TDEs) have recently been demonstrated to impact noncancerous cells, creating a tumor microenvironment that supports tumor growth and metastasis [21]. TDEs released into the extracellular milieu and circulation contain the PD-L1 protein [22, 23]. Exosomal PD-L1 is considered to be a useful predictor in anti-PD-1 therapy [24] and has the ability to prevent T cells from destroying breast cancer cells and promote tumor growth [25]. However, the biological mechanism by which exosomal PD-L1 induces immunosuppression during the progression of GC remains unclear.

In our exploration of PD-L1 function in the TME of GC, we compared soluble PD-L1, and PD-L1 is largely enriched in exosomes. Moreover, compared with those of normal individuals, the serum exosomes of GC patients contain higher levels of PD-L1, and higher levels of exosomal PD-L1 are related to poor prognosis in GC patients, suggesting that exosomal PD-L1 might be an applicable prognostic indicator for GC. In addition, we found that circulating exosomal PD-L1 was strongly correlated with the proportion of MDSCs in the peripheral blood of GC patients. In vivo experiments, revealed that exosomal PD-L1 promoted the expansion of MDSCs and the progression of GC. Mechanically, exosomal PD-L1 might activate the IL-6/STAT3 signaling pathway, which increases the number of MDSCs. These results reveal a new mechanism by which PD-L1 promotes the progression of GC and may provide a new strategy for immunotherapy against GC through the use of exosomal PD-L1 as a treatment target.

scheme 1

Schematic illustration depicting the intrinsic relationship between GC cells and MDSCs in the tumor microenvironment

Materials and methods

Patients and clinical samples

Thirty female and sixty male GC patients who were treated at the Affiliated Hospital of Nanjing University of Chinese Medicine between 2018 and 2022 were included in this study. In addition, 72 physically fit individuals were chosen for this study’s typical control group. Table 1 provides all of the patients’ clinical data, which included their vital status, age, gender, pTNM stage, tumor size, lymph node metastases, vascular invasion, and chemotherapy conditions. All patients provided the informed permission, and the Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine approved this study, which was carried out in accordance with the Declaration of Helsinki’s principles (2021NL-133-03).

Cell lines and cell culture

MFCs, murine GC cell lines, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured at 37 °C and 5% CO2 in RPMI 1640 (Gibco, Grand Island, United States) medium supplemented with 10% fetal bovine serum (FBS) (Evergreen Company, China). The cells were subcultured or the media was replaced when they had grown to a confluence of 80–90%.

MDSCs are produced by bone marrow (BM) progenitor cells

In accordance with earlier techniques, BM cells were isolated [26]. Briefly, BM cells were obtained from the femurs and tibias of male C57BL/6 mice (18–22 g, 6 weeks old; purchased from Tianjin Institute of Hematology). A 70 μm cell filter was used to filter the cell suspension. Red blood cell lysis buffer (Beyotime, C3702) was incubated at room temperature for 4 min after the isolated cells were centrifuged at 4 °C for 6 min (600 × g). Subsequently, PBS was used to terminate the process of lysis, and the cells were collected. After that, the BM cells were plated at a density of 5 × 105 cells/mL in 6-well plates. On the basis of an earlier in vitro investigation, 100 µg/mL exosomes were chosen for further induction [26, 27]. The granulocyte and macrophage colony-stimulating factor (GM-CSF, 20 ng/mL) (Peprotech, 031955), 10% FBS supplied without exosomes, and RPMI 1640 medium were used to cultivate the BM cells. Exosomes (100 µg/mL) were subsequently added for intervention. Using flow cytometry, the cell phenotype was identified following 4 days of incubation.

Lentivirus generation and transfection

The PD-L1 sequence was amplified from the full-length cDNA of mice by Shanghai GeneChem Company and subcloned and inserted into the GV493 vector. The vector element was hU6-MCS-CBh-gcGFP-IRES-puromycin. The vector was co-transfected into 293T cells via lipo3000 (Thermo) according to the manufacturer’s protocol. The PD-L1 shRNA expressing lentivirus was harvested and stored at -80 °C for 3 days. Western blotting experiments revealed that PD-L1 gene expression was sufficiently, downregulated, and the dilution was set to 20 MOI for subsequent experiments.

