Establishment of xenografts

Six-week-old, female, athymic Rj:NMRI-Foxn1^nu/nu mice were supplied by Janvier Labs. Xenografts were established by bilateral implantation of a human tumor fragment subcutaneously as described previously [34]. In brief, fresh tumor samples from donor patients were cut into fragments of ±10 mm³. A 0.5–1 cm skin incision was made on each flank of an immunodeficient mice, anesthetized using a 3% isoflurane mixture in oxygen, and one tumor piece was inserted subcutaneously on each side of the animal. Wounds were then closed with 6–0 silk sutures (Mersilk, Ethicon). Mice were sacrificed once tumor volumes reached approximately 700–1000 mm³. After removal of the tumors, fragments were retransplanted to a next generation of mice (n = 2–3). This process of ‘passaging’ was repeated to maintain the model. With every passage, morphology was checked and immunohistochemistry was performed to characterize the models. A model was considered as ‘established’ when stable histological and molecular features were confirmed for at least two passages. The Medical Ethics Committee of the University Hospitals Leuven approved the collection of human tumor tissue and its usage for xenograft work (approval number: {"type":"entrez-protein","attrs":{"text":"S53483","term_id":"1078473","term_text":"pir||S53483"}}S53483). All donor patients gave written informed consent to the usage of their tumor tissue. The Animal Ethics Committee of KU Leuven approved the creation of the PDX models and the in vivo experiments (approval number: P175-2015), which were performed according to its guidelines and Belgian regulations.