2.3. Animal tumor models and treatment protocols

Xenograft pancreatic tumor models were generated by subcutaneous implantation of 2×10⁶ MiaPaCa2 or BxPC3 cells resuspended in 40 µl of serum-free medium into 6-week old male NOD/SCID mice. Vismodegib was administered orally once a day at different doses (40mg/Kg or 100mg/Kg, as indicated) from day 35 in MiaPaCa2 and day 4 in BxPC3 post-implantation, 10 days before the initiation of chemotherapy. Gemcitabine (50 mg/kg) was administered by intraperitoneal (i.p.) injection when tumors reached an average size of ~200 mm³; from day 45 in MiaPaCa2 and day 13 in BxPC3 post-implantation, every 72 hours. Orthotopic xenograft breast tumors were generated by implantation of 5×10⁵ MCF10CA1a cells resuspended in 40 µl of serum-free medium into the mammary fat pad of 6-week old female CD1 nude immunodeficient mice. Vismodegib (40 mg/kg) was administered orally from day 4 post-implantation. In both models, Doxil (3mg/kg) and Abraxane (20 mg/kg) were administered by intravenous (i.v.) injections on day 14 and 21 post-implantation [8, 21–23]. During the course of each experiment, tumor growth was monitored daily and the planar dimensions (x, y) were measured using a digital caliper. Tumor volume was calculated using the volume of an ellipsoid and assuming that the third dimension, z, is equal to xy. All in vivo experiments were conducted in accordance with the animal welfare regulations and guidelines of the Republic of Cyprus and the European Union under a license acquired by the Cyprus Veterinary Services (No CY/EXP/PR.L1/2014), the Cyprus national authority for monitoring animal research.

To study alterations in the tumor microenvironment, right before the end of each treatment protocol, animals were anesthetized by i.p. injection of Avertin (200mg/kg) and interstitial fluid pressure was measure using the wick-in-needle technique [3, 24, 25]. Next, mice were injected intracardially with 100µl biotinylated lectin (1mg/ml, Vector Labs), which was allowed to distribute throughout the body for 7 minutes [7–9]. Finally, mice were sacrificed via CO2 inhalation and tumors were excised for measurement of mechanical properties and/or histological analysis.