Isolation and identification of exosomes

Plasma exosomes

Firstly, 400 µL plasma samples were centrifuged at 4 °C for 30 min at 10,000 × g to remove cells and their fragments. The ExoQuick (System Biosciences, EXOQ5TM-1) reagent (0.25 volume) was added to the samples according to the manufacturer’s instructions. After incubation at 4 ℃ for 16 h, the mixture of plasma and reagents was centrifuged at 1,500 × g for 30 min to precipitate the exosomes. Finally, 100 µL of PBS was used to resuspend the exosome particles, which were subsequently stored at -80 °C.

Cell culture supernatant

The cell culture supernatant was centrifuged at 800 × g for 5 min, 2,000 × g for 10 min, 10,000 × g for 30 min, and 100,000 × g for 80 min. The supernatant was discarded, and the exosome particles were resuspended in PBS and ultracentrifuged at 100,000 × g for 80 min. All the experiments were carried out at 4 °C. The exosomes were subsequently resuspended in PBS and stored at 4 °C (1–7 days) or -80 °C for a long period of time.

Identification

Exosomes suspension (20 µL) was placed on copper with no liquid for 3 min, and 2% phosphotungstic acid solution was used to counterstain them for 10 min. The exosomes were dried under an incandescent lamp for 2 min and photographed via transmission electron microscopy (TEM, Tecnai-12; Philips, Netherlands). The size and concentration of the particles were detected via nanoparticle tracking analysis (NTA, Particle Metrix, Germany). The expression of CD9, CD63 and TSG101, makers of exosomes, was examined via western blotting.

ELISA

A human PD-L1 ELISA kit (JiyinmeiBio, JYM1968Hu) was used for quantitation of the PD-L1 in plasma following the manufacturer’s instructions. Exosome-bound buffer was used to resuspend exosome particles isolated from plasma for ELISA. The total protein content of the exosomes was determined via a BCA protein analysis kit (Beyotime Biotechnology, Shanghai, China). The content of PD-L1 in exosomes was determined via a human PD-L1 ELISA kit. Specifically, 100 µL exosomes were added to each well and incubated for 2 h. Then, human PD-L1 detection antibody was added to each well and incubated for 2 h. 100 µL of horseradish peroxidase-conjugated streptavidin (streptavidin-HRP) was added to each well in 1% BSA PBS at pH 7.2 ~ 7.4, added (R&D, Catalog # DY995), and incubated for 20 min. Then, 100 µL of a 1 : 1 mixture of Color Reagent A (H2O2) and Color Reagent B (tetramethylbenzidine) (R&D, Catalog # DY999) was added to the substrate solution and incubated at room temperature for 20 min. The reaction was terminated with 2 N H2 SO4 (R&D, Catalog # DY994). The standard curve was made by human PD-L1 standard. The absorbance was measured at a wavelength of 490 nm via a microplate reader. All operations were performed in strict accordance with the instructions.

Flow cytometry

The tumor tissues were washed with ice-cold PBS, and then the tissue was cut into 0.1 mm3 pieces. The cells were isolated by incubating the tissues with digestion buffers including RPMI 1640, 5% FBS, 1 mg/mL collagenase IV (BioFroxx, 2091MG100), 0.2 mg/mL DNaseI (SigmaAldrich, D2821) and 0.2 mg/mL hyaluronidase (BioFroxx, 1141MG100). The suspension was shaken at 37 °C for 1 h, filtered through a 70 μm cell filter, and centrifuged at 800 × g at 4 °C for 5 min to collect the cells. The single-cell suspensions (1 × 107 cells/mL) obtained as described above were incubated with the fluorescent dye-conjugated antibodies AF488-CD45 (Elabscence, E-AB-F1136L), PE/Dazzle, TM 594-CD11b (BioLegend, 101256) and APC-Gr-1 (BioLegend, 108412) at 4 °C for 30 min. The cells were analyzed via FACSVerse (BD Biosciences, MA). Human MDSCs were isolated by incubating the peripheral blood of GC patients with the fluorescent dye-conjugated antibodies PE-CY7-CD11b (BD Pharmingen, 557743), PE-CD14 (BioLegend, 301806), FITC-CD15 (BioLegend, 323004), and APC-HLA-DR (BioLegend, 307610). The cells were analyzed via a FACSVerse (BD Biosciences, MA) after washing. FlowJo software was used to analyze the results.

