The effect of exosome derived from gastric cancer cell line MKN-28, MKN-45, SGC-7901 on CD8+ T cell

Abstract

Objective To detect exosome from gastric cancer cell line MKN-28, MKN-45, SGC-7901 on CD8+ T cell. Methods CD8+ T cell was isolated from health person peripheral blood by density gradient centrifugation and immunomagnetic bread, exosome of SGC-7901, MKN-45 and MKN-28 cell lines was obtained by ultracentrifugation. After coculture exosome and CD8+ T cell with different concentration for 48 h, CD8+ T cell was collected, and analyzed cell cycle by flow cytometry, cell apoptosis by Annexin V-propidium iodide (PI) stain, expression of cytokine by enzyme linked immunosorbent assay (ELISA), gene expression by real-time quantitative polymerase chain reaction (Real-time PCR). Results Exosome was isolated successfully from three gastric cancer cell lines and expressed molecular surface markers TSG101 and Alix. CD8+ T cells were isolated in peripheral blood mononuclear cell (PBMC) with a purity of over 98%. The effect of MKN-28 exosome on apoptosis was dose dependent. After stimulation with 100 mg/L MKN-45 exosome for 48 h, CD8+ T cells secreted IL-2, IL-6, IL-10, IFN-γ [(305.44±42.07) ng/L vs. (129.89±11.07) ng/L, P<0.01; (344.39±25.34) ng/L vs. (149.67±15.23) ng/L, P<0.01; (413.31±38.76) ng/L vs. (35.19±24.95) ng/L, P<0.01; (303.78±44.98) ng/L vs. (277.67±40.66) ng/L, P<0.05], and the difference was statistically significant. After SGC-7901 exosome stimulated, CD8+ T cells secreted IL-4, IL-6, IL-10, IFN-γ [(318.61±41.56) ng/L vs. (238.89±35.84) ng/L, P<0.05; (501.06±55.05) ng/L vs. (149.67 ±15.23) ng/L, P<0.01; (486.75±71.20) ng/L vs. (35.19±24.95) ng/L, P<0.01; (621.00±55.14) ng/L vs. (277.67±40.66) ng/L, P<0.01], the difference was statistically significant. After MKN-28 exosome stimulated, CD8+ T cells secreted IL-2, IL-6, IL-10, IFN-γ [ (411.56±48.62) ng/L vs. (129.89±11.07) ng/L, P<0.01; (403.83±52.29) ng/L vs. (149.67±15.23) ng/L, P<0.01; (678.31±78.02) ng/L vs. (35.19±24.95) ng/L, P<0.01; (751.88± 63.16) ng/L vs. (277.67±40.66) ng/L, P<0.01], and the difference was statistically significant. The expression of IL-10 secreted by CD8+ T cells stimulated by 100 mg/L MKN-28 exosome was about 19.53 times compared to the control group. After MKN-45 exosome stimulated, CD8+ T cells expressed forkhead/winged helix transcription factor P3 (Foxp3), interleukin (IL)-10, interferon (IFN)-γ, phosphatase and tensin homologue deleted on chromosome ten (PTEN), signal transducer and activators of transcription 3 (STAT3) (3.46±0.39 vs. 1.00±0.09, P<0.01; 3.29±0.39 vs. 1.01±0.16, P<0.01; 2.24±0.30 vs. 1.00±0.06, P<0.01; 2.14±0.12 vs. 1.00±0.08, P<0.01; 1.24±0.12 vs. 1.00±0.07, P<0.05). After SGC-7901 exosome stimulated, CD8+ T cell expressed Eomes, Foxp3, IL-10, IFN-γ, 2B4, CD160, GATA3, STAT3, PTEN (2.35±0.17 vs. 1.01±0.13, P<0.01; 3.19±0.21 vs. 1.00±0.09, P<0.01; 2.38±0.31 vs. 1.01±0.16, P<0.01; 5.44±0.71 vs. 1.00±0.06, P<0.01; 3.20±0.31 vs. 1.00±0.12, P<0.01; 3.19±0.13 vs. 1.01±0.16, P<0.01; 1.35±0.17 vs. 1.00±0.07, P<0.05; 1.44±0.18 vs. 1.00±0.07, P<0.05; 1.31±0.15 vs. 1.00±0.08, P<0.05) mRNA; after MKN-28 exosome stimulated, CD8+ T cell expressed Eomes, Foxp3, IL-10, IFN-γ, STAT3, PTEN, CD160, 2B4 (3.36±0.18 vs. 1.01±0.13, P<0.01; 4.35±0.30 vs. 1.00±0.09, P<0.01; 5.40±0.63 vs. 1.10±0.16, P<0.01; 6.43±0.84 vs. 1.00±0.06, P<0.01; 1.54±0.28 vs. 1.00±0.07, P<0.05; 0.78±0.11 vs. 1.00±0.08, P<0.01; 1.68±0.42 vs. 1.01±0.16, P<0.05; 1.31±0.22 vs. 1.01±0.12, P<0.05) mRNA expression. The gene expression of CD8+ T cells stimulated by MKN-28 exosome was relatively more changes, such as Foxp3, IL-10 and IFN-γ. Conclusion Gastric cancer cell line exosome stimulated CD8+ T cells to induce cell apoptosis, and change gene expression and cytokine secretion, but the proportion of negative regulatory molecules was much higher. Cancer exosome interacted with CD8+ T cells, which inhibited its immune function, resulting in tumor immune escape, which may be an important factor to promote disease progression. Key words: Gastric cancer; Exosome; CD8+ T cell; Gene expression; Cytokine