A Novel Image Cytometric Analysis Method For T Cell-Mediated Cytotoxicity Of 3d Tumor Spheroids

Abstract Cell-mediated cytotoxicity assays have been frequently performed to characterize cytotoxic potential of immune cells, antibodies, and drug compounds. Traditionally, these assays are performed using release assays such as Cr51 (radioactivity), Calcein (fluorescence), or LDH (enzymatic). However, release assays have limitations such as the handling of hazardous material and the indirect measurement of cell death. These assays are commonly performed in 2D cultures, which have difficulty identifying qualifying drug candidates for clinical testing, thus there has been an interest of performing cytotoxicity assays in 3D tumor models. Traditional 3D spheroid analysis methods require the use of standard microscopy, which is time-consuming and subjective. In this work, we demonstrate a novel method of analyzing T cell-mediated cytotoxicity on 3D tumor spheroids in the presence of absence of ImmTAC molecules, which can promote higher T cell killing. In this experiment, MDA-MB-453 GFP expressing breast cancer cells are used to form tumor spheroids in an ultra-low attachment plate. The spheroids are then treated with primary T cells at different E:T ratios, and different concentrations of ImmTAC. The results showed a dose response effect of T cell killing with the addition of ImmTAC molecules by measuring spheroid size in GFP fluorescence and viability using propidium iodide. The captured bright-field and fluorescent images also clearly showed the cytotoxicity effect from the combination of T cells and ImmTAC. The ability to screen cytotoxic effects of immune cells, antibody, and drug compounds on 3D tumor spheroids can provide an alternative tumor model for identifying more qualified cancer drug candidates for drug discovery campaigns.