D luciferin

All animal studies were conducted in accordance with the institutional guidelines approved by the Animal Research Ethics Committee of Zhejiang University.
For the xenograft tumor model, approximately 1 × 10⁶ ccRCC cells suspended in 100 μL PBS were subcutaneously inoculated in the right flank of 5-week-old BALB/c nude mice. After 4 weeks, the xenograft tumors were collected and tumor volume was calculated according to the following formula: volume  =  (width² × length)/2.
For the ccRCC orthotopic implantation model, approximately 1 × 10⁶ ccRCC cells suspended in 30 μL Matrigel were injected under the renal capsule of 5-week-old BALB/c nude mice. After 6 weeks, the anesthetized mice were intraperitoneally injected with D-luciferin (Yeason) and imaged using an in vivo imaging system to detect tumor growth and metastasis. The mice were then sacrificed, and the lung, liver, spleen, and intestine tissues were harvested, imaged, and subjected to IHC staining and H&E staining.
For the lung metastasis model, approximately 5 × 10⁵ ccRCC cells suspended in PBS were injected into the tail vein of 5-week-old mice. After 6–8 weeks, mice were anesthetized and lung metastasis was imaged as above. Lung tissues were further harvested, imaged, and subjected to H&E staining.

Before the imaging of mice, depilatory cream was applied to the chest, abdomen, and hind limbs by using cotton swabs; and five minutes later, the covering hair were removed by scrubbing the mice with cotton swabs dipped in water. D-Luciferin in PBS was intraperitoneally injected into mice at a dose of 150 mg/kg (Cat. 40902ES02, Yeasen Biotechnology, Shanghai, China). Thirteen minutes later, animals were anesthetized in an anesthesia machine containing 2% isoflurane and imaged in the IVIS Lumina Series III with the isoflurane anesthesia gas on (PerkinElmer, Hopkinton, MA, USA). Regions of interest (ROI) were created on the image, and total flux was measured.
Some mice were euthanized by cervical dislocation, and 13 types of organs or tissues were collected, washed once with cold PBS, weighed, fragmented, and frozen at −80 °C for luciferase activity assay and virus genome determination, including heart, liver, spleen, lung, kidney, brain, trachea, esophagus, stomach, small intestine, large intestine, Peyer’s patches, and quadriceps femoris muscles. Especially, the stomach, small intestine, and large intestine were cut open with ophthalmic scissors to remove the contents.

The six-week-old male nude mice used in this study were purchased from Nanjing Medical University Animal Center. For intracranial GBM xenograft experiments, PG7 cells lentivirally transduced with firefly luciferase (Fluc) were implanted into the frontal subdural region. The IVIS Imaging System (Caliper Life Sciences) was used to measure intracranial tumor growth. Each mouse was intraperitoneally injected with 10 mg D-luciferin (YEASEN, Shanghai, China) before imaging. The Living Images software package (Caliper Life Sciences) was used to analyze the integrated flux of photons in each region. The procedures were approved by the Animal Management Rule of the Chinese Ministry of Health (documentation 55, 2001) and the Nanjing Medical University Animal Experimental Ethics Committee (Ethics number: IACUC-1907006).

The target cells H460GL, MKN-28GL, SMMC-7721GL, HeLaGL and H460-MSLNGL (10^4 cell/well) were incubated with CAR T or negative control T cells at the indicated ratios in triplicate wells of U-bottomed 96-well plates. Target cell viability was monitored 24 h later by adding 100 μl/well of the substrate D-Luciferin (potassium salt) (YEASEN, 40901ES03) at 150 μg/ml. Background luminescence was negligible (< 1% of the signal from the wells with only target cells). The viability percentage (%) was equal to the experimental signal/maximal signal× 100, and the lysis percentage was equal to the 100–viability percentage.

Target tumor cells K562-PDL1-GL, HeLa-GL, and H460-MSLN-GL (10⁴ cell/well) were incubated with CAR T cells or negative control T cells at the indicated E:T ratios in triplicate wells of U-bottomed 96-well plates at 37 °C for 24 h. Residual tumor cells were quantified by bioluminescent imaging (BLI) of the plate after adding 100 µl/well D-luciferin (potassium salt) (YeaseN, 40901ES03) at 150 µg/ml. The percentage of viable cells was equal to the experimental signal/maximal signal×100, and the percentage of cell lysis was equal to 100-percentage of viable cells. Both anti-PD-L1 mAb (atezolizumab) (Selleck, A2004) and anti-PD-1 mAb (Pembrolizumab) (Selleck, A2005) used at 20 μg/ml concentration. Anti-IL2 mAb (Biolegend, 500301), anti-IFN-γ mAb (ThermoFisher, 16-7318-81) and anti-CD70 mAb (Abcam, ab213102) used at 10 μg/ml concentration.

To detect tumor sizes dynamically, bioluminescence imaging were performed on fourteen days after HepG2-fLuc cells were injected, 72 h after each treatment and 7 days after the last treatment. For the bilateral xenograft model, Bioluminescence imaging techniques were performed on fourteen days after HepG2 cells were injected and on the 9th day after treatment. Mice were injected intraperitoneally 150 mg/kg D-luciferin (Yeasen Biotech, 40902ES), sedated with isoflurane and imaged using an IVIS Spectrum (PerkinElmer).

hADSCs at passage 4 were transfected with an adenoviral vector containing a firefly luciferase reporter gene (Ad-Luc) (Shanghai Genechem Co., Ltd., Shanghai, China) to analyze the in vivo homing and survival rates. Transfected cells were injected via the tail vein at a concentration of 5 × 10⁵ cells per mouse directly after BDL surgery. Mice under anesthesia were injected intraperitoneally with D-luciferin and sodium salt (Yeasen, Shanghai, China) and imaged using an IVIS Lumina XRMS III in vivo imaging system (PerkinElmer, Waltham, MA, USA) at the indicated time points.