LILRB2-containing small extracellular vesicles from glioblastoma promote tumor progression by promoting the formation and expansion of myeloid-derived suppressor cells

The gene expression profile of LILRB2 in GBM and normal brain samples was obtained from GEPIA (http://​gepia.​cancer-pku.​cn/​), while the gene expression profile of LILRB2 in different grades (WHO II, WHO III and WHO IV) of glioma was obtained from CGGA (http://​www.​cgga.​org.​cn/​).

The collected tissues were fixed immediately in 4% paraformaldehyde solution and embedded in optimal cutting temperature (OCT) compound. The samples were cut into Sects. (10 μm) with a freezing microtome (Leica CM 1950; Leica Biosystem, Heidelberg, Germany). The sections were permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin (BSA), followed by incubation with primary antibody and second antibody and staining with a DAB kit (Servicebio, China). The samples were imaged by DM6 microscopy (Leica, Germany). The antibodies used in IHC were as follows: ILT4 (1:200, Santa Cruz); secondary antibody: anti-mouse-HRP (1:1000, CST). The mean density (integrated option density/area) of the images was measure by Image Pro Plus software.

The murine glioblastoma cell line GL261 and the human glioblastoma cell lines U87 and U251 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All these cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) containing 10% FBS (Gibco) and maintained at 37 °C in 5% CO₂.

The pirb-overexpressing lentivirus, pirb-overexpressing lentivirus with red fluorescence protein and control lentivirus were purchased from Vigene Bioscience (Shandong, China). GL261 cells were plated in 6-well plates at a density of 5 × 10⁵ cells/well overnight, and the lentivirus was added to DMEM (MOI: 50). After 72 h, the infected cells were screened by puromycin (MCE, American) at a concentration of 1 μg/mL for 2 weeks. Thus, GL261 cells transfected with control lentivirus (GL261-LV-nc), GL261 cells transfected with pirb-overexpressing lentivirus (GL261-pirb⁺), GL261 cells transfected with pirb-overexpressing lentivirus with red fluorescence protein (GL261-pirb-RFP) were harvested.

Small interfering RNA (siRNA) targeting pirb was purchased from Bioneer (Korea). Lipofectamine RNAiMAX Reagents (Thermo Fisher, USA) were used in the transfection of siRNA in GL261 cells. The expression of pirb was detected by WB. Thus, GL261 cells transfected with siRNA of control (GL261-nc), and GL261 cells transfected with siRNA of pirb (GL261-pirb⁻) were harvested.

EV-depleted FBS was obtained by 18 h ultracentrifugation at 100,000 g [25]. After the cells were cultured in EV-depleted media for 72 h, conditioned media were collected and centrifuged at 400 × g for 10 min and 2000 × g for 15 min. The MVs were collected by centrifugation at 15,000 × g for 30 min, and then, the supernatant underwent ultracentrifugation at 100,000 × g for 75 min at 4 °C (Optima XPN-100 Ultracentrifuge, Beckman Coulter Life Sciences) to collect sEVs. The precipitate was resuspended in sterile PBS, and the suspension was ultracentrifuged again (100,000 g, 75 min, 4 °C) to collect the sEVs. Thus, the sEVs derived from GL261 cells (GL261-sEVs), the sEVs from GL261-pirb + (GL261-pirb-sEVs), the sEVs from GL261-pirb-RFP (GL261-pirb-RFP-sEVs) were harvested. The obtained sEVs were stored at − 80 °C.

Blood from control mice and tumor-bearing mice was collected, mixed with EDTA solution, and centrifuged at 500 g to collect the supernatant. Then, the supernatant was mixed with PBS of the same volume, and the sEVs were collected by ultracentrifugation (100,000 g, 75 min, 4 °C).

Cells and sEV samples were lysed with a Radio Immunoprecipitation Assay (RIPA, Beyotime Biotechnology, China) with phenylmethanesulfonyl fluoride (PMSF) (10 μL/1 mL) (Beyotime Biotechnology, China). The protein concentration was measured by a BCA assay (Beyotime Biotechnology, China). Then, the protein solution was mixed with loading buffer (Beyotime Biotechnology, China) and boiled at 95 °C for 10 min. Proteins were separated with SDS‒PAGE (Shanghai Epizyme Biomedical Technology, China) and transferred to a 0.45 μm polyvinylidenedifluoride (PVDF) membrane (Millipore, American). After that, the membranes were blocked with 5% (w/v) nonfat milk (Beyotime Biotechnology, China) for 90 min and incubated with primary antibody followed by secondary antibody. The primary antibodies used for immunoblotting were anti-pirb (1:1000, R&D Systems, MAB2754), anti-actin (1:1000, Invitrogen, MA5-15,739), anti-GM130 (1:1000, Abcam, ab52649), anti-CD63 (1:1000, Abcam, ab134045), anti-CD9 (1:1000, Abcam, ab92726), and anti-Alix (1:500, Santa Cruz, sc-53540). The secondary antibodies used for immunoblotting were anti-mouse IgG, HRP-linked antibody (1:2000, CST, 7076) and anti-rabbit, IgG, HRP-linked antibody (1:2000, CST, 7074).

