Gastric cancer-derived exosomal indoleamine 2,3 dioxygenase 1 drives immunosuppression in gastric cancer by suppressing dendritic cell maturation

Abstract

BACKGROUND

Indoleamine 2,3 dioxygenase 1 (IDO1) restrains the functions of dendritic cells (DCs) and T cells, reinforcing the immunosuppressive microenvironment. Owing to its established role as an oncogene in gastric cancer, understanding how IDO1 modulates antitumor immunity is critical.

AIM

To elucidate the roles of gastric cancer-derived exosomal IDO1 in regulating DC maturation and antitumor immune response in gastric cancer.

METHODS

IDO1 protein expression in gastric cancer tissues and cell lines was examined. Exosomes from gastric cancer line (MKN45) were isolated. Bone marrow-derived DCs were exposed to lipopolysaccharide and then treated with unmodified or IDO1-silenced exosomes. The maturation status of DCs was subsequently analyzed by measuring the surface expression of key markers cluster of differentiation (CD) 80, CD86, and major histocompatibility complex class II (MHC II) and the secretion of cytokines interleukin (IL)-12p70 and IL-10. Furthermore, a subcutaneous tumor mouse model was established and injected with unmodified or IDO1-silenced exosomes. Tumor growth, DC maturation, and T cell activation were also systematically evaluated.

RESULTS

IDO1 was upregulated in gastric cancer tissues and enriched in gastric cancer cell-derived exosomes. Treatment with these exosomes suppressed DC maturation, as evidenced by significant reductions in the expression of CD80, CD86, and MHC II at approximately 63%, 62%, and 70%, respectively; approximately 60% decrease in IL-12p70; and more than 178% increase in IL-10. IDO1 silencing in exosomes reversed these effects. In vivo administration of unmodified exosomes inhibited DC maturation (CD80, CD86, and MHC II expression levels reduced by 55%, 64%, and 65%, respectively) and T-cell activation, thereby promoting tumor growth in mice (tumor weight increased by 1.9 folds). Treatment with IDO1-silenced exosomes restored these effects and attenuated tumor growth (tumor weight reduced by approximately 67%).

CONCLUSION

Gastric cancer-derived exosomal IDO1 drives immunosuppression in gastric cancer by suppressing DC maturation. Our findings deepen the comprehension of gastric cancer immune evasion and propose IDO1 as a promising therapeutic target.

Key Words: Gastric cancer; Exosomal indoleamine 2,3 dioxygenase 1; Dendritic cells; Cytotoxic T cells; Immunosuppression

Core Tip: Gastric cancer-derived exosomal indoleamine 2,3 dioxygenase 1 (IDO1) suppresses dendritic cell (DC) maturation, thereby facilitating tumor immune evasion. This study demonstrates that IDO1 is upregulated in gastric cancer tissues and selectively enriched in tumor-derived exosomes. These IDO1-carrying exosomes are internalized by DCs, leading to downregulation of maturation markers (cluster of differentiation 80, cluster of differentiation 86, major histocompatibility complex class II) and an immunosuppressive cytokine shift (decreased interleukin-12p70, increased interleukin-10). Silencing IDO1 in exosomes reverses these effects and attenuates tumor growth in vivo. Our findings identify exosomal IDO1 as a key mediator of tumor-DC crosstalk and a promising therapeutic target in gastric cancer.

INTRODUCTION

Gastric cancer is among the top causes of cancer-related death worldwide[1]. According to the latest epidemiological data, approximately 960000 new cases and 360000 deaths occurred worldwide in 2022[2]. Incidence rates of gastric cancer among people under 50 have been increasing at an accelerated pace[3]. Gastric carcinogenesis is a step-by-step formation of disease[4]. The risk factors are related to Helicobacter pylori infection, virus, high salt dietary habit, overweight people, smokers and genetic defects, etc. Advances in diagnostic technologies, surgical techniques and multimodal therapies have improved the five-year survival rates of early-stage patients[5]. However, because late-detection (80 percent of cases) leads to an adverse events rate remain high[6]. Clinical management at present includes surgery and other treatments. Postoperatively recurrence is still prevalent, resulting in poor clinical outcome and therapy results[7]. Understanding whether the underlying molecular pathologies that cause this condition differ significantly from other diseases may inform the development of new therapies or drug targets.

