Establishment of the patient-derived xenograft (PDX) model

The tumor mass, immediately after being explanted from the patient, was collected and transferred to Hank's Balanced Salt Solution, then cut using a sterile scalpel in pieces smaller than 1 cm, and collected in cryovials to be frozen directly at −80 °C and moved after a few days in liquid nitrogen. The development of the PDX model, after approval of the Ethical Committee (Prot. N. 254/C.E), was conducted at the Biogem Animal House in Ariano Irpino (Avellino, Italy), in accordance with the National Academy of Sciences guidelines. Tissue fragments were implanted subcutaneously in the flanks of 4–5-week-old CD1 immunodeficient nude female mice. Each mouse was given drinking water ad libitum and a complete pellet diet (GLP 4RF21, Mucedola) during the study. Mice were monitored daily for clinical signs and mortality, and BW records were assessed weekly. Tumor growth was controlled every 2 weeks with Mitutoyo forceps. Experiments ended 8 weeks after the tumor implant, sacrificing animals with tumor masses greater than 15% of body weight (BW) and/or with a body weight loss (BWL) of 10%. All animals were weighed every 2–3 days during the experimental period. The BWL was determined as follows: body weight loss percent (% BWL max) = 100 − (mean BW day x/mean BW day 1 × 100), where BWx is the mean BW at the day of maximal loss during the experiment, and BW1 is the mean BW on the first day of the experimental period. At the end of the study, the mice were sacrificed by cervical dislocation, and the tumor masses were photographed and collected. Biopsies of 100 mm³ were implanted, and after a week from implantation, to allow the engraftment of the masses, the mice were divided into groups of ten animals and treated as follows: (1) vehicle only; (2) LY3039478 (8 mg/kg); (3) gemcitabine as a chemotherapeutic control (125 mg/kg). The Tumor Volume formula (mm³) = [length (mm) × width (mm)²]/2 was used, where width and length are the shortest and longest diameters.