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

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.

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.

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.

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.

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.