胃癌间充质干细胞促进胃癌细胞PD-L1表达的特征观察

Characterization of gastric cancer mesenchymal stem cells promoting PD-L1 expression in gastric cancer cells

摘要:

目的 探讨胃癌来源的间充质干细胞（GCMSC）促进胃癌细胞程序性死亡配体1（PD-L1）表达的具体特征，为进一步研究GCMSC促进肿瘤细胞PD-L1表达机制及胃癌的靶向治疗提供依据。方法 采用细胞贴壁法从胃癌组织中分离出GCMSC，流式细胞仪分析GCMSC表面标志物，采用成脂和成骨分化实验评价GCMSC的多向分化能力。流式细胞术分选PD-L1阴性和PD-L1阳性胃癌细胞。收集GCMSC条件培养基（GCMSC-CM）处理胃癌细胞，分别采用实时荧光定量聚合酶链反应（qRT-PCR）和Western blotting检测胃癌细胞PD-L1的mRNA和蛋白表达。采用酶联免疫吸附试验（ELISA）检测胃癌患者血清游离PD-L1和白细胞介素-8（IL-8）水平。结果 成功分离鉴定GCMSC，结果发现GCMSC-CM处理24、48和72 h后，SGC-7901细胞的PD-L1表达均上调（P <0.05），GCMSC-CM处理MGC-803细胞 24、48和72 h后，MGC-803细胞PD-L1表达也上调（P <0.05）。然而，去除GCMSC-CM后48 h，SGC-7901和MGC-803的PD-L1表达又逐渐恢复。当IL-6或IL-8在GCMSC-CM中被阻断时，与GCMSC-CM组比较，这种促进作用被逆转，尤其是IL-8的作用更明显（P <0.05）。胃癌患者血清游离PD-L1与IL-8呈正相关（r =0.347，P <0.05）。流式细胞术结果显示，经GCMSC-CM再次处理后，PD-L1阳性胃癌细胞PD-L1表达能够再次升高（P <0.05），而PD-L1阴性的胃癌细胞PD-L1的表达仍然不升高（P >0.05）。结论 GCMSC-CM促进胃癌细胞中PD-L1表达，并依赖GCMSC-CM的持续作用。胃癌细胞具有异质性，其PD-L1表达对GCMSC具有不同的反应性。

Abstract:

Objective To investigate the specific characteristics of gastric cancer-derived mesenchymal stem cells (GCMSCs) in promoting programmed death ligand-1 (PD-L1) expression in gastric cancer cells. This study aims to provide a basis for further research into the mechanism by which GCMSCs promote tumor cell PD-L1 expression and targeted therapy for gastric cancer.Methods GCMSCs were isolated from gastric cancer tissues using an adherent culture method. Flow cytometry was used to analyze the surface markers of GCMSCs, and adipogenic and osteogenic differentiation experiments were conducted to evaluate the multi-directional differentiation ability of GCMSCs. Flow cytometry was used to sort PD-L1-negative and PD-L1-positive gastric cancer cells. Conditioned medium from GCMSCs (GCMSC-CM) was collected and used to treat gastric cancer cells. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression of PD-L1 in gastric cancer cells. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of free PD-L1 and interleukin-8 (IL-8) in the serum of gastric cancer patients.Results GCMSCs were successfully isolated and identified. The results showed that PD-L1 expression in SGC-7901 cells was upregulated after treatment with GCMSC-CM for 24, 48, and 72 hours (P < 0.05). PD-L1 expression in MGC-803 cells also increased after treatment with GCMSC-CM for 24, 48, and 72 hours (P < 0.05). However, PD-L1 levels gradually returned to baseline after 48 hours of GCMSC-CM removal in SGC-7901 (P < 0.05) and MGC-803 (P >0.05) cells. When IL-6 or IL-8 was blocked in GCMSC-CM, the promoting effect was reversed compared to the GCMSC-CM group, especially with IL-8 inhibition (P < 0.05). Serum levels of free PD-L1 and IL-8 in gastric cancer patients were positively correlated (r = 0.347, P < 0.05). Flow cytometry results showed that PD-L1 expression in PD-L1-positive gastric cancer cells could be upregulated again after re-treatment with GCMSC-CM (P < 0.05), while PD-L1 expression in PD-L1-negative gastric cancer cells remained unchanged (P > 0.05).Conclusion GCMSC-CM promotes PD-L1 expression in gastric cancer cells, depending on sustained exposure to GCMSC-CM. Gastric cancer cells exhibit heterogeneity in their response to GCMSCs regarding PD-L1 expression.