For instance, immune checkpoint inhibitors can restore cytotoxic T cell functions and exert persistent anti-tumour effect through eliminating obstacles for them in the tumour microenvironment (TME)[8]. In addition to T cells, accessory cells that constitute cellular immunity. Among them, dendritic cells (DCs) have the strongest antigen presentation function[9]. Following antigen capture, DCs process and display these substances through major histocompatibility complex (MHC) class I and MHC-II molecules on their surfaces. Peptide-MHC complexes then bind to cluster of differentiation (CD) 8 and CD4 molecules on the surface of CD8⁺ and CD4⁺ T cells, providing the initial signal required for T-cell activation[10]. A second stimulus is given by the costimulatory molecule on the surface of DCs, including CD86, CD80 and CD40; it then binds to T-cell receptors to activate T cells[10]. The third type is stimulated cytokines secreted by DCs, including interleukin (IL)-12p70 (a type of IL) and interferon (IFN), which stimulate T-cell proliferation and differentiate into effectors[11]. Abundant costimulatory molecules are present on mature DCs to act as master coordinators of adaptive immunity and have strong anti-tumour properties[12]. The main way of connecting innate and adaptive immunity; matured DC serves to ensure that cancer can avoid immune regulation or attack in the process. Thus, matured DC is expected to improve treatment outcomes by increasing immunosuppressive responses.

DC is regulated in various aspects inside the TME; but it has a strong effect due to changes induced by tumours. The tumour cell-modulated alterations in DC biology include its recruitment, differentiation, activation and antigen-presentation ability[13]. Besides traditional ways of connecting cells directly or secreting dissolved factors, some researchers have recently found that tumor-secreted exosomes play a crucial role in mediating cellular communications and immune modulation within the TME[14,15]. Exosomes are extracellular vesicles that carry specific proteins, lipids and nucleotides inside the cell to transfer materials or information among cells[16]. The gastric cancer-derived exosome-suppressed DC maturation to promote immune evasion and cancer progression[17]. It is still unknown which molecules inside exosomes directly affect DC dysfunction. Thus, this research area has been lacking in exploration of how gastric cancer achieves immune tolerance.

Indoleamine 2,3 dioxygenase 1 (IDO1) is considered a critical enzyme in the trypotphan pathway and an oncogenic factor in many human tumours[18]. IDO1 as an immunological barrier has inhibited anticancer effect in some extent through suppressing activities of antitumour immune cells[19,20]. Specifically, IDO1 promotes tryptophan to kynurenine in the TME via metabolic reactions and activates the aryl hydrocarbon receptor signalling pathway. Activation triggers the differentiation of immune-tolerant DCs and regulatory T cells to strengthen the immunosuppressive microenvironment[21]. IDO1 knockdown of DCs increases expression of tumour necrosis factor-alpha and IL-12p70 subunits but decreases IL-10 levels; therefore, activated DCs promote T-cell mediated anti-tumour immune response[22]. IDI-1 up-regulation is considered a potential diagnostic biomarker of gastric cancer[23]. Upregulated IDO1 was positively associated with a worse chemotherapy outcome and lymph node metastasis in gastric cancer patients[24]. The DC maturation process is crucial for triggering antitumor immunity; meanwhile, IDO1 regulation by other factors has been established. However, whether tumour-derived exosomal-IDO1 affects the function of DC-mediated anti-tumour immune response in gastric cancer as well as how it participates in DC dysfunction in the TME remain controversial.

We determined the role of extracellular IDO1-expressing gastric cancer-derived exosomes in modulating DC maturation and immunosuppressive effects on tumour progression. By identifying exosomal-IDO1 as an essential mediator of tumour-inflammatory-dendritic cell dysfunction, we sought to clarify the primary molecular pathway by which gastric cancer affects immune response. This discovery can provide new immuno-therapeutic targets in reversing antitumor immunity at these sites.

MATERIALS AND METHODS.

Clinic samples

A total of 35 patients with gastric cancer who underwent surgical resection were enrolled from the First Affiliated Hospital of Nanchang University. The cohort comprised 21 males and 14 females, with a median age of 58 years (range 35-76 years). In accordance with the American Joint Committee on Cancer 8^th edition staging system, the pathological stages of the patients were as follows: (1) Stage I (n = 8); (2) Stage II (n = 12); and (3) Stage III (n = 15). Regarding histology, 23 cases were intestinal-type adenocarcinoma and 12 cases were diffuse-type adenocarcinoma according to Lauren classification. The inclusion criteria were as follows: (1) Pathological confirmation of gastric adenocarcinoma; (2) Completeness of clinical data and availability of tissue specimens; and (3) No prior antitumor treatment. The exclusion criteria were as follows: (1) A history of other gastric disorders, additional malignancies, severe systemic diseases, and significant infectious diseases; and (2) Any prior antitumor therapy. During surgical resection, paired tumor and adjacent nontumor tissues were collected from each patient. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University.

