Endoplasmic Reticulum Stress Causes Liver Cancer Cells to Release Exosomal miR-23a-3p and Up-regulate Programmed Death Ligand 1 Expression in Macrophages

Abstract

Endoplasmic reticulum (ER) stress promotes tumor cell escape from immunosurveillance. However, the underlying mechanisms remain unknown. We hypothesized that ER stress induces hepatocellular carcinoma (HCC) cells to release exosomes, which attenuate antitumor immunity by modulating the expression of programmed death ligand 1 (PD-L1) in macrophages. In this study, we demonstrated that expression of several ER stress markers (glucose-regulated protein 78, activating transcription factor 6, protein kinase R-like ER kinase, and inositol-requiring enzyme 1α) was up-regulated in HCC tissues and negatively correlated with the overall survival and clinicopathological scores in patients with HCC. Expression of ER stress-related proteins positively correlated with CD68⁺ macrophage recruitment and PD-L1 expression in HCC tissues. High-throughput sequencing analysis identified miR-23a-3p as one of the most abundant microRNAs in exosomes derived from tunicamycin (TM)-treated HCC cells (Exo-TMs). miR-23a-3p levels in HCC tissues negatively correlated with overall survival. Treatment with Exo-TMs up-regulated the expression of PD-L1 in macrophages in vitro and in vivo. Bioinformatics analysis suggests that miR-23a-3p regulates PD-L1 expression through the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway. This notion was confirmed by in vitro transfection and coculture experiments, which revealed that miR-23a-3p inhibited PTEN expression and subsequently elevated phosphorylated AKT and PD-L1 expression in macrophages. Finally, coculture of T cells with Exo-TM-stimulated macrophages decreased CD8⁺ T-cell ratio and interleukin-2 production but increased T-cell apoptosis in vitro. Conclusion: ER-stressed HCC cells release exosomes to up-regulate PD-L1 expression in macrophages, which subsequently inhibits T-cell function through an exosome miR-23a-PTEN-AKT pathway. Our findings provide insight into the mechanism how tumor cells escape from antitumor immunity.

FIG. 1.. ER stress is activated in human HCC.

(A) Representative low and high expression of ER stress markers GRP78, PERK, ATF6, and IRE1α in HCC tissue samples (scale bar = 200 μm). (B) Representative western blot analyses of GRP78, PERK, ATF6, and IRE1α protein expression in human HCC tissues. (C) qPCRdetected the mRNA levels of GRP78, PERK, ATF6, and IRE1α in 16 cases of freshly resected human HCC tissues. *P < 0.05, **P < 0.01.

FIG. 2.. Activation of ER stress correlates with poor survival in human HCC.

Kaplan-Meier curves demonstrated that the overall survival in patients with overexpression of (A) GRP78, (B) ATF6, (C) PERK, and (D) IRE1α was significantly shorter than those with low expression levels (n = 55). Log-rank test was used to evaluate statistical significance. **P < 0.01 as indicated.

FIG. 3.. ER stress is associated with macrophage infiltration and PD-L1 expression in HCC patients.

(A) Representative images of the expression pattern of CD68 protein in HCC tissues with GRP78^high and GRP78^low groups (scale bar = 200 μm for the upper panel; scale bar = 50 μm for the lower panel). Quantitative analysis of CD68-positive areas in the GRP78^high and GRP78^low group is shown in the right panel. (B) Representative images of the expression pattern of PD-L1 protein in HCC tissues with GRP78^high and GRP78^low groups (scale bar = 200 μm for the upper panel; scale bar = 50 μm for the lower panel). (C) qPCR analyses of PD-L1 mRNA levels in 16 cases of freshly resected HCC tissues. (D) Western blot analyses of PD-L1 protein levels in human HCC tissues. (E) The expression pattern of PD-L1 on CD68⁺ cells in GRP78^low and GRP78^high HCC tissues was measured by immunofluorescence (scale bar = 100 μm). (F) Log-rank test evaluated overall survival and PD-L1 expression in 55 HCC patients. Data are presented as the means ± SD (error bar). *P < 0.05, **P < 0.01 as indicated.

