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.