Cell culture and transfection

Gastric cancer cell lines (MKN45, AGS, HGC27, and BGC-823) and human gastric epithelial cell line GES-1 were obtained from American Type Culture Collection (Manassas, VA, United States). The cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin in 5% carbon dioxide at 37 °C. Short hairpin RNA targeting IDO1 (sh-IDO1) and short hairpin RNA targeting negative control (sh-NC) were obtained from RiboBio (Guangzhou, Guangdong Province, China) and transfected into gastric cancer cells using Lipofectamine^TM 3000 (Invitrogen, United States) in accordance with the manufacturer’s instructions. Stable transfectants were selected using puromycin (2 μg/mL) for 14 days.

Generation of bone marrow-derived DCs

Male C57BL/6 mice (6-8 weeks old) were euthanized via cervical vertebra dislocation. A single-cell suspension of bone marrow cells was obtained from femurs and tibias using a 70 μm cell strainer. These cells were cultured in complete RPMI 1640 medium enriched with 20 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (RD System, United States) and 10 ng/mL recombinant mouse IL-4 (RD System, United States). The medium was replaced every 2 days. After 6 days, bone marrow-derived DCs were harvested and treated with 1 μg/mL lipopolysaccharide (LPS) (Sigma-Aldrich, United States) for 24 hours to induce maturation.

Isolation and identification of gastric cancer cell-derived exosomes

Exosomes were isolated from MKN45 cells by ultracentrifugation as previously described[25]. In brief, cells were cultured in Dulbecco’s modified eagle medium containing exosome-depleted fetal bovine serum for 48 hours. The conditioned medium (CM) was collected and subjected to a series of centrifugal steps at 4 °C: (1) 1000 × g for 5 minutes; (2) 2000 × g for 20 minutes; (3) 5000 × g for 30 minutes; and (4) 10000 × g for 30 minutes. The resulting supernatant was filtered through a 0.22 μm pore membrane and subsequently ultracentrifuged at 100000 × g for 70 minutes to pellet the exosomes. The pellet underwent a second round of ultracentrifugation (100000 × g, 70 minutes) for purification. Finally, the purified exosomes were resuspended in phosphate buffered saline (PBS) and stored at -80 °C for further use. Exosome morphology was visualized by transmission electron microscopy (Hitachi, Japan). Size distribution was assessed via nanoparticle tracking analysis (Malvern Instruments, United Kingdom)[26]. The protein concentration of isolated exosomes was quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, United States). Specific exosomal markers (CD63, CD9, CD81), and negative control calnexin were detected using Western blot analysis.

Exosome uptake by DCs

Exosome uptake by DCs was assessed using a PKH26 dye (Sigma-Aldrich, United States) in accordance with the established procedures[27]. In brief, exosomes were treated with 2 μmol/L PKH26 for 5 minutes and quenched with 1% bovine serum albumin. The PKH26-labeled exosomes were then purified via ultracentrifugation and co-incubated with DCs for 24 hours at a concentration of 100 μg/mL. Following incubation, nuclei were stained with 4’,6-diamidino-2-phenylindole. Exosome uptake was visualized under a confocal fluorescence microscope (Zeiss, Germany).

Western blot analysis

Proteins were isolated using radioimmunoprecipitation assay lysis buffer (Solarbio, Shanghai, China). The extracted proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes (Millipore, United States). After being blocked with 5% skimmed milk, the membranes were incubated overnight at 4 °C with primary antibodies, followed by incubation with secondary antibodies at 37 °C for 1 hour. Protein bands were detected using an enhanced chemiluminescence kit (Pierce, United States) and quantified with ImageJ software. The primary antibodies were listed below: IDO1 (1:1000, ab137384), CD63 (1:1000, ab134045), CD9 (1:1000, ab236630), CD81 (1:1000, ab109201), calnexin (1:20000, ab92573), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, ab8245), all purchased from Abcam (Cambridge, United Kingdom).

