Experimental pulmonary metastasis assay in vivo

Balb/c-nude female mice were obtained from Orient Bio Inc. (Seungnam, Republic of Korea). All animal experiments were performed at Gachon University following IACUC (Institutional Animal Care and Use Committee)-approved protocols (No.LCDI-2012-0069). For the experimental metastatic model, MDA-MB-231-luc cells (shCT and shCCR1, 1 x 10⁶ cells in 100 μl PBS) were injected into the lateral tail veins of 7-week-old Balb/c-nude mice (n = 10 each group). After 8 weeks, D-luciferin (15 mg/kg; PerkinElmer, Waltham, MA, USA) was intraperitoneally injected 10 min prior to imaging acquisition. To monitor pulmonary metastasis, in vivo bioluminescent imaging was conducted once a week using the Spectrum IVIS (PerkinElmer, Waltham, MA, USA). The BLI signal intensity (photons/sec) was quantified and calculated as the sum of all detected regions per second using Living Image software (Xenogen, Alameda, CA, USA).