Four-week-old female nude mice and six-week-old C57BL/C mice were purchased from SLAC Laboratory Animal Company (Shanghai, China), and all animal experimental protocols were approved by the Animal Research Committee of the Soochow University (Approval No. SUDA20210708A03).

For analysis of the function of pirb in GBM, a subcutaneous tumor model was constructed with GL261 cells (3 × 10⁶, 100 μl) or GL261-pirb⁺ cells (3 × 10⁶, 100 μl) on the flanks of C57BL/C mice (n = 5). The tumor volumes were measured by calipers every four days and recorded. Four weeks later, these mice were killed, and the tumors were collected.

For survival analysis, an orthotopic brain tumor model of C57BL/C mice was constructed (n = 5). GL261 cells (1 × 10⁵, 1 μL) and GL261-pirb⁺ cells (1 × 10⁵, 1 μL) were injected into the right striatum of C57BL/C mice by a stereotactic instrument. Then, the survival time of these mice was recorded.

Subcutaneous tumor models were constructed on both sides of the C57BL/C mice. GL261 cells (3 × 10⁶, 100 μl) and GL261-pirb⁺ cells (3 × 10⁶, 100 μl) were injected into the left flanks and the right flanks of C57BL/C mice, respectively (n = 5). The tumor volumes were measured by calipers every four days and recorded for four weeks.

For analysis of the function of sEV-pirb-induced MDSCs in GBM, a subcutaneous tumor model was constructed with GL261 cells (3 × 10⁶, 100 μl) in the right flank of C57BL/C mice. MDSCs (1 × 10⁶) were injected intravenously when the tumor volume reached 100 mm³. Three days later, the tumors were harvested. The presence of MDSCs in tumors was detected by IF.

For analysis of the function of pirb-sEVs, GL261 cells (3 × 10⁶, 100 μl) were injected into the flanks of C57BL/C mice to construct subcutaneous xenograft tumor models. The tumor-bearing mice were divided into three groups when the tumor volume reached 100 mm³. The control group (n = 3) was treated with PBS (100 μL), while the other mice were treated with GL261-sEVs (1 × 10¹¹, 100 μL) or GL261-pirb-sEVs (1 × 10¹¹, 100 μL). The treatment was given every four days 3 times, and the tumor volume was measured with calipers. The mice were killed sixty days after the intervention, and the tumors were collected. Tumor volumes were calculated with the formula: tumor volume = L × W²/2.

For the proliferation assay, GL261-nc, GL261-pirb⁺ and GL261-pirb⁻ cells were plated on a 96-well plate at a density of 8000 cells/well. Then, the cell viability was measured at 24, 48 and 72 h by a Cell Counting Kit-8 (CCK8; Dojindo) assay. For the cytotoxicity test, GL261 cells were plated on a 96-well plate at a density of 8000 cells/well. After 24 h, GW4869 (10 μM) was added to the DMEM culture medium. Cell viability was measured at 72 h according to the manufacturer’s protocol. The absorbance of each cell sample was detected at 450 nm using a plate reader (Bio-Rad, USA).

ICB therapy is becoming a promising treatment for tumors, and the outcome is closely related to the immune TME [27]. Evidence has shown that immunosuppressive proteins are pivotal in the regulation of the immune TME [28, 29]. We found that LILRB2 was highly expressed in human GBM tissues compared to normal human brain tissues (Fig. 1A) and positively related to pathological grade (Figure S1A). Subsequently, a greater number of LILRB2-positive cells was observed in GBM than in normal human brain tissues, as detected by IHC (Fig. 1B, Figure S1B). Next, we found that the expression level of LILRB2 in GBM was closely related to the prognosis of GBM patients. Higher expression of LILRB2 indicated shorter DFS and OS both in GBM patients (Fig. 1C) and high-grade glioma patients (Figure S1C), demonstrating that LILRB2 is an oncogene that indicates a dismal prognosis in GBM patients.

