Living image acquisition and analysis software

Sixteen days after tumor establishment, xenogen imaging was performed to confirm viable engraftment. A charge-coupled device camera (Xenogen IVIS-100, Xenogen Corporation, Almeda, CA) was used to image the firefly luciferase cancer cells. The nude mice were firstly anesthetized with isoflurane, then D-luciferin substrate suspended in PBS at 4.29 mg was administered per each mouse, while the whole procedure was performed in an induction chamber. The light emission was measured over an integration time of 10 s. A living image acquisition and analysis software (PerkinElmer, Inc., Waltham, MA) served for the resultant tumor flux analysis. Only after confirmation of viable engraftment by xenogen imaging, photothermal ablation was performed on breast cancer tumors.

Experiments were conducted in accordance with the recommendations of EEC (86/609/CEE). Experiments were approved by the Animal Experimental Ethics Committee of our Institution (Institut Gustave Roussy, France, permit n°26-2012-13). We used the RPMI 8226-Luc-GFP cell model described previously [23] (link). Six-week old non-obese diabetic/severe combined immunodeficiency/interleukin 2 receptor γ chain −/− or NSG mice were injected in the caudal vein with 5×10⁶ MM cells. Three days later, mice received either vehicle (n = 5) or were injected i.p. with 3 mg/kg DZNep twice a week (n = 5) until the end of the experiment. In a second series, using the same protocol of cells injection and engraftment, treatments started ten days later. NSG mice received vehicle (n = 5), bortezomib 0.4 mg/kg twice a week (n = 5), DZNep 1.5 mg/kg every two days (n = 5) or bortezomib in combination with DZNep 1.5 mg/kg every two days (n = 5). For bioluminescence imaging (BLI), mice were injected with 75 mg/kg of D-luciferine (Promega) and then analyzed with a Xenogen Optical In Vivo System (IVIS) coupled to Living Image Acquisition and Analysis software (Perkin Elmer, Waltham, MA). Ventral plus dorsal luminescence were determined by quantifying photon flux through the whole mouse and quantified. Mice were imaged at successive time points and were euthanized at the end of the treatment.