Gao Feng-Xia, Wu Jian, Ren De-Lian
Department of Immunology, Southwest Medical University, Luzhou 646000, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2019 Jan;50(1):40-47.
To investigate the effect of epithelial-mesenchymal transition (EMT) on biological activity of natural killer (NK) cells in esophageal squamous cell carcinoma (ESCC).
Western blot selected EMT EC9706 and non EMT KYSE150 from six esophageal cancer cell lines. NK cells were collected from 10 cases of healthy volunteers. According to different co-culture conditions, the medium was divided into 1640 normal medium stimulus-NK group (NC group), EC9706 cell supernatant stimulus-NK group (EC9706 group) and KYSE150 cell supernatant stimulus-NK group (KYSE150 group). The expression of NK cells surface/intracellular molecules was detected by flow cytometry after co-culture of NK cells with different conditioned medium for 72 h. 10 ng/mL transforming growth factor-β (TGF-β) treated KYSE150 cell line for 7 d to induce EMT. KYSE150 EMT and KYSE150 supernatant were collected and co-cultured with NK cells, respectively. 72 h later, flow cytometry was used to detect the expression of surface/intracellular molecules of NK cells, degranulation ability of CD107a, killing effect of target cell K562, proliferation and apoptosis of NK cells.
Two EMT-treated and four non-EMT-treated esophageal squamous cell carcinoma lines were selected from the six strains, and one EC9706 and one KYSE150 respectively were selected for subsequent experiments. The purity of NK cells was more than 90% and Tcells <1%. After the supernatant of esophageal squamous cell carcinoma cells was stimulated, the surface activation type and inhibitory type receptors of NK cells in the three groups were stimulated. The effector molecule results showed that: compared with NC group and KYSE150 group, the expressions of activated type receptors NKP30, NKG2D and NKP44 in EC9706 group were decreased, and the release of granulozyme was decreased ( <0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( <0.05). NKP46, CD226, CD16 expressions and perforin release showed no statistically significant difference among the three groups. Compared with KYSE150 supernatant stimulation, the expressions of activated receptors NKP30, NKG2D and NKP44 decreased after KYSE150 EMT supernatant stimulation, and perforin release decreased. The degranulation of CD107a and the killing effect of target cell K562 were decreased, and the proliferation index of NK cell Ki67 was decreased ( <0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( <0.05). The expressions of NKP46, CD226 and CD16, granulozyme release and apoptosis of NK cells were not statistically significant.
EMT of esophageal cancer cells can escape the immune surveillance by inhibit the activity of NK cells and reduce the release of effective molecules.
探讨上皮-间质转化（EMT）对食管鳞状细胞癌（ESCC）中自然杀伤（NK）细胞生物学活性的影响。
通过蛋白质免疫印迹法从六种食管癌细胞系中筛选出发生EMT的EC9706细胞和未发生EMT的KYSE150细胞。从10例健康志愿者中采集NK细胞。根据不同的共培养条件，将培养基分为1640正常培养基刺激-NK组（NC组）、EC9706细胞上清刺激-NK组（EC9706组）和KYSE150细胞上清刺激-NK组（KYSE150组）。将NK细胞与不同条件培养基共培养72小时后，采用流式细胞术检测NK细胞表面/细胞内分子的表达。用10 ng/mL转化生长因子-β（TGF-β）处理KYSE150细胞系7天以诱导EMT。分别收集KYSE150 EMT细胞和KYSE150细胞上清，并与NK细胞共培养。72小时后，采用流式细胞术检测NK细胞表面/细胞内分子的表达、CD107a的脱颗粒能力、靶细胞K562的杀伤效果、NK细胞的增殖和凋亡情况。
从六种细胞系中筛选出两种发生EMT处理的和四种未发生EMT处理的食管鳞状细胞癌系，分别选取一种EC9706细胞和一种KYSE150细胞用于后续实验。NK细胞纯度>90%，T细胞<1%。食管鳞状细胞癌细胞上清刺激后，三组NK细胞表面激活型和抑制型受体均受到刺激。效应分子结果显示：与NC组和KYSE150组相比，EC9706组激活型受体NKP30、NKG2D和NKP44的表达降低，颗粒酶释放减少（P<0.05）。抑制性受体NKG2A和CD158b的表达增加（P<0.05）。三组间NKP46、CD226、CD16的表达及穿孔素释放差异无统计学意义。与KYSE150上清刺激相比，KYSE150 EMT上清刺激后激活型受体NKP30、NKG2D和NKP44的表达降低，穿孔素释放减少。CD107a的脱颗粒及靶细胞K562的杀伤效果降低，NK细胞Ki67增殖指数降低（P<0.05）。抑制性受体NKG2A和CD158b的表达增加（P<0.05）。NKP46、CD226和CD16的表达、颗粒酶释放及NK细胞凋亡差异无统计学意义。
食管癌细胞的EMT可通过抑制NK细胞活性、减少有效分子释放来逃避免疫监视。