Flow cytometry analysis

For cell phenotype analysis, bone marrow-derived DCs or single-cell suspensions from tumors were stained with the following fluorescent dye-labeled anti-mouse antibodies (BioLegend, United States) at 4 °C for 20 minutes: (1) Anti-CD80 (104707); (2) Anti-CD86 (105007); (3) Anti-MHC II (116407); (4) Anti-CD45 (147715); (5) Anti-CD11c (117329); (6) Anti-Lineage Cocktail (133301); (7) Anti-CD3 (100217); (8) Anti-CD4 (100449); and (9) Anti-CD8a (100711). For the assessment of intracellular proteins, the cells were fixed and permeabilized using a fixation/permeabilization buffer (421403, BioLegend, United States) and stained with PE anti-IFN-γ (505807), and anti-granzyme B (515403). Appropriate isotype controls were used to set gating thresholds. Fluorescence analysis was conducted using FACSCalibur cytometer (BD Biosciences, United States) and FlowJo version 10.8 software.

Enzyme-linked immunosorbent assay

IL-12p70 and IL-10 concentrations in the cell supernatants were detected using commercial enzyme-linked immunosorbent assay kits (Abcam, United States) in accordance with the manufacturer’s protocols. Each sample was assayed in triplicate, and the absorbance was measured at 450 nm using a microplate reader (Bio-Rad, United States).

Animal experiments

Male C57BL/6 mice (6-8 weeks, 18-20 g) were purchased from the First Affiliated Hospital of Nanchang University. Thirty-two mice were used in this study and housed under specific pathogen-free conditions with a 12 hours light/dark cycle and free access to food and water. For the establishment of a subcutaneous tumor model, 5 × 10⁵ MKN45 cells in 100 μL of PBS were injected subcutaneously into the right flank of mice. When the tumor volume reached approximately 100 mm³ (approximately 7 days post-injection), the mice were randomly allocated into the following four groups (n = 8 per group): (1) PBS control; (2) MKN45-exo; (3) MKN45-exo (sh-NC); and (4) MKN45-exo (sh-IDO1). Exosomes were suspended in 200 μL of PBS and administered via tail vein injection every other day over a 2-week period. Control animals received an equal volume of PBS. Tumor volume was monitored on a weekly basis and calculated using the formula: V = (length × width²)/2. On day 28, all mice were euthanized, and tumors were harvested for further analysis. All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Nanchang University.

Immunohistochemistry

For immunohistochemical analysis, 4 μm-thick sections were prepared from formaldehyde-fixed, paraffin-embedded tumor tissues. After deparaffinization and rehydration, the sections were treated with 10 mmol/L sodium citrate under microwave heating for antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. The sections were probed with primary antibodies against IDO1 (1:100, ab137384) or Ki67 (1:200; ab16667, Abcam) at 4 °C overnight, followed by incubation with the secondary antibody for 1 hour. Immunostaining was developed using a 3,3’-diaminobenzidine kit (Beyotime, Shanghai, China). Images were acquired under a Nikon light microscope and analyzed with Image J software.

Preparation of single-cell suspension from tumors

Tumor tissue fragments (approximately 1 mm³) were subjected to a 2 hours enzymatic digestion at 37 °C in RPMI 1640 containing 0.1% collagenase type IV, 0.01% hyaluronidase, and 0.002% DNase I (Sigma-Aldrich, United States). The resulting cell mixture was then filtered through a 100 μm strainer to obtain a single-cell suspension. The cells were washed twice with PBS and resuspended in flow cytometry staining buffer for subsequent analysis.

Statistical analysis

Data were presented as the mean ± SD, and statistical analyses were performed using SPSS 22.0. For biological replication, sample sizes were set as n = 6 for cell-based assays and n = 8 for animal studies, with each experiment repeated independently at least three times. All data were tested for normality and homogeneity of variance using Shapiro-Wilk test and Levene’s test, respectively. Comparisons between two groups were conducted using Student’s t-test, and those among multiple groups were assessed by one-way analysis of variance and subsequent Tukey’s post-hoc test. P < 0.05 was considered statistically significant.

RESULTS

IDO1 upregulation is negatively correlated with the infiltration of CD11c⁺ DCs in gastric cancer

According to the Gene Expression Profiling Interactive Analysis database, IDO1 levels were higher in stomach adenocarcinoma than in normal tissues (Figure 1A). This upregulation was consistently validated in our clinical cohort, where IDO1 protein levels were significantly elevated in tumor tissues compared with those in adjacent normal tissues (Figure 1B and C), suggesting the potential oncogenic role of IDO1. Immunohistochemical staining also confirmed enhanced IDO1 expression in gastric cancer specimens (Figure 1D). IDO1 upregulation was detected across several gastric cancer cell lines (Figure 1E and F). In addition, immunofluorescence results showed that IDO1 protein level was negatively correlated with the infiltration level of CD11c⁺ DCs in tumor specimens (Figure 1G and H). Our results demonstrate that IDO1 upregulation is negatively correlated with the infiltration of CD11c⁺ DCs in gastric cancer. This negative correlation implies that IDO1 may contribute to immune evasion by limiting DC recruitment or retention within the TME.

