Cell Assay: To examine the relationship between a cancer stem cell (CSC) subpopulation and antibody-dependent cell-mediated cytotoxicity (ADCC) activity, UM-Chor1 cells are left untreated or treated with 50 ng/mL of IFN-gamma for 24 h. Cells are then plated as targets at 50, 000 cells/well in 6-well round-bottom culture plates and incubated with 2 ug/mL of Avelumab at room temperature for 30 min. NK cells are added at 2500, 000 cells/well at an effector-to-target (E:T) ratio of 50:1. After 4 h, tumor cells are harvested and stained with antibodies for flow cytometry.
Animal Studies: Female C57BL/6 mice are used in this study. Subcutaneous tumor injections are carried out by inoculating C57BL/6 mice with 1x105 MB49 parental cells on the right shaved flank. Tumor growth is measured with calipers and 8 days post-inoculation mice are assigned to treatment groups. Tumor-bearing mice are treated with Avelumab (400 ug per 100 uL) and injected i.p. three times, 3 days apart. Since Avelumab is a human IgG1, three injections have to be compressed within a 7 to 9 day window (i.e., days 9, 12, and 15 post-tumor inoculation) to avoid the onset of neutralizing mouse anti-human Ig.