Western blotting

The cells and exosomes were lysed via RIPA buffer (Beyotime) containing a protease/phosphatase inhibitor cocktail for 15 min to obtain total protein. A BCA protein assay kit (Beyotime) was used to determine protein concentrations. Thereafter, the proteins were separated via polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The Western blotting steps were carried out in strict accordance with published methods [28]. The membranes were incubated with the primary antibody and placed in a 4 ℃ refrigerator overnight. The following antibodies were used: anti-iNOS (Proteintech, 22226-1-AP, 1:5000), anti-PD-L1 (Proteintech, 66248-1-Ig, 1:3000), anti-Arg-1 (Proteintech, 16001-1-AP, 1:2000), anti-GADPH (Proteintech, 60004-1-Ig, 1:10000), anti-CD9 (Proteintech, 20597-1-AP, 1:2000), anti-CD63 (Proteintech, 25682-1-AP, 1:2000), anti-TSG101 (Proteintech, 28283-1-AP, 1:3000), anti-STAT3 (Proteintech, 10253-2-AP, 1:5000), anti-p-STAT3 (Cell Signaling Technology, 9145 S, 1:1000), and anti-IL-6 (Cell Signaling Technology, 12912 S, 1:1000). The membranes were further incubated with an HRP-labeled secondary antibody (#7074, 1:2000, CST) at room temperature for 1 h. Finally, the protein signal was detected via the enhanced chemiluminescence (ECL) method. ImageJ software (NIH, Bethesda, MD, United States) was used to quantify the expression level of each protein.

Establishment of a xenograft tumor model

A total of 615 strain mice (male, 6–8 weeks old, weighing 20 ± 1 g) were purchased from Tianjin Institute of Blood. Before the experiment, all the purchased mice were allowed to adapt to the feeding conditions in a pathogen-free environment within one week. For the subcutaneous tumor formation experiments, 5 × 107/200 µL MFC cells were digested, resuspended in sterilized PBS, and then injected into the right axilla of each mouse. After the tumor volume reached approximately 100 mm3, the mice were randomly divided into three groups: the PBS group, PD-L1 Negative Control group (PD-L1-NC-Exo group) and PD-L1 Knock Down group (PD-L1-KD-Exo group), with 6 mice in each group. The mice were treated with PBS, or 20 µg PD-L1-NC or PD-L1-KD exosomes via intratumoral injection two times a week. After 21 days, the mice were euthanized by an overdose of pentobarbital sodium (100 mg/kg i.p.), and the spleen and tumors were isolated and weighed. Animal experiments were performed under specific pathogen-free conditions and protocols were approved by the Experimental Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine (No. 2021DW-35-01).

Immunofluorescence staining

Tumor tissues from the mice were analyzed via primary antibodies against CD11b (Servicebio, GB11058, 1:5000), anti-Gr-1 (Servicebio, GB11229, 1:5000), and anti-PD-L1 (Servicebio, GB12339, 1:2000). The sections were observed and photographed via a fluorescence microscope.

Statistics

The results were analyzed with GraphPad Prism 8.0 via Student’s t test (unpaired, two-tailed) or one-way analysis of variance (ANOVA), followed by Tukey’s post hoc tests. The data are expressed as the mean ± SEM of three independent experiments. The overall survival of patients was determined via Kaplan–Meier analysis. The correlation between gene expression was determined via Pearson’s correlation analysis. The log-rank test was used for statistical analysis. *P < 0.05 was considered statistically significant.