Figure 1 Upregulation of indoleamine 2,3 dioxygenase 1 was negatively correlated with the infiltration of cluster of differentiation 11c⁺ dendritic cells in gastric cancer. A: GEPIA database (http://gepia.cancer-pku.cn/) showed upregulation of indoleamine 2,3 dioxygenase 1 (IDO1) in stomach adenocarcinoma; B-D: Western blot analysis (B and C) and immunohistochemical staining (D) were conducted to assess of IDO1 protein levels in tumor and adjacent tissue specimens from gastric cancer patients (n = 35 per group); E and F: IDO1 protein expression in gastric cancer cell lines was examined using Western blot analysis; G: Immunofluorescence analysis of IDO1 and cluster of differentiation 11c protein levels in tumor specimens; H: Upregulation of IDO1 was negatively correlated with the infiltration of cluster of differentiation 11c⁺ dendritic cells in gastric cancer. Data were presented as mean ± SD. ^aP < 0.01. CD: Cluster of differentiation; DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IDO1: Indoleamine 2,3 dioxygenase 1; STAD: Stomach adenocarcinoma.

IDO1 is highly enriched in exosomes derived from gastric cancer cells

Exosomes were isolated from MKN45 cells to investigate the functional role of IDO1 in the TME of gastric cancer. Transmission electron microscopy imaging showed that the isolated vesicles exhibited classical cup-shaped morphology (Figure 2A). Nanoparticle tracking analysis indicated a predominant size range of 30-140 nm (Figure 2B), consistent with exosomal characteristics. Exosomal identity was further validated by Western blot, which detected the strong expression of exosomal markers CD63, CD9, and CD81 and the absence of the negative control protein calnexin (Figure 2C), confirming the successful isolation of high-purity exosomes from MKN45 cells. Furthermore, IDO1 was highly expressed in total CM and exosomes but was nearly absent in exosome-depleted CM (Figure 2D and E), indicating that IDO1 is selectively enriched within the exosomes derived from MKN45 cells. In addition, PKH26 probe was used to label the exosomes to evaluate exosome uptake. PKH26-labeled exosomes were incubated with DCs. Fluorescence microscopy confirmed the successful uptake of the labeled exosomes by DCs (Figure 2F), demonstrating that these IDO1-carrying exosomes can be directly internalized to DCs.

Figure 2 Indoleamine 2,3 dioxygenase 1 was highly enriched in exosomes derived from gastric cancer cells. Exosomes were isolated from MKN45 cells. A: Exosome morphology was examined using transmission electron microscopy; B: Nanoparticle tracking analysis was employed to determine size distribution; C: The presence of exosomal markers was confirmed using Western blot analysis; D and E: Indoleamine 2,3 dioxygenase 1 expression in exosomes was measured using Western blot analysis; F: Uptake of PKH26-labeled exosomes by dendritic cells was visualized using microscopy. Data were presented as mean ± SD. ^aP < 0.01. CD: Cluster of differentiation; CM: Conditioned medium; DAPI: 4’,6-diamidino-2-phenylindole; exo: Exosomal; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IDO1: Indoleamine 2,3 dioxygenase 1.

Gastric cancer-derived exosomes suppress the maturation and immunostimulatory function of DCs by delivering IDO1

MKN45 cells were transfected with sh-IDO1 or sh-NC, and exosomes were subsequently isolated. Western blot analysis confirmed successful IDO1 silencing in MKN45 cells (Figure 3A) and exosomes (Figure 3B). Bone marrow-derived DCs were stimulated with LPS to induce maturation and incubated with 100 μg/mL unmodified exosomes, IDO1-silenced exosomes, and negative control exosomes to investigate the role of gastric cancer-derived exosomal IDO1 in modulating DC maturation within the TME. Treatment with unmodified exosomes upregulated IDO1 protein levels in DCs, while IDO1-silenced exosomes normalized IDO1 expression to baseline level (Figure 3C). Flow cytometry revealed that LPS exposure significantly upregulated the expression of maturation markers including CD80, CD86, and MHC II (Figure 3D-G), indicating that LPS successfully induced DC maturation. However, treatment with unmodified exosomes significantly reduced the expression of these markers. This inhibitory effect was largely attenuated upon IDO1 silencing in exosomes (Figure 3D-G), suggesting that the suppression of DC maturation is specifically mediated by exosomal IDO1. Furthermore, the LPS-treated DCs showed enhanced secretion of IL-12p70 and IL-10 (Figure 3H), with a particularly notable increase in the IL-12p70/IL-10 ratio (Figure 3I). Treatment with unmodified exosomes skewed this balance toward an immunosuppressive state by suppressing IL-12p70 secretion and enhancing IL-10 production, leading to a significantly decreased IL-12p70/IL-10 ratio. Consistent with the maturation marker results, this shift in cytokine balance was abrogated upon IDO1 silencing in exosomes. Overall, these findings suggest that gastric cancer-derived exosomes suppress the maturation and immunostimulatory function of DCs specifically by delivering IDO1.

