Ivis lumina 2

Tumor growth was monitored weekly by bioluminescence imaging (BLI) using an IVIS Lumina II in vivo imaging system (Perkin Elmer, Waltham, MA, USA). Mice were anesthetized by isoflurane inhalation and subcutaneously injected with 150 μg of in vivo-grade VivoGlo™ luciferin (Promega, Madison, WI, USA) dissolved in 100 μL of DPBS per mouse. Images were acquired after an incubation time of 15 min. Data were recorded and analyzed using Living Image in vivo Imaging Software (Perkin Elmer). Total flux (photons/s) was used for measurement and statistical analysis of the tumor burden using a uniform region of interest in all mice.

Vascular leakage was visualized on indocyanine green (ICG)-based fluorescence imaging. ICG (0.5 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) dissolved in Kolliphor HS 15 (Sigma-Aldrich, St. Louis, MO, USA) was injected intravenously into anaesthetized mice (100/10 mg/kg ketamine-xylazine i.p.) on days 2 and 7 following CFA administration. Fluorescence imaging was performed 20 minutes post injection using the IVIS Lumina II (PerkinElmer, Waltham, MA, USA; auto acquisition time, f/stop = 1, binning = 2, excitation: 745 nm, emission filter: >800 nm). Data were analyzed and ROIs were drawn around the ankle joints. Fluorescence was expressed as total radiant efficiency ([photons/s/cm2/sr]/[μW/cm2]) [51 (link)].

Neutrophil myeloperoxidase (MPO) activity was assessed with Luminol-derived bioluminescence. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) sodium salt (150 mg/kg, Gold Biotechnology, Olivette, MO, USA) dissolved in sterile phosphate-buffered saline (PBS, 20 mg/ml) was injected intraperitoneally into anaesthetized mice (100/10 mg/kg ketamine-xylazine i.p.) on days 2 and 7 post CFA administration. Bioluminescence imaging was performed 10 minutes post injection using the IVIS Lumina II (PerkinElmer, Waltham, MA, USA; 60s acquisition, f/stop = 1, binning = 8). Identical regions of interest (ROIs) were applied around the ankles and luminescence was expressed as total radiance (total photon flux/s) [51 (link), 52 (link)].

Bioluminescent imaging was performed using an IVIS Lumina II in vivo imaging system (IVIS, PerkinElmer). For live imaging, 3 mice/group were anesthetized using isoflurane inhalation (at 2.5% concentration), followed by intraperitoneal (ip.) injection of D-luciferin (150 mg/kg) in Dulbecco’s Phosphate Buffered Saline (D-PBS). Mice were then placed in a light-tight chamber for imaging and the chamber was under continuous exposure to 1–2% isoflurane. Imaging times ranged from 8 to 10 min, depending on the tumor model and tumor sizes. For ex vivo lung and other tissue imaging, mice were given the substrate 150 mg/kg D-luciferin by intraperitoneal (ip.) injection in DPBS right before necropsy. Tissues of interest (lung and lymph nodes) were freshly excised, placed into 24-well cell culture plates with D-PBS containing 300 μg/ml D-luciferin, and imaging time was up to 20 min. Luminescence and images collected were integrated and quantified as total photon counts or photons/second using Living Image® software.

The control plasmid and the lumican-specific shRNA plasmid-transfected bone metastatic A549/luc and LLC/luc cells (1 × 10⁵ cells/mouse) were injected I.V. and I.C. for the establishment of lung and bone metastases, respectively, as described previously [23 (link)]. Colonization of the bone and lung metastases was observed using the IVIS^TM live-imaging system (IVIS Lumina II, PerkinElmer, SantaClara, CA, USA). To assess the autocrine effect of lumican on the establishment of bone metastasis of lung cancer cells, bone metastatic or parental LLC/luc cells, cultured with and without recombinant lumican (100 ng/mL) at 37 °C for 24 h, were I.C. injected into mice (1 × 10⁶ cells/mouse). The total bone marrow cells were harvested from the mice one day after the I.C. injection, and the cDNA was synthesized as described above. The gene expression for luciferase was determined by real-time PCR following the manufacturer’s protocol of the SYBR^® Green PCR Master Mix Kit. The primer sequences that were used are as follows: 5′-GCTGGGCGTTAATCAGAGAG-3′ and 5′-GTCGAAGATGTTGGGGTGTT-3′ for firefly luciferase; 5′-TTCCAGTATGACTCCACTCA-3′ and 5′-ATCACGCCACAGCTTTCCAG-3′ for mouse GAPDH. The gene expression for luciferase was analyzed using a 7500 Fast Real-Time PCR System and the identity of products obtained after the PCR reaction was confirmed by agarose gel electrophoresis.