Gastric cancer mesenchymal stem cells derived IL-8 induces PD-L1 expression in gastric cancer cells via STAT3/mTOR-c-Myc signal axis

Abstract

The expression of PD-L1 in tumor cells is one of the main causes of tumor immune escape. However, the exact mechanism for regulating PD-L1 expression in gastric cancer (GC) cells remains unclear. Our previous studies have shown that mesenchymal stem cells (MSCs) exert broad immunosuppressive potential, modulating the activity of cells either in innate or adaptive immune system to promote tumor progress. This study aims to investigate whether GCMSCs regulate the PD-L1 expression in GC cells and explore the specific molecular mechanism. The results have shown that GCMSCs enhanced PD-L1 expression in GC cells resulting in the resistance of GC cells to CD8⁺ T cells cytotoxicity. However, this resistance was attenuated with IL-8 inhibition. Further studies proved that IL-8 derived from GCMSCs induced PD-L1 expression in GC cells via c-Myc regulated by STAT3 and mTOR signaling pathways. Our data indicated that blocking IL-8 derived from GCMSCs may overcome the immune escape induced by PD-L1 in GC cells and provide a potential strategy to enhance the immunotherapy efficiency in GC.

Fig. 1. The expression of PD-L1 in GC tissues and GC cells.

The PD-L1 expression of GC tissues and corresponding adjacent normal tissues was detected by western blot (a) and immunohistochemistry (b). Bar = 50 μm. The PD-L1 expression of GC cells was tested by western blot (c) and flow cytometry (d)

Fig. 2. The expression of PD-L1 in GC cells treated with GCMSC-CM.

The PD-L1 expression of SGC-7901 and MGC-803 treated with BMMSC-CM or GCMSC-CM for 24 h was detected by western blot (a) and flow cytometry (b) (median was shown). Cells expressing different levels of PD-L1 (red fluorescence) were shown above corresponding flow cytometry histograms. c The sPD-L1 levels in culture supernatants of SGC-7901 and MGC-803 treated with BMMSC-CM or GCMSC-CM for 24 h were detected by ELISA. d The PD-L1 expression of SGC-7901 and MGC-803 treated with GCMSC-CM for 12, 24, 48, 72 and 96 h was detected by western blot. e The PD-L1 level of MGC-803 treated with GCMSC-CM for 24 h was evaluated by immunofluorescence. Bar = 50 μm. Data in c represents the mean ± SD of three repeated experiments (n = 3). GCMSCs were isolated from three different GC patients. *P < 0.05, **P < 0.01

Fig. 3. C-Myc played a role in GCMSC-CM-mediated PD-L1 up-regulation in GC cells.

The expression of PD-L1 and c-Myc in SGC-7901 (a) and MGC-803 (b) treated with GCMSC-CM was detected by western blot. The concentration of JQ1 was 10 μM. c Cytotoxicity assay of MGC-803. MGC-803 was pre-treated with JQ1 and then treated with GCMSC-CM for 24 h. The survival cells were stained with crystal violet. Bar = 100 μm

Fig. 4. GCMSCs-derived IL-8 up-regulated PD-L1 expression in GC cells.

a Cytokine array for screening different cytokines between BMMSC-CM and GCMSC-CM. b Each neutralizing antibody was added in GCMSC-CM and incubated at room temperature for 1 h and GCMSC-CM was then used to treat SGC-7901. c Immunofluorescence for the PD-L1 expression in SGC-7901 treated with GCMSC-CM. The concentration of IL-8 neutralizing antibody was 5 μg/ml (R&D Systems). Bar = 10 μm

Fig. 5. IL-8 derived from GCMSCs induced PD-L1 expression in GC cells via c-Myc regulated by STAT3 and mTOR signaling pathways.

a The expression of PD-L1 and c-Myc in GC cells treated with GCMSC-CM was detected by western blot. b The phosphorylation and activation of STAT3 in GC cells was detected by western blot. c GC cells were treated with 5 μM of a STAT3 inhibitor (Stattic) or 0.1 μM of an mTOR inhibitor (Rapamycin). d The expression of PD-L1 and the phosphorylation of AKT and S6 ribosomal protein in GC cells was detected by western blot. The concentration of IL-6/IL-8 neutralizing antibody was 5 μg/ml

Fig. 6. IL-8 in GCMSC-CM promoted GC cells resistance to cytotoxic effect of CD8⁺ T cells.

a, b Cytotoxicity assay of GC cells. GC cells were treated with BMMSC-CM or GCMSC-CM for 24 h and then used for cytotoxicity assay. c, d Quantification of survival GC cell numbers. e, g Cytotoxicity assay of GC cells. IL-8 neutralizing antibody was added in GCMSC-CM and incubated at room temperature for 1 h and GCMSC-CM was then used to treat GC cells. After 24 h treatment, GC cells were used for cytotoxicity assay. f, h Quantification survival GC cell numbers. The concentration of PD-L1 neutralizing antibody was 2 μg/ml. Bar = 100 μm. Data in c, d, f and h represent the mean ± SD of three repeated experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant

Fig. 7. Schematic of proposed network of GCMSCs-mediated GC cells PD-L1 up-regulation and resistance to CD8⁺ T cells cytotoxicity.

GCMSCs-derived IL-8 induced PD-L1 expression via c-Myc regulated by STAT3 and mTOR signaling pathways in GC cells. Combination with IL-8 inhibition enhanced the efficacy of PD-L1 antibody and promoted the cytotoxic effect of CD8⁺ T cells