To explore the functional role of LILRB2 in GBM, we generated pirb-overexpressing GL261 cells (GL261-pirb), and the expression of pirb in GL261-pirb was confirmed by WB analysis (Figure S1D). Then, a subcutaneous tumor model was established in C57BL/C mice (Figure S1E). The mean GL261-nc tumor volumes reached 358.85 mm³ at 28 days after implantation, while the mean tumor volumes of GL261-pirb increased by 100.35% compared with that of the GL261-nc group (Fig. 1D). Consistently, the mean tumor weight of GL261-pirb increased by 120.10% compared with that of GL261-nc (Figure S1F). As the immune microenvironment in the brain is different from that in other places, the function of pirb was further evaluated by survival analysis in an intracranial orthotopic tumor model. The survival time of the control group was significantly longer than that of the GL261-pirb⁺ group (Fig. 1E). All these results indicated that LILRB2/pirb is an oncogene that promotes GBM progression, resulting in a short survival time.

The biological function of pirb in GBM cells was evaluated. First, siRNA was used to downregulate pirb expression in GL261 cells and verified by WB (Figure S2A). Then, a CCK8 assay was conducted in GL261-LV-nc, GL261-pirb⁺, GL261-nc and GL261-pirb⁻ cells. The results of the CCK8 assay showed that the viability of GL261 cells was significantly inhibited by upregulating pirb and promoted by downregulating pirb (Fig. 2A), which was in contrast to the in vivo results. Afterward, cell apoptosis was measured by flow cytometry. We found that there was no significant difference in cell apoptosis when the expression of pirb in GL261 cells changed (Fig. 2B, Figure S2B). Next, the migration and invasion of GL261-LV-nc, GL261-pirb⁺, GL261-nc and GL261-pirb⁻ cells were evaluated. We found that there was no significant difference in migration and invasion (Fig. 2C-D, Figure S2C-D). Based on the paradoxical results presented, we speculated that pirb may function through immunity.

Previous studies have shown that LILRB2 functions as an immunosuppressive protein to induce T-cell senescence and suppress tumor immunity [30, 31]. CD8 + T cells, the main antitumor effector, CD4 + T cells, an important regulator, MDSCs, which play a central role in the regulation of immunosuppression, and Tregs, the main effector in the suppression of immunity, were selected and detected in GBM tissues. Thus, immunofluorescence staining (IF) was performed in human GBM tissues. Significantly high expression of CD11b and CD33 was observed in GBM tissues with high LILRB2 expression, indicating that higher LILRB2 expression in GBM tissue was related to more MDSCs (Fig. 2E). Subsequently, the expression of Foxp3 was detected, and the results indicated that more Treg cells were observed in GBM tissues with higher LILRB2 expression (Fig. 2F). In addition to MDSCs and Treg cells, T cells, including CD4 + T cells and CD8 + T cells, have an important role in tumor rejection [32]. Fewer infiltrated CD8 + T cells and CD4 + T cells were observed in human GBM tissues with high LILRB2 expression (Fig. 2G). Thus, LILRB2 is an immunosuppressive protein correlated with the immunosuppressive TME.

As the previous studies indicated that LILRB2/pirb acted as a suppressor of immunity [30, 33, 34]. Thus, immunes cells were detected in the TME and circulation. The Tregs detected by flow cytometry were gated as shown in Figure S3A [35]. We found that more MDSCs were observed in GL261-pirb⁺ group than in GL261-LV-nc group (Fig. 3A). Consistently, more Tregs were detected in GL261-pirb⁺ group than in GL261-LV-nc group (Fig. 3B). We found more CD8 + and CD4 + T cells in the GL261-LV-nc group than in the GL261-pirb group (Fig. 3C, D). Next, the relationship between LILRB2 and immune cells in GL261-LV-nc and GL261-pirb⁺ orthotopic tumors was evaluated by IF. More MDSCs and Tregs in the GL261-pirb⁺ tumor sites, while fewer CD8 + T cells and CD4 + T cells were observed (Figure S3B).

