4 chamber glass bottom confocal plates

For cell-in-cell count, 3000–10,000 tumor cells per one cm² were plated in a 4 Chamber glass-bottom confocal plates (Cellvis) or 96-well flat bottom (Corning) and let adhere for several hours. Cells were then incubated overnight with immune cells or exosomes. Cells were then washed with PBS, trypsinized and moved to coverslip for counting under fluorescent microscope. Percentages of cell-in-cells structures were calculated by counting 200 cells in three independent samples, three fields for each sample.

For inhibition assays, B16F10 were plated in 4 Chamber glass-bottom confocal plates (Cellvis) 3000 cells/chamber and let adhere for several hours. Cells were incubated for 24–48 hr in complete DMEM, with or without 50 μg/mL cycloheximide, 120 mM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) (Sigma-Aldrich), 200 nM Latrunculin B (Lat-B, Sigma-Aldrich), 2 μM Stattic (Sigma-Aldrich), 4 μM Gefitinib, 5 μM XAV-939 (Sigma-Aldrich) or the blocking antibodies anti-E-cadherin (clone 36) and anti-N-cadherin (clone 13A9). In addition, gp100-reactive T cells (E:T ratio of 5:1), or T cells-derived secreted granules (10–50 μL of concentrated secreted granules per well) were added to the culture. Cell-in-cell structures were counted under fluorescent microscope for cell-in-cell per 200 cells, three times for each chamber.