Exosomes derived from endoplasmic reticulum-stressed liver cancer cells enhance the expression of cytokines in macrophages via the STAT3 signaling pathway

Abstract

Previous studies have shown that endoplasmic reticulum (ER) stress serves an important role in shaping the immunosuppressive microenvironment by modulating resident immune cells. However, the communication between ER-stressed tumor cells and immune cells is not fully understood. Exosomes have been reported to play a vital role in intercellular communication. Therefore, in order to investigate the role of ER stress-related exosomes in liver cancer cells mediated macrophage function remodeling, immunohistochemical analysis, western-blotting immunofluorescence and cytokine bead array analyses were performed. The results demonstrated that glucose-regulated protein 78 (GRP78) expression was upregulated in human liver cancer tissue. Moreover, 69.09% of GRP78-positive liver cancer tissues possessed macrophages expressing CD68⁺ (r=0.55; P<0.001). In addition to these CD68⁺ macrophages, interleukin (IL)-10 and IL-6 expression levels were increased in liver cancer tissues. It was also demonstrated that exosomes released by ER-stressed HepG2 cells significantly enhanced the expression levels of several cytokines, including IL-6, monocyte chemotactic protein-1, IL-10 and tumor necrosis factor-α in macrophages. Furthermore, incubation of cells with ER stress-associated exosomes resulted inactivation of the Janus kinase 2/STAT3 pathway, and inhibition of STAT3 using S3I-201 in RAW264.7 cells significantly reduced cytokine production. Collectively, the present study identified a novel function of ER stress-associated exosomes in mediating macrophage cytokine secretion in the liver cancer microenvironment, and also indicated the potential of treating liver cancer via an ER stress-exosomal-STAT3 pathway.

Keywords: endoplasmic reticulum stress; exosome; inflammation; liver cancer; macrophage.

Figure 1.

GRP78 expression is positively associated with poor prognosis in patients with liver cancer. (A) Representative images of the low, moderate and high expression levels of the ER stress marker protein GRP78 in liver cancer tissue samples. Magnifications, ×100 for upper panel; ×400 for lower panel. (B) Percentage of liver cancer tissues of negative, low, moderate and high expression levels of GRP78. (C) Western blot analysis of GRP78 protein expression in three human liver cancer tissue samples, and (D) semi-quantitative analysis of band intensity using Scion Image 4.0.3.2 relative to GAPDH intensity. (E) Kaplan-Meier curves of overall survival in patients stratified into high- and low-GRP78 expression groups. GRP78, glucose-regulated protein 78; ER, endoplasmic reticulum; Cum survival, cumulative survival.

Figure 2.

Upregulation of GRP78 is associated with macrophage infiltration and an inflammatory microenvironment in liver cancer. (A) Representative images of CD68 protein expression in GRP78^high or GRP78^low liver cancer tissues. (B) Representative images of IL-6 and IL-10 expression levels in liver cancer tissues with GRP78^high or GRP78^low expression levels. Immunofluorescence was used to measure the expression pattern of IL-6 (C) and IL-10 (D) in CD68⁺ cells in GRP78-positive liver cancer tissues. Scale bar, 50 µm. GRP78, glucose-regulated protein 78; IL, interleukin.

Figure 3.

Characteristics of ER stress-associated exosomes. (A) HepG2 cells were treated with 0, 1.25, 2.5 and 5 µM TM for 24 h, and (B) GRP78 protein expression was measured and semi-quantitatively analyzed, relative to β-actin intensity. (C) HepG2 cells were treated with 2.5 µM TM for 12, 24 and 48 h, and (D) GRP78 protein expression was measured and semi-quantitatively analyzed, relative to β-actin intensity. (E) Representative transmission electron microscope images of Exo-TM. Scale bar, 100 nm. (F) Expression levels of CD63, TSG101, CD81, β-actin and Calnexin were measured using cell lysates or exosomes by western blotting. *P< 0.05, **P< 0.01. ER, endoplasmic reticulum; TM, tuniamycin; GRP78, glucose-regulated protein 78; Exo-TM, exosomes from the supernatants of HepG2 cells treated with 2.5 µM tunicamycin; TSG101, tumor susceptibility gene 101; Exo-con, exosomes from the supernatants of control HepG2 cells.

Figure 4.

Incubation with Exo-TM increases expression of cytokines in macrophages in vitro. (A) Confocal microscopy was used to measure the incorporation of PKH67-labeled exosomes into RAW264.7 cells. Cells were incubated with Exo-con and Exo-TM for 24 h, and (B) IL-6, (C) IL-10, (D) MCP-1 and (E) TNF-α levels were measured using a CBA inflammatory factor kit. *P< 0.05, **P< 0.01 with indicated groups. Exo-Con, exosomes from the supernatants of control HepG2 cells; Exo-TM, exosomes from the supernatants of HepG2 cells treated with 2.5 µM tunicamycin; IL, interleukin; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor-α; CBA, cytokine bead array.

Figure 5.

Exo-TM enhances cytokine expression by activating the JAK2/STAT3 pathway. RAW264.7 cells were treated with Exo-con or Exo-TM for 24 h. (A) Protein expression levels of the JAK2/STAT3 pathway were measured by western blot analysis. Blots presented in (A) were semi-quantitatively analyzed for comparing the (B) p-STAT3/STAT3 and (C) p-JAK2/JAK2 ratios, relative to β-actin intensity. (D) Western blot analysis of the protein expression levels of members of the JAK2/STAT3 signaling pathway in RAW264.7 cells treated with Exo-TM and S3I-201. Blots presented in (D) were semi-quantitatively analyzed for comparing the (E) p-STAT3/STAT3 and (F) p-JAK2/JAK2 ratios, relative to β-actin intensity. *P< 0.05, **P< 0.01 with indicated groups. Exo-Con, exosomes from the supernatants of control HepG2 cells; Exo-TM, exosomes from the supernatants of HepG2 cells treated with 2.5 µM tunicamycin; TM, tunicamycin; JAK2, Janus kinase 2; p-, phosphorylated.

Figure 6.

Inhibition of STAT3 decreases Exo-TM-induced cytokine expression. RAW264.7 macrophages were treated with Exo-con or Exo-TM with or without S3I-201 for 24 h. (A) IL-6, (B) IL-10, (C) MCP-1 and (D) TNF-α levels were measured using a CBA inflammatory factor kit. *P< 0.05, **P< 0.01 with indicated groups. Exo-Con, exosomes from the supernatants of control HepG2 cells; Exo-TM, exosomes from the supernatants of HepG2 cells treated with 2.5 µM tunicamycin; IL, interleukin; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor-α; CBA, cytokine bead array.