Results

Exosomal PD-L1 was associated with poor prognosis in GC patients

First, the Oncomine database was used to evaluate the expression of PD-L1 in various tumor and normal tissue types. The results revealed that PD-L1 was highly expressed in a number of cancer types, such as head and neck squamous cell carcinoma (HNSC), breast invasive carcinoma (BRCA), cholangio carcinoma (CHOL), esophageal carcinoma (ESCA), kidney chromophobe (KICH), and stomach adenocarcinoma (STAD) (Fig. 1A), suggesting that PD-L1 was crucial for the emergence of several tumor types. Additionally, we quantified the levels of soluble and exosomal PD-L1 in the peripheral blood of GC patients and normal individuals. The clinical characteristics of the 90 GC patients are summarized in Table 1. The results revealed that exosomes contained a large amount of PD-L1 (Fig. 1B-C), and compared with normal individuals, GC patients presented a much greater level of enrichment of exosomal PD-L1 (Fig. 1D). The expression specificity of soluble and exosomal PD-L1 was evaluated via a receiver-operating characteristic (ROC) curves in GC patients. Exosomal PD-L1 displayed an area under the curve (AUC) of up to 0.7474, which was greater than that of soluble PD-L1 (0.5696, Fig. 1E). Finally, Kaplan-Meier model analysis was carried out to examine the correlation between soluble and exosomal PD-L1 and the prognosis of patients with GC. However, soluble PD-L1 did not significantly predict the survival of GC patients (Fig. 1F). Notably, overall survival was considerably shorter in patients with higher exosomal PD-L1 levels than in those with lower exosomal PD-L1 levels (P = 0.0042, Fig. 1G). These results suggested that exosomal PD-L1, rather than soluble PD-L1, was significantly associated with poor prognosis in patients with GC.

Fig. 1
figure 1

Exosomal PD-L1 was positively associated with poorer prognosis in GC patients. (A) The expression level of PD-L1 (CD274) in different types of tumor tissues and normal tissues in the TIMER database. (B-C) The levels of soluble PD-L1 and exosomal PD-L1 in normal individuals and GC patients. (D) The levels of exosomal PD-L1 in normal individuals and patients with GC. The significance of the difference was tested with an unpaired t test. (E) Receiver operating characteristic (ROC) curve analysis revealed high expression specificity of exosomal PD-L1 in GC patients. AUC, area under the curve. (F-G) Kaplan‒Meier (KM) survival curve for patients classified as showing either high or low expression of soluble PD-L1 or exosomal PD-L1 in GC according to the ROC curve cutoff value. The significance of the prognostic value was tested by a log-rank test. (*P < 0.05, **P < 0.01, ***P < 0.001)

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Table 1 Characteristics of GC patients
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Exosomal PD-L1 can be used to distinguish the clinicopathological characteristics of GC patients

A comparison analysis was carried out using patients with various stages of GC. We observed significantly higher levels of exosomal PD-L1 in patients with vascular invasion, lymph node metastasis or advanced-stage disease (stage III/IV) than in patients with no vascular invasion, without lymph node metastasis or with early-stage disease (stage I/II) (P < 0.01, P < 0.001 and P < 0.0001; Fig. 2A–C), respectively. Significant correlations between soluble PD-L1 and these parameters were not detected (Fig. 2E-G). In addition, we did not detect any significant correlations between the levels of exosomal and soluble PD-L1 and other parameters (Fig. S1A–J). ROC curve analysis was performed to evaluate the ability of exosomal PD-L1 and soluble PD-L1 to predict the early or advanced stage of GC. As expected, the AUC of the exosomal PD-L1 group reached 0.7481 (Fig. 2D), which was greater than that of the soluble PD-L1 group (0.5460, Fig. 2H). Overall, these results showed that GC patients with varying levels of exosomal PD-L1 presented distinct patterns of clinical and pathological characteristics. Exosomal PD-L1 was explicitly enriched in the advanced stage (stage III/IV) and may function as a potential malignancy biomarker of GC.

Fig. 2
figure 2

Associations between exosomal and soluble PD-L1 and the clinicopathological characteristics of patients with GC. (A-C, E-G) Correlations between exosomal PD-L1 and soluble PD-L1 and vascular invasion, lymph node metastasis and stage in GC patients. (D, H) Receiver operating characteristic (ROC) curve analysis revealed high-expression specificity of exosomal PD-L1 in advanced stage GC. AUC, area under the curve. The significance of the difference was tested with an unpaired t test and one-way ANOVA

