Image analysis

Videos were processed and analyzed using Fiji software (ImageJ 2.0.0). Figures and videos based on two-photon microscopy are shown as two-dimensional (2D) maximum intensity projections of 3D data. Tumor apoptotic events were manually quantified and scored as direct killing when a CAR T cell engaged the tumor cell before the detection of FRET loss. Indirect events corresponded to tumor cells undergoing FRET loss without any detectable interactions with a CAR T cell during the imaging period. To perform an unbiased analysis of the spatial localization of apoptotic tumors and CAR T cells, we developed an in-house Fiji script that computes a correlation score. Briefly, for each bone marrow mosaic of 2000 μm by 2000 μm, both CFP and GFP channels are first binarized using the Yen threshold. Then, for each region of interest (ROI) of 100 μm by 100 μm (spaced 50 μm apart), the mean CFP and GFP values are calculated and considered as a good proxy for apoptotic tumor and CAR T cell presence. For determining tumor cell area (CFP + YFP), apoptotic tumor cell area (CFP), and CAR T cell area (GFP) from in vivo images, each channel was binarized and subjected to surface area calculation.