15353-j-veluchamy

5 Towards UCB-NK cells treatment in colorectal cancer | 133 with 0.5 x 106 SW480 Gluc cells at day 0 and were randomized into 4 groups. Group A only received SW480 cells, group B received SW480 in combination with cetuximab intraperitoneally (i.p., 0.5 mg, days 1, 4, 7), group C received SW480 in combination with UCB-NK i.v. (1 x 107, days 1, 4, 7), and group D received SW480 cells in combination with UCB-NK i.v. (1 x 10 7 , days 1, 4, 7) and cetuximab i.p. (0.5 mg, days 1, 4, 7). Groups C and D received i.p. 0.5µg IL-15 + 7.5µg IL-15Rα every 2-3 days from day 0 till day 14. Further, three adult mice received i.v tail vein injection of 0.5 x 10 6 A431 Gluc cells at day 0 and were treated with 0.5mg cetuximab (i.p., 0.5mg days 1, 4, 7), was used as a cetuximab efficacy control. Treatment effects were monitored using blood Gluc levels and bioluminescence imaging (BLI). All manipulations of BRGS mice were performed under laminar flow conditions. Blood Gluc quantification in vitro Secreted Gluc was measured according to a protocol described previously 27. Ten µl of blood was collected by capillarity into EDTA containing Microvette® CB tubes. Blood samples were distributed in 96 well black plates then mixed with 100µl of 100mM Gluc substrate native coelenterazine in PBS (P.J.K. GmbH; Kleinblittersdorf, Germany) and 5 minutes later light emission was quantified. Blood that was withdrawn before tumor inoculation served to determine a baseline value. Measurements were done twice a week until day 35. Gluc activity was measured using IVIS spectrum luminescence detector (PerkinElmer, Villebon-sur-Yvette, France). Data obtained were quantified using Living Image 4.0 software (PerkinElmer, Villebon-sur-Yvette, France). Bioluminescence imaging in vivo Mice were anesthetized using isofluorane gas in an induction chamber at a gas flow of 2.5 pm. Retro orbital injection of coelenterazine (4mg/kg body weight) was administered and mice were placed in the anaesthesia manifold inside the imaging chamber and imaged within 5 mins following substrate injection. Mice were placed into the light chamber and overlay images were collected for a period of 15min using IVIS spectrum in vivo imaging system (PerkinElmer, Villebon-sur-Yvette, France). Images were then analysed using Living Image 4.0 software (PerkinElmer, Villebon-sur-Yvette, France). Ethics statement Animals were housed in isolators under pathogen-free conditions with humane care and anaesthesia was performed using inhalational isoflurane anaesthesia to minimize suffering. Experiments were approved by the Institut Pasteur’s ethical committee for animal use in research, Comité d’étique en expérimentation animale (CETEA) #89, protocol reference # 2007–006 and validated by the French Ministry of Education and Research (Reference # 02162.01).