Nmri nude mice

In collaboration with TRACE and after approval by the University Hospital KU Leuven Medical Ethical Committee (S54185) and written informed consent from the patient, PDX model MEL006R (BRAFi resistant) was established from an in‐transit metastasis resected as part of standard‐of‐care melanoma treatment at the University Hospital KU Leuven. The procedures involving mice were performed in accordance with the guidelines of the IACUC and KU Leuven and carried out within the context of approved project applications P147/2012, P038/2015 and P098/2015. Fresh tumor tissue was collected in transport medium (RPMI1640 medium supplemented with penicillin/streptomycin and amphotericin B). Tumor fragments were subsequently rinsed in phosphate‐buffered saline supplemented with penicillin/streptomycin and amphotericin B and cut into small pieces of approximately 3 × 3 × 3 mm³. Tumor pieces were implanted in the interscapular fat pad of female SCID‐beige mice (Taconic). After reaching generation 4 (F4), tumor fragments were implanted in the interscapular fat pad of female NMRI nude mice (8 weeks, Taconic). Ketamine, medetomidine and buprenorphine were used for anesthesia. Because tumor growth of the BRAFi‐resistant PDX model (Mel006R) is very fast, this model is probably not appropriate to study the mechanisms of invasion and metastasis at least in the time window analyzed (20–60 days).

U87 MG human glioblastoma cancer cells (ATCC HTB-14, LGC Standards) were cultured in EMEM media supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Invitrogen) at 37 °C and 5% CO₂. Cells in their exponential growth phase and at 80–90% confluence were harvested by trypsinization and resuspended in 1:1 EMEM media and Matrigel (BD Biosciences) at 3 × 10⁷ cells/mL. Subcutaneous tumors were established in female NMRI nude mice (Taconic, Denmark) by inoculation of 3 × 10⁶ cells in 100 μL on each flank above the hind limbs in the subcutaneous space. All animals received the same level of care and were distributed amongst the test groups at random, and all inoculations and injections occurred on the same dates. Upon excision, the average tumor mass was 46 ± 17 mg. All animal experiments were performed under a protocol approved by the National Animal Experiments Inspectorate of Denmark. The study was performed in compliance with the ARRIVE 2.0 criteria. A compliance evaluation is included as supplementary information.

Eight different human BTC cell lines were tested for tumorigenicity in immunodeficient NMRI nude mice (TFK-1: metastasized extrahepatic biliary adenocarcinoma, EGI-1: extrahepatic biliary adenocarcinoma, Mz-ChA-1: gallbladder adenocarcinoma abdominal wall metastasis, Mz-ChA-2: gallbladder adenocarcinoma liver metastasis, Sk-ChA-1: malignant ascites in extrahepatic biliary adenocarcinoma, BDC: bile duct cancer, CC-SW-1: intrahepatic biliary adenocarcinoma, GBC: gallbladder mucosal carcinoma [19 (link)]). Tumor cells (10⁷ cells in 50 μl medium mixed with 50 μL Matrigel™ Basement Membrane Matrix (Dulbecco)) were injected subcutaneously into the left flank of two NMRI nude mice (Taconic, Bomholdtgard, Denmark) per cell line, respectively. Cells were cultured in Dulbecco›s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum in 5% CO₂ at 37°C. All animal procedures were performed in accordance with the local animal welfare committee (Gen0030/15).
Tumor size (length x width) was measured by calipers once weekly for up to 125 days. Individual tumor volumes (V) were calculated by the formula: V = (length x [width]²)/2.

To evaluate tumor accumulation of L-OHP after liposome or free-drug administration, Balb/cJRj mice carrying CT26 tumors or NMRI nude mice (Taconic, Denmark) carrying FaDu tumors (5 × 10⁶ cells in 100 μL of RPMI media inoculated subcutaneously on the flank) were injected intravenously with 8 mg of L-OHP/kg. Liposomes containing encapsulated L-OHP were injected intravenously (8 mg L-OHP/kg). After the mice were sacrificed, tumors were dissected and snap-frozen. To measure platinum content in tumors, 50 to 100 mg of tissue was added to 15-ml Falcon tube and weighed before being digested overnight at 65°C in 500 μl of HNO₃, 50 μl of HCl, and 300 μl of H₂O₂. Ten milliliters of MQ (mill-q) water was added, and after weighing, the samples were further diluted in 2% HCl. The platinum content in the tissues was measured by ICP-MS.

PDX models were previously established and characterised at the TRACE Platform (UZ/KU Leuven). Tumour tissue fragments from PDX tumours were implanted interscapularly in female NMRI nude mice (minimum 6 weeks old) (Taconic) or NOD‐Prkdc^em26Cd52Il2rg^emCd22 mice engrafted with human CD34+ hematopoietic stem cells (HSCs) from three donors (C‐AFP, 760 and C‐AED) with different human leukocyte antigen (HLA)‐A2 serotypes (minimum 24 weeks old) (TransCure bioServices).

BxPC-3 cells (ATCC CRL-1687, LGC Standards) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen) at 37°C and 5% CO₂. Cells were tested negative for mycoplasma and a panel of murine pathogens. Cells in their exponential growth phase were harvested by trypsinization at 80-90% confluence and resuspended in complete medium. Tumors were generated in female NMRI nude mice (Taconic) on the flank above the hind limb by subcutaneous injection of 5 × 10⁶ cells in 100 μL. Tumor growth was monitored twice a week by caliper measurements and tumor volume was calculated using the formula: 0.52 x length x (width)². All animal experiments were performed under a protocol approved by the Danish National Animal Experiments Inspectorate.