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Fig. 3
figure 3

Exosomal PD-L1 was strongly associated with the levels of MDSCs in GC patients. (A) MDSCs gating process in the peripheral blood of GC patients. (B-C) Correlation analysis between exosomal PD-L1 and PMN-/M-MDSCs. (D-E) Kaplan-Meier curves of OS of GC patients based on the PMN- and M-MDSCs frequencies. Graphics were made by an online website Sangerbox. (F) Correlation of PD-L1 expression with infiltrating levels of immune cells of GC tissues in TISIDB database. (G) Correlation of PD-L1 expression with CD11b (ITGAM) in TIMER database

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Exosomal PD-L1 was significantly associated with the aggregation of MDSCs in GC patients

To investigate the key factors that exosomal PD-L1 affected the progress of GC patients, we extracted MDSCs from the peripheral blood of GC patients (Fig. 3A). MDSCs were divided into polymorphonuclear (PMN-MDSCs, CD11b+CD14−CD15+) and monocytic (M-MDSCs, CD11b+CD14+HLA-DRlow) (Fig. 3A). As expected, the level of exosomal PD-L1 was similar to those for frequencies of PMN-MDSCs (r = 0.4944, P < 0.0001; Fig. 3B) and M-MDSCs (r = 0.3663, P = 0.005; Fig. 3C) in the peripheral blood of GC patients. Furthermore, negative correlation was found between patient OS and the proportion of PMN-MDSCs (P = 0.02, Fig. 3D), but not M-MDSCs (P = 0.08, Fig. 3E). In addition, integrated tumor-immune system interaction storage portal (TISIDB) network server and tumor immune estimation resource (TIMER) were used to evaluate the correlation between PD-L1 and MDSCs in tumor microenvironment. The correlation between PD-L1 and CD11b (a marker of MDSCs), MDSCs, B cells, NK cells, Treg cells, Th1, Th2, Th17 cell and macrophage was analyzed (Fig. 3F), indicating PD-L1 had a strong correlation with MDSCs (r = 0.59, P < 0.0001) and CD11b (r = 0.47, P < 0.001; Fig. 3G) in STAD, which was consistent with the above results. These results suggested that the exosomal PD-L1 was highly associated with MDSCs, which might be a main reason of leading to poor prognosis in GC patients.

Exosomes promoted the proliferation of MDSCs and enhanced its immunosuppressive properties in vitro

To determine the effect of GC-derived exosomes on MDSCs, we further extracted exosomes from the supernatant of the GC cells. Exosomes are 40–150 nm extracellular vesicles with double membranes and are positive with CD9, CD63 and TSG101 (Fig. 4A-C). Next, in order to observe the effect of exosomes on MDCSs, BM cells were extracted from C57BL/6J male mice. A certain proportion of BM cells in mice have the phenotype of MDSCs [29], which can be generated from BM progenitors as described previously [26]. To investigate whether GC-derived exosomes could stimulate MDSCs (CD11b+Gr-1+) expansion, BM-derived MDSCs were cultured with exosomes derived from supernatants of GC cells. Compared with the PBS group, exosomes significantly promoted the proliferation of MDSCs (Fig. 4D-E) (P < 0.05), and the number of MDSCs (CD11b+Gr-1+) were significantly increased (Fig. 4F-G) (P < 0.001). As the main immunosuppressive factors of MDSCs, iNOS and Arg-1 were detected by Western blot, the results showed that the expression of iNOS (P < 0.05) and Arg-1 (P < 0.05) were increased after exosomes intervention. (Fig. 4H-I). The above results implied that GC-derived exosomes induced the expansion of MDSCs and mediated their immunosuppressive properties.

Fig. 4
figure 4

Gastric cancer cell-derived exosomes can promote the expansion and immunosuppression of MDSCs. (A) Transmission electron microscopy image of exosomes. (B) NTA analysis confirmed the exosomes. (C) Western blot of the exosome-related markers in GC cell-derived exosomes. (D-E) The represent images of MDSCs after PBS or exosome intervention. (F-G) Percentages of CD11b+Gr-1+ MDSCs detected by flow cytometry. (H-I) The expression levels of iNOS and Arg-1 in MDSCs after PBS or exosome intervention. Data are expressed as the mean ± SEM. *P < 0.05, ***P < 0.001

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Exosomal PD-L1 promoted MDSC expansion via activation of IL-6/STAT3 pathway