The local antitumor immune response can be influenced by continuous communication of the periphery. Thus, a thorough understanding of immune responses to cancer must encompass immune cells (CD4 + T cells, CD8 + T cells, MDSCs and Tregs) across the peripheral immune system in addition to within the TME. The percentage of CD8 + T cells in the spleen of the GL261-pirb tumor-bearing mice decreased significantly compared to that in the GL261-nc tumor-bearing mice from 10.10% ± 0.25% to 4.49% ± 0.44% (P < 0.0001, Fig. 3E). In contrast, the percentage of MDSCs in the spleen of the GL261-pirb tumor-bearing mice was 4.54% ± 0.21%, which was significantly higher than the 2.60% ± 0.21% in the control group (P < 0.0001, Fig. 3F). Consistently, a significant increase in Tregs was detected in the spleen of the GL261-pirb tumor-bearing mice compared to the GL261-nc mice (Fig. 3G). Moreover, the percentage of CD4 + T cells in the GL261-pirb tumor-bearing mice was significantly lower than that in the GL261-nc tumor-bearing mice (Fig. 3H). These results indicated that pirb overexpression in GBM cells suppresses immunity by suppressing CD8 + T cells and inducing MDSCs in the TME and circulation.

Accidently, we found that the tumor progression of GL26-nc can be affected by GL261-pirb⁺ in the same mouse. Briefly, a subcutaneous tumor model was constructed using both sides of C57BL/C mice with GL261-nc and GL261-pirb⁺, as shown in Figure S4A-B. Statistical analysis showed that the GL261-nc tumor volume in Fig. 1C was 358.85 ± 168.58 mm³, while the GL261-nc tumor volume in Fig. 4A was 962.40 ± 353.43 mm³ (P = 0.0087), which implied that pirb in G261-pirb regulates the function of GL261-nc in some way. Because of the tumor-derived factors, immunity in GBM patients was substantially suppressed. Current studies have demonstrated that tumor cell-derived sEVs transmit oncoproteins between tumor cells, stromal cells and immune cells to regulate the function of recipient cells, promoting tumor progression and suppressing the immune system [36, 37]. Thus, we speculated that pirb can be secreted by GBM cells and regulate the function of immune cells.

Thus, GL261-pirb-RFP (overexpressed with red fluorescence protein, RFP) was constructed, and the overexpression of pirb in GL261 cells was confirmed by WB (Figure S4B). Currently, study have shown that extracellular vesicle can be mainly divided into small extracellular vesicle and macrovesicle [38]. After that, the conditioned medium (CM) of GL261-nc and GL261-pirb-RFP cells was harvested and cultured with GL261 cells after removal of cell debris, as shown in the schematic diagram in Fig. 4B. RFP fluorescence was observed in GL261 cells cultured with CM from GL261-pirb-RFP cells but not in GL261-nc cells (Fig. 4C). Then, the components of the CM were isolated by differential centrifugation and cultured with GL261 cells to determine the way in which pirb was transferred. We found that there was no RFP fluorescence in GL261 cells after incubation with MVs from GL261-pirb-RFP (GL261-pirb-RFP-MV) or supernatant (without MVs and sEVs) from GL261-pirb-RFP, while RFP fluorescence was observed in GL261 cells cultured with sEVs from GL261-pirb-RFP (GL261-pirb-RFP-sEVs) (Fig. 4D). Therefore, we concluded that pirb can be secreted by tumor cells and carried by sEVs.

Furthermore, sEVs from U251, U87 and GL261 cells were isolated and verified according to the guidelines (Figure S4D-F). The presence of pirb in GL261-sEVs and LILRB2 in U251-sEVs was detected by WB but not in the MVs of GL261 or U251 cells (Fig. 4E). The presence of LILRB2 in sEVs was also confirmed by the result of U87 cells (Figure S4G). In addition, sEVs from the blood of the tumor-bearing mice and the control mice were isolated, and sEVs secreted by GL261-pirb tumors contained more pirb than sEVs from GL261-nc tumors (Fig. 4F). Finally, we found that the secretion of pirb was inhibited by GW4869, a kind of medicine used to inhibit the secretion of sEVs [39], both in U251 and GL261 cells (Fig. 4G). The sEV amount and cell number were detected by nanoflow cytometry and CCK8 assays, respectively. We found that the secretion of sEVs in GL261 and U251 cells was inhibited after incubation with GW4869 (10 μM) (Fig. 4H). Moreover, sEVs from the same volume of CM of GL261-nc and GL261-pirb were isolated. We found that more pirb was secreted by GL261-pirb cells than by GL261-nc cells, and the increase in pirb was independent of the secretion rate of sEVs (Fig. 4I-J). These results suggest that LILRB2/pirb can be transmitted between cells via sEVs.

