Xenogen ivis 200 imaging system

Four-week-old male BALB/c nude mice (eight in each group) were bought from Taipei’s National Laboratory Animal Center and orthotopically injected with JJ012 or JJ012/NGF cells (5 × 10⁶, resuspended in 100 μL of a medium containing 50% serum-free DMEM/α-MEM and 50% Matrigel) according to a previous protocol [27 (link)]. The tumor growth in the tibiae was monitored each week by bioluminescence imaging using a Xenogen IVIS imaging system 200 (PerkinElmer; Waltham, MA, USA). At 12 weeks, the mice were euthanized by CO2 inhalation. The lungs were removed and fixed in 10% formalin for a further analysis. All animal experiments satisfied the protocols specified by China Medical University’s Institutional Animal Care and Use Committee (IACUC Approval No. 104-154-N).

Metastatic lung cancer was established by injecting 6×10⁶ A549 or A549-luc-C8 (A549luc) cells into the tail vein of athymic nude or SCID/beige mice. After 1-3 weeks the mice were randomized in groups of 12 and treated by nose-only (Inexpose System, Scireq-EMKA Technologies) or whole body (AirFamily system, Pic indolor) aerosol with AvidinOX (6.5 mL of 3 mg/mL solution) followed, after 4 h, by nebulized PBS (antibody vehicle) or bCet. Treatments were repeated for 4-8 consecutive weeks and death events recorded. Control groups were i.v. administered 1 mg/mouse Cetuximab. Tumor bioluminescence imaging (BLI) was recorded at different time points by Xenogen IVIS Imaging System 200 (Perkin Elmer), 15 min after i.p. injection of luciferin (150 μg/mouse). Details on nose-only and whole body equipment and dose calculation in Supplementary Figure S5B,C.

JJ012/vehicle or JJ012/visfatin cells (5  ×  10⁶) were orthotopically injected into 4-week-old male BALB/c nude mice (Taipei’s National Laboratory Animal Center), according to a previous protocol [20 (link)]. Tumor growth in the tibiae was examined each week by bioluminescence imaging by Xenogen IVIS imaging system 200 (PerkinElmer, MA, USA). After 12 weeks, the mice were sacrificed by CO₂ inhalation. The lungs were then excised for further examination. All animal procedures were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of China Medical University (CMUIACUC-2019-079).

All animal experiments satisfied the protocols specified by China Medical University’s Institutional Animal Care and Use Committee (IACUC Approval No. 104-154-N). Four-week-old male BALB/c nude mice were purchased from Taipei’s National Laboratory Animal Center and randomly assigned to one of the following orthotopic implant groups: JJ012, JJ012(S10), or JJ012(S10)/CCN6 shRNA. Under isoflurane anesthesia (1.5–2.5%), JJ012, JJ012(S10), or JJ012(S10)/CCN6 shRNA cells (5 × 10⁶, resuspended in 50 μL of medium containing 50% serum-free DMEN/α-MEM and 50% Matrigel) were orthotopically implanted into the left leg tibia of each mouse. Tumor growth in the tibiae was monitored for 8 weeks by bioluminescence imaging using a Xenogen IVIS imaging system 200 (PerkinElmer, MA, USA). Prior to imaging, the mice were anesthetized with isoflurane (1.5–2.5%) and then intraperitoneally injected with D-Luciferin potassium salt 150 mg/kg. Images were analyzed using Living Image 4.0 software (Caliper, Alameda, CA). At 8 weeks, the mice were euthanized by CO₂ inhalation. The lungs were removed and fixed in 10% formalin for further analysis.