Effect of Epithelial-to-mesenchymal Transition on Biological Activity of NK Cells in Esophageal Squamous Cell Carcinoma

Objective To investigate the effect of epithelial-mesenchymal transition (EMT) on biological activity of natural killer (NK) cells in esophageal squamous cell carcinoma (ESCC). Methods 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. Results 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 T CM(155mmcells <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 ( P<0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( P<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 ( P<0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( P<0.05). The expressions of NKP46, CD226 and CD16, granulozyme release and apoptosis of NK cells were not statistically significant. Conclusion EMT of esophageal cancer cells can escape the immune surveillance by inhibit the activity of NK cells and reduce the release of effective molecules.