To further investigate the role of exosomal PD-L1 in MDSCs expansion, lentiviral GV493 vector was used to stably knockdown PD-L1 in MFC cells, respectively(Fig. 5A). The strong green fluorescence after lentivirus transfection was observed by fluorescence microscope (Fig. 5B). Western blotting veritified that PD-L1 expression was significantly decreased in MFC cells and exosomes (P < 0.001, P < 0.01; Fig. 5C-D). Subsequently, MDSCs were treated with exosomes, and we found that the expansion of MDSCs were increased significantly in PD-L1-NC-Exo group (P < 0.001) compared with PBS group and were significantly diminished in PD-L1-KD-Exo group (P < 0.05) compared with PD-L1-NC-Exo group (Fig. 5E-F).

Fig. 5
figure 5

Exosomal PD-L1 increased the MDSCs expansion through IL-6/STAT3 signaling pathway. (A) Lentivirus transfection of MFC cell lines. (B) Fluorescence images of PD-L1 after lentivirus transfection of MFC cells. (C-D) Western blotting analysis of the PD-L1 content in GC cells and exosome from the culture supernatant of GC cells. (E-F) Percentages of CD11b+Gr-1+MDSCs detected by flow cytometry. (G-H) Western blotting analysis of proteins in the IL-6/STAT3 signaling pathway. Data are expressed as the mean ± SEM. *P < 0.05, ***P < 0.001

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It has been reported that the IL6-STAT3-DNMT1/3b epigenetic pathway silences the TNFα-RIP1 necrosis pathway to increase the survival rate of MDSC, thereby maintaining the accumulation of MDSC in tumor-bearing hosts [30], which led us to speculate that the expansion of MDSCs might also be related to the IL-6/STAT3 signaling pathway. Western blotting results showed that the expression of IL-6 (P < 0.05) and p-STAT3 (P < 0.05) was increased significantly in the PD-L1-NC-Exo group compared with PBS group, and was decreased significantly in the PD-L1-KD-Exo group (P < 0.05) compared with PD-L1-NC-Exo group (Fig. 5G-H). The above results implied that GC-derived exosomes induced the expansion of MDSCs might be mediated by exosomal PD-L1 through IL-6/STAT3 signaling pathway.

Exosomal PD-L1 promoted MDSCs expansion in GC cells-derived xenograft model

To further validate that the exosomal PD-L1 promoted MDSCs expansion, the experiments were next applied in vivo. The tumor weight in PD-L1-NC-Exo group (P < 0.001) was significantly higher compared with PBS group, and was decreased significantly in PD-L1-KD-Exo (P < 0.001) compared with PD-L1-NC-Exo group(Fig. 6A-B). Immunofluorescence results showed that the levels of PD-L1 in the intratumoral content of MDSCs (CD11b+Gr-1+) in PD-L1-NC-Exo group were higher compared with the PBS group, and were decreased in PD-L1-KD-Exo group compared with PD-L1-NC-Exo group (Fig. 6D). Moreover, flow cytometry analysis also showed the frequency of MDSCs (CD45+CD11b+Gr-1+) was significantly increased in PD-L1-NC-Exo group (P < 0.01) compared with the PBS group, and were significantly decreased (P < 0.05) in PD-L1-KD-Exo group compared with PD-L1-NC-Exo group (Fig. 6E-F). Furthermore, it was found that the proportion of PD-L1+MDSCs (PD-L1+CD45+CD11b+Gr-1+) was significantly increased in PD-L1-NC-Exo group (P < 0.01) compared with the PBS group, and were significantly decreased (P < 0.05) in PD-L1-KD-Exo group compared with PD-L1-NC-Exo group (Fig. 6E, G). The same phenomenon was observed in the spleen of mice (Fig. S2). Altogether, it was confirmed that exosomal PD-L1 might remodel the tumor microenvironment (TME) by promoting MDSCs expansion and finally enhanced tumor growth.