Previous studies have shown that the function of immunity can be regulated by proteins on sEVs [40]. Then, flow cytometry was performed to evaluate the function of pirb on sEVs after spleen cells were treated with GL261-nc-sEVs or GL261-pirb-sEVs. We found that the percentage of CD8 + T cells was downregulated by GL261-nc-sEVs, while the immunosuppressive ability of G261-pirb-sEVs was stronger than that of GL261-sEVs (Fig. 5A). The association between LILRB2 and MDSCs was evaluated by bioinformatics analysis on the GEPIA website. CD33, ITGAM, S100A8 and S100A9 were selected as gene signatures of MDSCs [41]. We found a positive correlation between LILRB2 and CD33 (R = 0.82)/ITGAM (R = 0.75)/S100A8 (R = 0.67)/S100A9 (R = 0.69) (Figure S5A). Furthermore, the formation and expansion of MDSCs were activated by GL261-sEVs and GL261-pirb-sEVs. We found that the immunosuppressive ability of GL261-pirb-sEVs was stronger than that of GL261-sEVs (Fig. 5B). Then, confocal microscopy analysis showed that the fluorescence of DiI merged with the fluorescence of CD11b, indicating that GL261-sEVs may interact with the membrane of the splenocyte in some way or internalized by recipient cells (Fig. 5C). The presence of RFP and DiO fluorescence signal in splenocytes indicated the interaction between sEVs-pirb with splenocytes or the internalization of sEVs-pirb by splenocytes (Fig. 5D). The function of sEVs-pirb was further analyzed by a tumor cell killing assay (Fig. 5E). We found that the antitumor effect of CD8 + T cells was inhibited by GL261-sEVs and GL261-pirb-sEVs, but the immunosuppressive ability of GL261-pirb-sEVs was significantly stronger than that of GL261-sEVs. Moreover, the proliferation of CD8 + T cells was detected in the presence of MDSCs induced by GL261-sEVs or GL261-pirb-sEVs. As shown in Fig. 5F, the proliferation of CD8 + T cells was inhibited under the suppression of MDSCs induced by GL261-pirb-sEVs, which is consistent with previous results.

Next, the migration of MDSCs was evaluated in a transwell plate in the presence of GL261-sEVs or GL261-pirb-sEVs. We found that the migration of MDSCs was promoted by GL261-pirb-sEVs compared to GL261-sEVs (Fig. 5G, Figure S5B). Furthermore, MDSCs induced by GL261-pirb-sEVs were isolated, and these MDSCs were intravenously injected into the GL261 tumor-bearing mice, as shown in the schematic diagram (Figure S5C). The tumor samples were harvested 3 days after the injection. Then, IF was performed to detect MDSCs in the tumor samples. We found more MDSCs present in the tumor sites than in the controls after the injection of MDSCs (Fig. 5H). Therefore, we believe that sEV-pirb-induced MDSCs in the circulation can be a source of MDSCs in the TME. All these results indicated that sEV-pirb can be an immunosuppressor to suppress antitumor immunity by forming an immunosuppressive TME.

To verify the function of sEVs-pirb in vivo, we generated subcutaneous tumor models in C57BL/6 mice with GL261 cells (Fig. 6A). Injection of GL261-nc-sEVs (1 × 10¹⁰ particles/mL, 100 μL) significantly promoted the progression of GBM compared with that of the control group (control vs. GL261-nc-sEVs: 151.21 ± 22.45 mm³ vs. 270.02 ± 70.62 mm³, P = 0.05), while the protumor ability of GL261-pirb-sEVs was significantly stronger than that of GL261-sEVs (GL261-nc-sEVs vs. GL261-pirb-sEVs: 270.02 ± 70.62 mm³ vs. 547.08 ± 145.49 mm³, P = 0.0413) (Fig. 6B). Consistently, the mean tumor weight of GL261-sEVs increased 94.32% compared with that of the control, and the mean tumor weight of GL261-pirb increased by 84.49% compared with that of GL261-sEVs (Fig. 6C).

In addition, tumor samples were collected, and a significant decrease in CD8 + T cells and an increase in MDSCs were observed in the tumor sites treated with GL261-sEVs, while fewer tumor-infiltrating CD8 + T lymphocytes and more MDSCs were observed in the tumor sites treated with GL261-pirb-sEVs than in those treated with GL261-sEVs (Fig. 6D). Accordingly, the immune cells in the spleen of the tumor-bearing mice were also detected by flow cytometry. As shown in Fig. 6E, the percentage of CD8 + T cells in the control group was 10.78% ± 1.1%, decreased to 5.45 ± 0.52 in the GL261-sEV group, and decreased to 2.73% ± 0.21%. While the percentage of MDSCs in the control was 4.90% ± 0.17%, the percentage of MDSCs reached 6.82% ± 0.11% and 9.11% ± 0.22% in GL261-sEVs and GL261-pirb-sEVs, respectively. We found that pirb on sEVs can exert its function through the immune system by inducing MDSCs.