FIG. 4.. Exo-TM increases macrophage PD-L1 expression in vitro.

(A) PKH67-labeled exosomes were coincubated with mTHP-1 cells and examined by confocal microscopy (scale bar = 25 μm). Representative images are shown. Red arrows indicate PKH67-labeled exosomes. (B) Macrophages were coincubated with HepG2-derived Exo-con and Exo-TM, the protein levels of PD-L1 were determined by flow cytometry, and the mean fluorescence intensity (MFI) was statistically analyzed. Moreover, the impact of Exo-TM on macrophage PD-L1 protein expression was also determined by (C) western blot and (D) immunohistochemical analysis. PD-L1 mRNA was detected by (E) qPCR. Data are shown as the means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01 with indicated groups.

FIG. 5.. Exo-TM up-regulates the expression of PD-L1 in vivo.

PKH67-labeled exosomes were injected to nude mice intravenously through the tail vein, and the incorporation of PKH67-labeled exosomes by peritoneal macrophages was detected by (A) confocal microscopy (scale bar = 25 μm) and (B) flow cytometric analysis, peritoneal macrophages were isolated from mice treated with PBS, Exo-con, and Exo-TM, and the expression of PD-L1 was evaluated by (C,D) flow cytometric analysis, (E) immunohistochemical assay, and (F) qPCR. Data are shown as the means ± SD (error bar) of at least three independent experiments. *P < 0.05, **P < 0.01 with indicated groups.

FIG. 6.. Exo-TM-treated macrophages inhibit T-cell function and induce T-cell apoptosis.

Macrophages were coincubated with Exo-con and Exo-TM for 48 hours, then washed with exosome-free medium, followed by coincubating with CD3⁺ T cells at a ratio of 1:20 for another 48 hours. (A) CD4⁺ and CD8⁺ T cells were measured by flow cytometry and statistically analyzed by (B) SPSS 16.0. (C) The level of IL-2 secreted by T cells was determined by a Cytometric Bead Array (CBA) inflammatory factor kit. (D) Annexin V /PI apoptosis detection kit was used to detect the ratio of apoptotic T cells and statistically analyzed by (E) SPSS 16.0. Data are shown as means ± SD (error bar) of at least three independent experiments. *P < 0.05, **P < 0.01 with indicated groups. Mock= T cells; Con= macrophages + T cells; Exo-con= Exo-con-treated macrophages + T cells; Exo-TM= Exo-TM-treated macrophages + T cells.

FIG. 7.. miR-23a-3p up-regulates PD-L1 expression in macrophages.

(A) The heat map of miRNAs expressed in Exo-con and Exo-TM. (B-D) Macrophages were transfected with miR-23a-3p mimics, inhibitors, and their corresponding controls (NC, INC) for 48 hours. (B) Western blot and (C) qPCR assay were used to analyze the protein and mRNA levels of PTEN, respectively. The expression of PD-L1 on macrophages following miR-23a-3p transfection was analyzed by (D) flow cytometry and (E) qPCR. Data are shown as the means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01 with indicated groups. NC=Negative control; INC= Inhibitor negative control.

FIG. 8.. Exo-TM up-regulates expression of PD-L1 in macrophages through the inhibition of PTEN and subsequent activation of AKT pathway.

mTHP-1 cells were incubated with Exo-TM or Exo-Con, and the expression of PTEN and AKT proteins (A), PTEN mRNA (B), miR-23a-3p (C) levels in mTHP-1 were determined. (D) Transfection of mTHP-1 cells with miR-23a-3p inhibitor significantly reversed Exo-TM-mediated down-regulation of PTEN and up-regulation of p-AKT proteins. (E,F) PTEN in mTHP cells was knocked down by transfection of PTEN siRNA (si-PTEN). PD-L1 protein and mRNA expression on mTHP-1 cells were measured by flow cytometry and qPCR, respectively. Data are presented as the means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01 as indicated. Abbreviation: siRNA, small interfering RNA.