Living image software

Upon arrival at Northwestern University, tissues were washed with PBS then imaged on an IVIS Lumina Series III (Perkin Elmer) using the fluorescent imaging settings. Whole organs or large pieces of tissue were imaged using the same exposure conditions for each fluorophore, respectively (Cy3 and Cy5). The lungs were further separated into individual lobes and imaged again under identical conditions. Small pieces of tissue (~2x2cm²) were cut from whole organs and re-imaged on the IVIS before being frozen in OCT in cryomolds. Imaging conditions were kept consistent between animals that received the same fluorophore and all resulting images were set to the same scale before analysis using the Living Image Software (Perkin Elmer).
Quantification of fluorescent signal was carried out using the Living Image Software. Regions of interest (ROIs) were created automatically by the software and adjusted manually to cover each tissue piece individually (Supplementary Figure 1A). Signal is reported as total radiant efficiency, which is a unit less measure that uses photons per second then normalizes based on wattage and intensity of the excitation light. The average radiant efficiency value normalizes the total value by the size of each ROI.

All experiments were performed in compliance with all relevant ethical regulations and in accordance with an Institutional Animal Care and Use Committee-approved protocol (No. 18080B). NCG mice (GuangDong GemPharmatech Co., Ltd.) aged 4–6 weeks were inoculated subcutaneously with 2.5 × 10⁶ tumour cells (Hep-3B-luc), and after the tumour grew to 50–100 mm³, Mock T, CD133 CAR-T and CD133 CAR-T and PD-1 s cells were intravenously injected into NCG mice with Hep-3B. For metastatic mouse models, the NCG mice were intravenously injected with 2.5 million Hep3B-luc cells. The metastasis was confirmed by bioluminescence imaging (BLI) and then treated with a tail veil injection of 5 million Mock T, CD133 CAR-T or CD133 CAR-T and PD-1 s cells. BLI of tumour growth was performed using a PerkinElmer IVIS imaging system with Living Image software. Finally, the tumour weight was evaluated. Mice were euthanized when the tumour volume reached 2000 mm³ or when they showed any sign of sickness. For establishment of an in situ xenograft tumour model, 2.5 million Hep3B-luc cells were directly injected into the liver of NCG mice. The tumour was confirmed by BLI, and 5 million Mock T, CD133 CAR-T or CD133 CAR-T and PD-1 s cells were injected into the tail vein. BLI of tumour growth was performed using a PerkinElmer IVIS imaging system with Living Image software.