Three-colour Flow Cytometric Method to Measure Antibody-dependent Tumour Cell Killing by Cytotoxicity and Phagocytosis

We have developed a three-colour flow cytometric method to assay the contributions of cytotoxicity and phagocytosis to antibody-dependent cell-mediated tumour cell killing. In this assay, tumour target cells are pre-labelled with CFSE, and mixed with effector cells and a tumour antigen-specific monoclonal antibody. After incubation of the cells with the antibody, effector cells are labelled with PE and dead cells with PI. Using flow cytometry, dead and phagocytosed tumour cells can be quickly and easily counted and the numbers summed to determine the total number of killed cells. One can thereby measure the phagocytic aspect of antibody-dependent cell-mediated tumour cell killing, otherwise only revealed by microscopic examination. The failure to detect phagocytosed, in addition to live and dead target cells, by standard assays may result in an underestimation of tumour cell killing and hence the potential of an antibody for immunotherapy of cancer. We illustrate the new method by analysing human monocyte-mediated cytotoxic and phagocytic cell killing of IGROV1 ovarian tumour cells by the ovarian tumour antigen-specific anti-folate binding protein monoclonal antibody, MOv18 IgE.