Figure 3 Gastric cancer-derived exosomes suppress the maturation and immunostimulatory function of dendritic cells by delivering indoleamine 2,3 dioxygenase 1. Bone marrow-derived dendritic cells were stimulated with lipopolysaccharide to induce maturation and incubated with 100 μg/mL unmodified exosomes, indoleamine 2,3 dioxygenase 1 (IDO1)-silenced exosomes, and negative control exosomes, respectively. A: MKN45 cells were transfected with short hairpin RNA targeting IDO1 or short hairpin RNA targeting negative control, and IDO1 protein levels were measured using Western blot analysis; B: IDO1 protein levels in isolated exosomes were measured using Western blot analysis; C: IDO1 protein levels in dendritic cells were assessed using Western blot analysis; D-G: Representative flow cytometry histograms showing the expression of maturation markers (cluster of differentiation 80, cluster of differentiation 86, and major histocompatibility complex class II) and quantification of the mean fluorescence intensity; H: The secretion levels of interleukin (IL)-12p70 and IL-10 were measured using enzyme-linked immunosorbent assay kits; I: IL-12p70/IL-10 ratio was calculated. Data were presented as mean ± SD. ^aP < 0.01. CD: Cluster of differentiation; exo: Exosomal; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IDO1: Indoleamine 2,3 dioxygenase 1; IL: Interleukin; LPS: Lipopolysaccharide; MFI: Mean fluorescence intensity; MHC: Major histocompatibility complex; PBS: Phosphate buffered saline; sh-IDO1: Short hairpin RNA targeting indoleamine 2,3 dioxygenase 1; sh-NC: Short hairpin RNA targeting negative control.

Gastric cancer-derived exosomal IDO1 promotes tumour progression in mouse model

To examine whether exosomal-IDO1 contributed to cancer development under physiological conditions, a subcutaneous tumour mouse model for MKN45 cells was established, and treated with unmodified exosomes or IDO1-silencing exosomes. The administration of unmodified exosomes significantly promoted tumour growth, evidenced by increased tumour volume and weight (Figure 4A-C). However, IDO1-silenced exosomes effectively attenuated this tumor-promoting effect, resulting in significantly decreased tumor volumes and weights (Figure 4A-C). These results indicate that exosomal IDO1 is essential for this enhanced tumour growth. Further immunohistochemical examination showed increased expressions of Ki67 and IDO1 in tumours derived from the un-modified-exosome groups (Figure 4D-G). Moreover, the increased levels of proteins were inhibited after treatment with IDO1 silenced-exosome. In summary, gastric cancer-derived exosomal IDO1 can promote tumour growth in mice.

Figure 4 Gastric cancer-derived exosomal indoleamine 2,3 dioxygenase 1 promotes tumor growth in mice. A subcutaneous tumor mouse model was established and treated with unmodified exosomes, indoleamine 2,3 dioxygenase 1-silenced exosomes, and negative control exosomes, respectively (n = 8 group). A: The excised tumors in four group were imaged; B: Tumor volumes were measured every 7 days; C: The weights of excised tumors were measured; D-G: Ki67 and indoleamine 2,3 dioxygenase 1 protein levels were examined using immunohistochemistry. Data were presented as mean ± SD. ^aP < 0.01. exo: Exosomal; IDO1: Indoleamine 2,3 dioxygenase 1; IHC: Immunohistochemistry; PBS: Phosphate buffered saline; sh-IDO1: Short hairpin RNA targeting indoleamine 2,3 dioxygenase 1; sh-NC: Short hairpin RNA targeting negative control.

Gastric Cancer-exogenous IDO1 induces tumour immunosuppression through the suppression of DC maturation and T-cell activation.

