Hsd athymic nude foxn1nu mice

Male immunodeficient Hsd:Athymic Nude-Foxn1nu mice (Envigo) were used in the experiments. All animal studies were approved in accordance with the Weizmann Institute’s Animal Care and Use Committee (IACUC) guidelines and regulations (approval number 00580120-3). All animals were kept in a daily controlled room at the Weizmann Institute of Sciences animal facility with a surrounding relative humidity level of 50 ± 10% and a temperature of 22 ± 1 °C, with a 12/12 cycle of dark and light phases.

After an acclimation period of one week, eight week old female Hsd:Athymic Nude-Foxn1^nu mice (Envigo) were subcutaneously injected in the right hind leg with 50 μL of PBS containing 1.1 million SiHa cells derived from an exponentially growing culture. Once the tumor reached a size of 50 mm³, mice were randomly assigned to experimental groups and treated every 4th day, five times in total. Cisplatin was dissolved in PBS and injected intraperitoneally two hours before hyperthermia treatment at a dose of 2 mg/kg. Ganetespib was dissolved in a 10/18 DRD solution (10% DMSO, 18% Cremophor RH 40 (Sigma), 3.6% dextrose (Sigma) and 68.4% water) and injected intraperitoneally one hour before hyperthermia treatment at a dose of 50 mg/kg. Hyperthermia treatment (42 °C for one hour) was applied by submerging the right hind leg in a thermostatically controlled water bath during anesthesia with 2% isoflurane. Incubation was extended by 10 min to accommodate for the time needed to reach the target temperature. Radiotherapy was performed within 10 min after hyperthermia by exposing the right hind leg to a dose of 3 Gy with an X-strahl γ-ray source at a dose rate of 3 Gy/min. Tumor volume and animal weight were monitored every other day. Mice were sacrificed when the tumor reached a size larger than 1000 mm³ or upon meeting humane endpoints. No blinding method was used during the study.

Tumors derived from U87MG cells were induced in Hsd:AthymicNude-Foxn1nu mice (Envigo RMS-Spain) by subcutaneous injection of 9 × 10⁶ cells in PBS supplemented with 0.1% glucose. Tumors were allowed to grow until an average volume of 250–300 mm³ and animals were assigned randomly to the different groups. Treatments were administered with a single peritumoral (local) injection, in 100 μl of PBS supplemented with 5 mg/ml BSA. Tumors were measured with external caliper, and volume was calculated as (4π/3) x (width/2)² (link) x (length/2). All procedures involving animals were performed with the approval of the Complutense University Animal Experimentation Committee according to Spanish and European official regulations.

Atm^fl/fl, LSL-Kras^G12D/+, and Ptf1a^Cre/+ were previously described [22 (link),23 (link),24 (link)]. Eight-week-old female Hsd:Athymic Nude-Foxn1^nu mice were purchased from Envigo (Indianapolis, IN, USA). Mice were housed and bred in a conventional health status-controlled animal facility. All animal care and procedures followed German legal regulations and were previously approved by the respective governmental review board of the state of Baden-Württemberg (permission no. 1369 and 1273). All the aspects of the mouse work were carried out following strict guidelines to insure careful, consistent, and ethical handling of mice.