8 m pore filter

For the migration assay, 2 × 10⁵ TE-8 cells or 1 × 10⁵ TE-9 cells in 300 µl of medium with 0.1% FBS were placed in the upper transwell inserts with an 8-µm pore filter (BD Falcon, Lincoln Park, NY) in 24-well plates. For the invasion assay, the same number of TE-8 or TE-9 cells in the same medium were placed in the inserts of a Corning® BioCoat™ Matrigel® Invasion Chamber (Corning, Tewksbury, MA) in 24-well plates, and then 800 µl of medium containing 100 ng/ml rhCCL3 or 1 × 10⁵ TAMs was placed in the lower chamber. Next, 20 µM LY294002, 20 µM PD98059, or 20 µg/ml Maraviroc was added to the upper chambers, and the neutralizing antibody against CCL3 (400 ng/ml) was added to the lower chambers. The plates were then incubated for 24 h (for the migration assay) or 48 h (for the invasion assay) at 37 °C in 5% CO₂. The cells were fixed in methanol for 1 min and stained with Diff-Quik® (Sysmex, Kobe, Japan). Cells on the upper side of the filters were removed with cotton-tipped swabs. Five images at ×200 magnification were obtained from each membrane with a CCD camera (Olympus, Tokyo, Japan), and the number of cells was counted. The percent migration or invasion was calculated by dividing the number of cells by that in the negative control (without rhCCL3, co-cultured TAMs, inhibitors, Maraviroc, or the neutralizing antibody of CCL3).

1 × 10⁵ ESCC cells were resuspended in 300 µL of RPMI-1640 without FBS after the direct co-culture and transferred to transwell inserts with an 8-µm pore filter (BD Falcon) for migration assays and the inserts of a Corning^® BioCoat^® Invasion Chamber (Corning, Tewlbury, MA, USA) for invasion assays (Upper chambers); 800 µL of RPMI-1640 with 0.5% FBS (for assays of TE-9 or TE-10) or 0.1% FBS (for assays of TE-11) were placed in 24-well plates (Lower chambers). For assays of TE-9 or TE-10, 0.5 µM of MMP9 inhibitor (ab142180, Abcam) dissolved in dimethyl sulfoxide (DMSO) was added to the lower chambers. For assays of TE-11, 0.1 µM of MMP9 inhibitor was added to the lower chambers. Upper chambers were placed onto lower chambers and the cells were cultured for 48 h. Subsequently, the cells were fixed and stained using Diff-Quik^® (Sysmex, Kobe, Japan). Cells on the upper surface of the inserts were removed thoroughly using cotton swabs and cells that migrated or invaded the lower surface were observed under a microscope. We randomly captured five and four images in migration and invasion assays per insert at 200× magnification, respectively, and counted the number of cells. Relative migration or invasion was calculated by dividing the number of cells by that in the control (monocultured and without MMP9 inhibitor).

Before the cell migration assay analysis, THP-1 cells (3 × 10⁵ cells/well) were treated with 200 nM TPA on the upper insert with an 8 µm pore filter (BD Falcon) to differentiate to macrophage-like cells. During the differentiation, the lower chamber was empty. For the assays, the medium in the upper insert was changed to RPMI-1640 with 1% FBS, and the insert was then exposed to the lower chamber in the presence and absence of rhCCL20 (R&D Systems). To verify the effect of CCL20 in CM, we applied CM in the lower chamber with and without CCL20 neutralizing antibody (#ab9829, Abcam) or control IgG (#ab37415, Abcam). The insert was exposed to the lower chamber for 24 h at 37°C in a CO₂ incubator. We then gently removed the remaining cells in the upper surface of the insert with cotton swabs. The numbers of migrated cells were stained using Diff-Quik^® (Sysmex, Kobe, Japan). Five images at ×200 magnification were obtained from each membrane with a charge-coupled device camera, and we then counted the stained cells.