DC maturation is necessary to initiate antigen-specific T-cell-mediated anti-tumour immunity[28]. We further examined the role of extracellular IDO1 in modulating DC differentiation and T-cell responses. Unmodified exosomes treatment significantly decreased the number of tumour infiltrating CD11b⁺DCs (Figure 5A and B). Meanwhile, it also inhibited the expression levels of key DC maturation markers, including CD80, CD86 and MHC-II in tumours under experimental conditions (Figure 5C-F). These inhibitory effects disappeared after silencing IDO1 in the exosomes. These data show that the reduction in DC infiltration and functional maturation were impaired by exosomal IDO1. In addition, the unmodified exosomes inhibited cytotoxic T cell response through reduced levels of IFN-γ and granzyme B in CD8⁺ T cells (Figure 5G-I). The loss of this function may be due to impaired DC maturation caused by exosomal IDO1. Together, these findings show that gastric cancer-derived exosomal IDO1 induces immune evasion in tumours through suppression of DC maturation and T-cell activation.

Figure 5 Gastric cancer-derived exosomal indoleamine 2,3 dioxygenase 1 promotes tumor immune escape by suppressing dendritic cell maturation and T cell activation. A and B: Representative flow plots showing the abundance of tumor-infiltrating cluster of differentiation (CD) 11c⁺Lin^- dendritic cells (DCs) and quantification of the percentages of CD11c⁺Lin^- DCs; C-F: Representative flow cytometry histograms showing the expression of CD80, CD86, and major histocompatibility complex class II on gated tumor-infiltrating CD11c⁺Lin^- DCs and expression of maturation markers calculated by mean fluorescence intensity; G-I: Representative flow plots showing the population of interferon-γ⁺ CD8⁺ T cells and granzyme B⁺ CD8⁺ T cells and quantification. Data were presented as mean ± SD. ^aP < 0.01. CD: Cluster of differentiation; DC: Dendritic cell; exo: Exosomal; IFN: Interferon; LPS: Lipopolysaccharide; MFI: Mean fluorescence intensity; MHC: Major histocompatibility complex; PBS: Phosphate buffered saline; sh-IDO1: Short hairpin RNA targeting indoleamine 2,3 dioxygenase 1; sh-NC: Short hairpin RNA targeting negative control.

DISCUSSION

Recently, DC-based immunotherapy has attracted increasing attention in cancer research worldwide, including DC vaccines and the activation of endogenous DCs both ex vivo and in vivo by tumour-associated antigens or immunomodulatory substances[29]. DCs are specialised antigen-presenting cells that have been utilised in the development of personalisation-based vaccines for delivering various tumour-associated antigens, such as neoantigens, which may enhance robust T-cell-mediated immunity[30]. Studies show that DC based-immune therapy is significantly effective against gastric cancer at present[31-34]. However, the immunosuppressive nature of the TME impedes normal DC maturation and function. The extent to which gastric cancer cells impair DC capacity was investigated in this study. The current study has identified the delivery of IDO1 by exosome as a new and strong immunosuppressive mechanism directly hindering DC maturation, which contributes to immune escape.

Through this study, we have revealed a new pathway in which gastric cancer- derived exosomal IDO1 inhibits DC maturation and creates an immunosuppressive TME that promotes tumour-immune evasion. At first, it is observed that the level of IDO1 expression in gastric cancer tissues is higher than that in normal stomach tissue. This finding has also been confirmed to be related to an adverse prognosis[23]. Additionally, IDO1 expression has a negative correlation with CD11b-containing DC infiltration, and therefore may have an immunoregulatory effect in TME. This concept is consistent with the role identification of IDO1 to regulate DCs and regulatory T cells that reduces tumour immunity[21]. As the intensity of this correlation indicates, there is a degree to which the IDO1-regulated immune suppression in gastric cancers acts via pathways that directly affect DC physiology. Mechanismally, we showed that IDO1 was enriched in exosomes released by gastric cancer cells. IDO1-bearing exosomes can be effectively taken up by DCs and cause their immature state. This was evidenced by downregulation of key surface molecules CD80, CD86, and MHC-II, as well as an altered cytokine secretion pattern favoring immune suppression, with reduced IL-12p70 and increased IL-10 levels. The most notable change in the IL-12p70/IL-10 ratio determines whether T-cell responses polarize toward an immune reaction or a tolerant state. IDO1-knockdown tumour-derived exosomes exerted a significant ameliorative effect on this immature state, suggesting that their anti-tumour role involves IDO1 within tumour-exogenous extracellular vesicles. As shown in reports of other studies, IDO1 knockdown can create an immune-activated microenvironment through the increase of IL-12 expression and the reduction of IL-10 concentration[22]. As an example, the selective IDO1 inhibitor epacadostat can activate CD4⁺ conventional DCs to boost the cytotoxic effects of tumour-associated antigens-specific T lymphocytes[35]. In this paper, we have identified that the exosomal IDO1-mediated transfer mechanism is an important way for tumour cell-derived IFNAR2 ligands to deliver into DCs in gastric cancer.

