Effect of esophageal squamous cell carcinoma cells on the biological activity of natural killer cells

Abstract:

Objective：To study the regulation and control of the phenotype and function of natural killer（NK） cells by the primary cells of esophageal squamous cell carcinoma（ESCC）. Methods：Fresh ESCC tissue was collected，from which primary ESCC cells were cultured in vitro by the modified tissue block enzymolytic method，and identified using a subcutaneous tumor-formation model in nude mice and the immunohistochemical technique. Human peripheral blood mononuclear cells were collected from healthy subjects，from which the NK cells were isolated by negative-selection magnetic beads，and co-cultured with the supernatant of the primary ESCC cells obtained above. The cultured cells were divided into groups of NC-NK，ESCC1-NK，and ESCC2-NK according to their respective culture conditions. Flow cytometry was used to determine the molecular expression both on the surface of and within the NK cells. The subcutaneous tumor-formation model and lung metastasis model were used to study the effect of primary ESCC cells on the killing effect of NK cells in nude mice. Results：Compared with the NC-NK group，the ESCC1-NK group and the ESCC2-NK group had significantly reduced expression of the activated receptors NKG2D（56.84±1.28 vs. 41.89±2.17 and 29.40±1.32；P=0.008 and 0.001，respectively），NKP30（45.79±1.90 vs. 19.54±1.27 and 26.59±1.47；P=0.001 and 0.003，respectively），and CD16（84.82±1.38 vs. 55.95±2.29 and 36.07±1.97；P=0.001 and 0.000，respectively） as well as significantly reduced expression of the effector molecule granzyme B（94.87±1.04 vs. 80.52±0.99 and 78.67±2.73；P=0.013 and 0.008，respectively） and the prolif－eration-associated antigen Ki67（70.15±2.35 vs. 52.31±1.74 and 50.02±2.68；P=0.017 and 0.011，respectively） in NK cells. There were no significant differences between the three groups in the expression of the activated receptors NKP46，CD226，and NKP44 as well as perforin and the inhibitory receptors NKG2A and CD158b. Animal experiments showed that there was a significant difference between the ESCC1-NK group and the NC-NK group in the volume of subcutaneous tumors（F=54.689，P=0.000） with a significantly greater number of lung metastases observed in the ESCC1-NK group（41.00±3.11 vs. 25.25±2.32，t=4.068，P=0.007）. Conclusion：Primary ESCC cells may suppress the tumor-killing effect of NK cells by inhibiting the activation and proliferation of NK cells and down-regulating the expression of the effector molecules in NK cells，thereby escaping the immune surveillance in the body.