Fig. 6
figure 6

Exosomal PD-L1 promoted MDSC expansion in vivo. (A) Representative images of xenograft tumors in mice implanted with MFC cells and receiving PBS, NC-PD-L1-exosome or KD-PD-L1- exosomes treatment (n = 6/group). (B) The tumor weights at the last measurement. (C) The body weights were measured every 4 days. (D) Immunofluorescence staining for DAPI (nucleus), CD11b, Gr-1, and PD-L1 in tumor tissue sections from the xenograft tumor model. (E-G) Percentages of MDSCs (CD45 + CD11b + Gr1+) and PD-L1 + MDSCs in tumors determined via flow cytometry. The significance of the difference was tested with one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

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Discussion

For many years, cancer has been the main threat facing humanity. Many factors contribute to the occurrence of cancer, such as social modernization [31], and the use of chiral pesticides, pollutants, and drugs [5, 9, 32]. In 2017, the PD-1 inhibitor pembrolizumab was approved by the FDA for the treatment of recurrent locally advanced or metastatic stomach or adenocarcinoma of the esophagogastric junction that expresses PD-L1 [33]. Because most GC patients are insensitive to immune checkpoints (ICIs) [34, 35]. Immune checkpoint blockade ( ICB ) has achieved important therapeutic effects in many cancers, but its clinical activity is limited to some patients [35]. Therefore, it is particularly important to have a deeper understanding of the role of PD-L1 in ICB treatment.

Over the past decade, an increasing body of research has demonstrated that exosomes can induce pleiotropic functional responses in recipient cells, leading to various health complications such as cancer, neurodegenerative illnesses, rheumatic diseases, and infectious diseases [21, 36,37,38]. Exosomal miR-200a is related to EMT-mediated GC metastasis [20]. As a type of small secreted membrane vesicles, TDEs played a crucial role in the development of tumors, particularly in controlling the immune response associated with the tumor [39, 40]. Many proteins and nucleic acid molecules in TDEs could serve as markers in monitoring the therapeutic responses in cancer patients [41, 42]. PD-L1 was found in microvesicles, and exosomal PD-L1 had the same membrane topology with the cell surface PD-L1, with its extracellular domain exposed on the surface [24], which indicated that PD-L1 in TDEs could be a potential target to develop a novel clinical therapeutic strategy to tumors.

It had been demonstrated that the development, differentiation, and accumulation of MDSCs in multiple tumor [43]. MDSCs are distinguished by their capacity to impede T-cell proliferation and function via multiple mechanisms, including the expression of NADPH oxidase (Nox2), arginase 1 (Arg-1), oxide synthase 2 (Nos2), and S100A8/9, which permit tumor cells to evade immune system attack [43]. Myeloid precursor cells’ development into MDSCs could be skewed by TDEs [44, 45]. Macrophage migration inhibitory factor (MIF) in both human and murine pancreatic cancer-derived exosomes was reported to have the key role in inducing MDSC formation in pancreatic cancer [46]. Inhibition of the IL-6/STAT3 pathway can alleviate immunosuppression caused by MDSCs [47]. Nevertheless, the cell-cell communication between GC cells and MDSCs mediated by exosomes was not clear. Our study provided ideas for the interaction between exosomes and MDSC in TME.

Circulating soluble PD-L1 was easy to measure in GC patients, and its level was related to the expression of PD-L1 in GC tissues, inflammatory cell infiltration and disease progression [48]. In addition, it was reported that PD-L1 was also enriched in exosomes, which might prevent T cells from killing one another in order to maintain its immunosuppressive effects in the GC microenvironment [49]. It could also induce the higher levels of T-cell dysfunction due to its higher stability and MHC I expression in GC [50]. To systemically combat anti-tumor immunity, melanoma cells could release PD-L1-positive extracellular vesicles into the tumor microenvironment and circulation [24]. Genetically blocking exosome biogenesis or deleting Pd-l1 reverses the phenotype by strongly promoting T cells activation, proliferation, and killing potential [51]. Here, through bioinformatic analysis, we found that exosomal PD-L1 existed in the peripheral blood was correlated with poor prognosis of GC patients. We further revealed that exosomal PD-L1 was higher in AJCC stage III and IV but not in AJCC stage I and II and was related to the poor prognosis of GC patients. The above results indicated that as a molecular marker, exosomal PD-L1 could predict the survival of GC patients, which might be mediated by inducing the expansion of MDSCs. However, the interaction between exosomal PD-L1 and MDSCs in the tumor microenvironment remains to be explored. In this study, we discovered that the high MDSC numbers could be induced by GC cell-derived exosome. Subsequent investigation revealed a strong correlation between PD-L1 existed in exosomal forms and tumor-infiltrating MDSC in STAD as well as the number of MDSCs in the peripheral blood of GC patients.

