Figure 2.

Interaction between neutrophils and tumor cells promotes tumor EMT and enhances tumor cell invasion. (A, B) FCM analyses showed that co-culture with MKN45 cells exerted no remarkable influence on purity and viability of isolated neutrophils. (C, D) After co-culture with MKN28 or MKN45 cells, the CMs of Edu-Neus were used to treat fresh MKN28 or MKN45 cells, and cell migration (C) and invasion (D) were increased significantly (con, MKN28 or MKN45 cells in complete RPMI1640; Neu, MKN28 or MKN45 cells treated with CMs of isolated neutrophils; Edu-Neu, MKN28 or MKN45 cells treated with CMs of educated-neutrophils) (×100). (E) RNA-seq showed forty-seven differential expressed genes among “cytokine activity” category in “molecular function” after co-culture with MKN45, and the four most up-regulated genes were GDF15, MIF, BMP4 and FAM3C. (F) Isolated peripheral neutrophils from healthy donors (1, 2 and 3) were co-cultured with MKN28 or MKN45 cells, respectively. Subsequently, these neutrophils were harvested, and their CMs were applied to treat fresh MKN28 or MKN45 cells. The expression of E-cad, ZO-1 and Claudin-1 was decreased, while the expression of Vim and N-cadherin (N-cad) was increased. (G) Immunofluorescence assays showed that treatment with the Edu-Neu's CM increased Vim expression, and decreased E-cad expression (×400). (H) The left lane showed H & E images of tumor tissues of high-TANs group and low-TANs group. The middle lane showed IHC images indicating presence of TANs and lymphatic vessels with CD66b and D2-40 (right-upper frame), respectively. The right lane showed E-cad expression in tumor tissues or lymphatic cancer emboli. E-cad expression in tumor tissues or lymphatic cancer emboli was decreased in high-TANs group compared with that in low-TNAs group, and E-cad levels in lymphatic cancer emboli were further decreased with regard to tumor tissues in high-TANs group. (Neu, neutrophil; Edu-Neu, tumor-educated neutrophil).