Pre-clinical validation of a pan-cancer CAR-T cell immunotherapy against nfP2X7

Supplementary Videos 1A&B: Real-time imaging of nfP2X7-M CAR-T cell adhesion to and transmigration through blood vessels in vivo. For intravital microscopy, 2x106 MDA-MB-231-LM2 cells expressing GFP were injected into the mammary fat pad of NSG mice and allowed to establish for 2 weeks. The mice then received an intravenous injection of 2x107 nfP2X7-M CAR-T cells fluorescently labelled with CellTracker Orange as well as Cy5 Dextran (for visualisation of blood vessels with active blood flow). The surgically exposed tumour was then placed under a Zeiss LSM710-NLO microscope with a 20x objective lens and the interaction between the CAR T cells and the blood vessels were visualised in real time. Representative videos showing nfP2X7-M CAR-T cells firmly adhered to a blood vessel (a) and transmigrating across a blood vessel (b). Representative of two independent experiments. Scale bars = 50 µm. Supplementary Videos 2A&B: Real-time imaging of nfP2X7-M CAR-T cells killing MDA-MB-231 breast cancer cells in vitro. MDA-MB-231 breast cancer cells were seeded into wells and allowed to grow overnight. nfP2X7-M CAR-T cells (a) or untransduced (UT) T cells (b) pulsed with Fluo-4 AM (green), and PI (red) were added into the wells at the same time. Images were taken at 10s intervals for at least 3 hours using a 40x objective lens on a Zeiss LSM700 and compiled for time lapse video. Representative of three independent experiments.