In vivo experiments
All procedures involving animals, were conducted in accordance with protocols approved by ethical committee of the University of Angers and the regional ethics committee on animal experimentations (Authorization APAFIS#13365-2018020217041961 v3). Futhermore, animal experiments were carried out in strict accordance with recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioral research with Animals (European directive 2010/63/UE).
Seven-week-old male C57BL/6 mice were housed at the university animal facility (Service Commun d’Animalerie Hospitalo-Universitaire - Université d’Angers, France). Syngenic allograft model of melanoma was obtained by injecting subcutaneously a suspension of 105 B16F10 melanoma cells in 100 µL of PBS 1X (Lonza, Verviers, Belgium) into the right flank of mice. For optimal measurement of the tumor and its vascularisation, body hair was removed manually. The mice were shaved with a razor and the remaining hair was removed with depilatory cream. Tumor volume was monitored
Mice were randomized and were treated when tumor volume was approximatively 100 mm3. Mice were treated twice a week (days 1, 3, 8, 10, 15 and 18) with 3 mg/kg/day mice anti-PD1 (BioXcell, Lebanon, USA) or NaCl 0.9%. Tumor volume was monitored according to different techniques such as (i) caliper with the formula: V = π∕6 × L × W2 (V « volume », L « length » and W « width ») and (ii) ultrasonography. In accordance with ethical rules, animals were sacrificed when the tumor volume was greater than 2500 mm3 or when mice showed signs of suffering.