All animal experiments were performed according to protocols approved by the Seoul National University Institutional Animal Care and Use Committee. Mice were fed standard mouse chow and water ad libitum and housed in temperature- and humidity-controlled facilities with a 12-h light/12-h dark cycle. For xenograft experiments, H1944 and A549 cells (1×106 cells/spot, diluted in equal amounts of PBS and Matrigel) were subcutaneously injected into the right flank of 6-week-old non-obese diabetic-severe combined immunodeficiency (NOD/SCID) mice. After the tumor volume reached 50-150 mm3, the mice were randomly grouped and treated with vehicle [10% DMSO in 60% polyethylene glycol (PEG) 400 solution] or LL6 (80 mg/kg) 6 days per week for 2 weeks. Tumor growth was determined by measuring the short and long diameters of the tumor with a caliper, and body weight was measured once or twice per week to monitor toxicity. Additionally, to evaluate the effect of LL6 on mutant Kras-driven lung tumorigenesis, 2-month-old KrasG12D/+ transgenic mice 48 were randomized and treated with vehicle or LL6 (80 mg/kg) for 8 weeks. The mice were euthanized, and tumor formation was evaluated and compared with that of the vehicle-treated control group. Microscopic evaluations of the H&E-stained lung tissue were also performed to measure mean tumor number (N) and volume (V) in a blinded fashion. The number and size of tumors were calculated in five sections uniformly distributed throughout each lung. In both animal experiments, the tumor volume and burden of each sample were calculated using the following formulas: Tumor volume (mm3) = (short diameter)2 × (long diameter) × 0.5; Tumor burden (mm3) = number of tumors × the average of tumor volume.

We used the IVIS-Spectrum microCT and Living Image (ver. 4.2) software (PerkinElmer, Alameda, CA, USA) for monitoring metastatic tumor formation in the lungs. The instrument was operated according to the manufacturer's instruction. To facilitate the detection of photons emitted from metastatic lung tumors, we performed ex vivo imaging analysis. In brief, mice were injected at 60 mg/kg with the 15 mg/mL stock of luciferin prior to anesthesia. After 10-15 min, mice were euthanized, and lung tissues were excised and placed into a 60 mm dish. Tissues were immediately imaged after exposure for 1 min. Regions of interest (ROIs) from displayed images were quantified as photons/second (ph/s) using the Living Image software (PerkinElmer).

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