单克隆抗体18H12抑制PAMC-82胃癌干细胞的自我更新和侵袭能力

目的：探讨靶向胃癌干细胞的功能性单克隆抗体18H12对胃癌干细胞自我更新和侵袭能力的作用。方法：以胃癌 细胞株PAMC-82为模型，用流式细胞术检测其亲本细胞和通过成球培养富集干细胞后细胞膜表面烯醇化酶1（enolase-1，ENO1） 的表达水平，用流式细胞术分选出ENO1+和ENO1-细胞，并检测其自我更新和侵袭能力。以商业化的ENO1抗原和抗体作为样 品，利用免疫共沉淀实验（co-immunoprecipitation，CoIP）验证靶向ENO1的18H12抗体能够准确识别ENO1。分别用18H12处理 PAMC-82细胞12、24、48 h后， 用甲基纤维素成球实验和Transwell小室法分别检测18H12对PAMC-82细胞自我更新和侵袭能力的影 响。结果：细胞膜表面的ENO1在PAMC-82球体细胞中的表达水平明显高于亲本细胞（P<0.01），ENO1可作为一个靶向胃癌干细 胞的潜在靶点。分选的ENO1+细胞的自我更新和侵袭能力明显强于ENO1-细胞和亲本细胞（P<0.05或P<0.01）。18H12抗体能够 准确识别ENO1，与商业化抗体识别的条带一致。单抗18H12能显著抑制PAMC-82细胞的自我更新和侵袭能力（均P<0.01）。结 论：单克隆抗体18H12能够显著抑制胃癌干细胞的自我更新和侵袭能力，有望成为靶向胃癌干细胞的候选抗体药物。

Monoclonal antibody 18H12 suppresses the self-renewal and invasion of PAMC-82 gastric cancer stem cells

Objective: To investigate the effect of 18H12, a functional monoclonal antibody that can target gastric cancer stem cells, on the self-renewal and invasion ability of gastric cancer cells. Methods: The gastric cancer cell line PAMC-82 was used as cell model, the expression of ENO1 (enolase-1) on the membrane surface of its parental cells and enriched stem cells by sphere culture was detected by Flow cytometry.Flowcytometrywasusedtoseparate ENO1+ cells and ENO1-cellstodetecttheirself-renewal ability and invasion ability. With the commercial ENO1 antigen and antibody as the samples, CoIP (co-immunoprecipitation) was used to verify whether 18H12 antibody targeting ENO1 could able to accurately recognize ENO1. After being treated with 18H12 for 12 h, 24 h and 48 h, the selfrenewal and invasion ability of PAMC-82 cells were detected by methylcellulose pelletization experiment and Transwell chamber assay, respectively. Results: Flow cytometry showed that the expression of ENO1 on the membrane surface of PAMC-82 sphere cells was significantly higher than that of its parental cells (P<0.01), so ENO1 could be a potential target for targeting gastric cancer stem cells. The self-renewal ability and invasion ability of the sorted ENO1+ cells were significantly stronger than those of the ENO1cells and the parental cells (P<0.05 or P<0.01). 18H12 antibody could accurately recognize ENO1, which was consistent with the commercial antibody recognition band. 18H12 could significantly inhibit self-renewal ability and invasion ability of PAMC-82 cells (P<0.01). Conclusion: Monoclonal antibody 18H12 can significantly inhibit the self-renewal and invasion of gastric cancer stem cells and is expected to be a candidate antibody drug targeting gastric cancer stem cells.