Fig. 4
TAM-derived CCL22 activates FAK/AKT signaling in ESCC cells. A Total protein lysates from KYSE410 cells treated with PBS or CCL22 (50 ng/ml) were analyzed using an array of antibodies against phosphorylation sites in 43 signaling kinases (left panel). Representative sites of upregulated phosphorylation in protein kinases in CCL22-treated KYSE410 cells are listed (right panel). B, C The indicated ESCC cells were incubated with CM from MDMs, pol-TAMs, or pri-TAMs with or without the anti-CCL22 antibody (50 μg/ml) or CCL22 (50 ng/ml). The activity of FAK and AKT was assayed by a FAK Tyr397 (B) or an AKT Ser473 (C) activation quantitative ELISA. ***P < 0.001 compared with control cells; ###P < 0.001 compared with untreated cells cocultured with pol-TAMs or pri-TAMs. D KYSE410 or KYSE510 cells (lower chamber) were cocultured with control medium, pol-TAMs, or CCL22 (50 ng/ml) (upper chamber) in a Transwell apparatus with a 0.4 μm pore size membrane for five days and then treated with different concentrations of VS-6063 (0.5–10 μM). AKT activity was assayed by an AKT Ser473 activation quantitative ELISA. ***P < 0.001 compared with the indicated ESCC cells treated with 0.5 μM VS-6063 in the absence of pol-TAMs or CCL22 (50 ng/ml); ###P < 0.001 compared with the indicated ESCC cells treated with 1 μM VS-6063 in the absence of pol-TAMs or CCL22 (50 ng/ml); $$$ P < 0.001 compared with the indicated ESCC cells treated with 2.5 μM VS-6063 in the absence of pol-TAMs or CCL22 (50 ng/ml). E Stromal CCL22 expression was associated with FAK Tyr397 and AKT Ser473 phosphorylation in 25 primary human ESCC specimens. Two representative specimens with low and high levels of CCL22 are shown. Magnification, ×10 as indicated. Percentages of specimens showing low or high CCL22 expression relative to the level of pFAK or pAKT. Statistical differences were evaluated using the chi-square test. F, G KYSE410 and KYSE510 cells (lower chamber) were cocultured with control medium, pol-TAMs, or CCL22 (50 ng/ml) (upper chamber) in a Transwell apparatus with a 0.4 μm pore size membrane for five days and then treated with different concentrations of VS-6063 (0.5–10 μM). F The invasion ability of the indicated ESCC cells was assessed using a quantitative 96-well Boyden chamber assay. G The growth ability of the indicated ESCC cells was evaluated using a 3D MTS assay. ***P < 0.001 compared with the indicated ESCC cells treated with 0.5 μM VS-6063 in the absence of pol-TAMs or CCL22 (50 ng/ml); ###P < 0.001 compared with the indicated ESCC cells treated with 1 μM VS-6063 in the absence of pol-TAMs or CCL22 (50 ng/ml); $$$ P < 0.001 compared with the indicated ESCC cells treated with 2.5 μM VS-6063 in the absence of pol-TAMs or CCL22 (50 ng/ml)