The IL-6/STAT3 signaling pathway was shown to be one of the critical pathways in mediating the expansion of MDSCs [26]. Blocking of IL-6 function mostly inhibited STAT3 phosphorylation and the MDSCs expansion in BM cells [26]. However, the mechanism of exosomes promoting MDSC proliferation still needs to be explored. In vitro study, we found that exosomes facilitated expansion of MDSCs through promoting the expression of IL-6 and activating STAT3 phosphorylation, while the expression of IL-6/p-STAT3 and the accumulation of MDSCs were inhibited when the exosomal PD-L1 was decreased. Next, exosomal PD-L1 was discovered to encourage MDSCs development both in vitro and in vivo and quicken tumor growth, suggesting that exosomal PD-L1 promoted tumor growth most likely by stimulating MDSC expansion. These results provided a evidence for immunosuppressive effects of MDSCs in GC.

Conclusion

In summary, we found that the higher levels of exosomal PD-L1 is related to poor prognosis for GC patients. Exosomal PD-L1 were also found to promote tumor growth and induce expansion of MDSCs though IL-6/STAT3 signaling in vivo and vitro experiments. These findings show that exosomal PD-L1 has the potential to become a prognostic and diagnostic biomarker for GC patients and provide a new strategy for immunotherapy of GC using exosomal PD-L1 as a target.

Data availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

MDSCs:

Myeloid-derived suppressor cells

GC:

Gastric cancer

TDEs:

Tumor-derived exosomes

ELISA:

Enzyme-linked immunosorbent test

TEM:

Transmission electron microscopy

CDX:

Cells-derived xenograft

Nox2:

NADPH oxidase

Arg-1:

Arginase 1

Nos2:

NADPH oxide synthase 2

ICIs:

Immune Checkpoints

MVB:

Multivesicular bodies

MIF:

Macrophage migration inhibitory factor

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Acknowledgements

Thanks to all participants involved in this research.

Funding

This study was supported by Natural Science Foundation of Jiangsu Province, China (No. BK20211392), Jiangsu Provincial Medical Youth Talent (QNRC2016641), Jiangsu Provincial Hospital of Traditional Chinese Medicine Academic Talent Program (Y2021RC29. Y2021RC45); A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); Postgraduate Research &Practice Innovation Program of Jiangsu Province (SJCX23_0780), Foundation of Administration of Traditional Chinese Medicine of Jiangsu Province, China (ZD202307).

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Author notes

  1. Huaizhi Li, Xu Chen and Shanshan Zheng contributed equally to this work.

Authors and Affiliations

  1. Jiangsu Province Key Laboratory of Tumor Systems Biology and Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, 155 Hanzhong Road, Nanjing, 210029, Jiangsu, China

    Huaizhi Li, Xu Chen, Shanshan Zheng, Bo Han, Xiang Zhang, Xiaoxia Zheng, Yujia Lu, Qingmin Sun & Jian Wu

  2. No.1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China

    Huaizhi Li, Xu Chen, Shanshan Zheng, Bo Han, Xiang Zhang, Xiaoxia Zheng & Yujia Lu

  3. Department of General Surgery, Yixing Traditional Chinese Medicine Hospital, Wuxi, 214200, Jiangsu, China

    Xufeng Hu

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Contributions

Jian Wu designed the experiments; Huaizhi Li, Xu Chen and Shanshan Zheng performed the majority of experimental work as well as data analysis and authored the manuscript;

Bo Han, Xiang Zhang, Xiaoxia Zheng and Yujia Lu performed and coordinated the samples collection as well as clinical information; Qingmin Sun assisted with the experiments; Xufeng Hu and Jian Wu supervised the project and improved the manuscript; All authors reviewed the results and approved the final version of the manuscript.

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Correspondence to Xufeng Hu or Jian Wu.

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Li, H., Chen, X., Zheng, S. et al. The expansion of MDSCs induced by exosomal PD-L1 promotes the progression of gastric cancer. J Transl Med 22, 821 (2024). https://doi.org/10.1186/s12967-024-05611-y

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