In vivo validity has been established by the above-described system through a subcutaneous tumour model; The administration of un-modified extracellular vesicles can promote tumour progression and suppress maturation of intra-tumoral DCs and T-cell activation. These immune suppressive effects were abolished after IDO1 knockdown in exosomes. In this in vivo rescue experiment, it has been confirmed that exosomal IDO1 is essential for observing the tumour-promoting effect; thus, other components of exosomes are unlikely to be the primary cause. Previous research findings support this conclusion; therefore, reducing IDO1 can promote anti-tumour immunity currently studied here. Knockdown of IDO1 in DCs via a lipid nanoparticle delivery system enhanced cell-based cancer immunotherapy[36]. IDO1 blockade enhanced the effect of CAR-T cells on tumour suppression by reducing tumour growth and extending survival in gastric tumour models[37]. IDO1 transfer via extracellular vesicle forms an immunosuppressive state via a new route. IDO1 expression in tumour-derived extracellular vesicles was enriched upon IFN-γ stimulation, resulting in the generation of myeloid-derived suppressor cells that inhibited T-cell activity[38]. In acute myeloid leukemia, engineered extracellular vesicles delivering siIDO1 enhanced CD8⁺ T cell function and antileukemia immunity[39]. Additionally, IDO1 blockade improved the therapeutic effect of claudin-18.2 targeted CAR-T cell therapy on gastric cancer through overcoming the metabolic suppression of T cells via kynurenine[38]. Converging lines of evidence demonstrate that IDO1 is a crucial node connecting tumour-associated immunosuppression and holds promise as the target for therapy development.

The following content summaries from our research results are listed below: Exosomal IDO1 is one of the main mediators for tumour-activated DLC2 cross-talks in gastric adenocarcinoma. IDO1 encapsulated within exosomes is transported to gastric cancer cells through exosome-mediated transfer and causes an immunosuppressive effect on DCs within the TME, making it difficult for T-cells to recognise antigens. A recent study found that in patients with gastric cancer who received immune checkpoint inhibitors, plasma extracellular vesicles-expressed IDO1 was positively correlated with immune-related adverse events[40]. This mechanism may help explain why the present immunotherapy is often ineffective for gastric cancer at present and provide a theoretical basis for inhibiting IDO1 to restore DC function[21,22]. Our results are in agreement with those obtained from other tumours showing that IDO1 knockdown can enhance DC function. However, we found that the extracellular vesicle path is required to transmit this phenomenon for gastric cancer. They have great value in hospitals. A combination strategy of IDO1 inhibitor-based therapy (small-molecule inhibitors, nanotechnology) with immune checkpoint blocker may improve the effect when the latter causes primary resistance through activating DCs to enhance T-cell activation and infiltration after priming. Additionally, interference strategies to block the internalization of exosomes into DCs or engineer target-exosomal transport systems that carry IDO1 inhibitors can precisely eliminate this immune evasion path.

Several limitations appear in the research. A total of 35 patients with gastric cancer comprised the clinical group in this study. Though meticulous following of the inclusion and exclusion criteria produced a homogeneous group composition, this led to potential selection bias, thus affecting generalization results and consequently decreasing the statistical power to detect differences among certain subgroups. Future research with large-scale multi-centre trials is needed to assess the clinical applicability of exosomal IDO1 in gastric cancer. Secondly, the subcutaneous tumour model may not completely reproduce the complex immunological microenvironment in the natural gastric site. In the future, further study is needed to improve its clinical applicability using orthotopic gastric cancer models. Third, the current study primarily examined the impact of exosomal IDO1 on DC maturation and T cell activation. The effects on other types of immune cells within the TME, such as macrophages and T cell subsets, should be investigated in future studies. Finally, there are still several problems with the clinical application of IDO1 targeted therapy, including improving the specificity and safety of drug delivery to DCs.

CONCLUSION

We established gastric cancer-derived exosomal IDO1 as an essential factor for immune-suppressive effects through impairment of DC maturation and T cell activation. In our study, we hope to supplement existing knowledge on immune evasion in gastric cancer and identify IDO1 as an immunotherapy target. Inhibitory strategies to inhibit exosome-associated IDO1 expression can promote immune regulation of tumour progression and provide new avenues for improving treatment outcomes of gastric cancer patients.