Nmri nu nu mice

All experiments were performed in accordance with animal care guidelines from the European Union and were approved by the appropriate authority. The animal project registered under the number (#2438) received a favorable assessment from the Ethics Committee and was approved by the French Higher Education and Research Minister. All procedures involving animals were performed under general anesthesia with inhaled isoflurane (Vetflurane; Virbac, France) using a Univentor 400 anesthesia unit (Genestil, Royaucourt, France). Mice were housed in filtered air cabinets with a 12 h light/dark cycle at 22–24 °C and 50% humidity, provided with food and water ad libitum, and manipulated following aseptic procedures. Female NMRInu/nu mice (Janvier, St Berthevin, France) aged 9–10 weeks were used, with a mean bodyweight of 30 ± 3 g. Mice were inoculated subcutaneously in the left flank with 8 × 10⁶ exponentially growing HT29 cells and the experiments were initiated 5–7 days after inoculation when the tumors reached 50 mm³ in volume.

Animal experiments were approved by the ethics committee of KU Leuven (P131/2014). Male NMRI Nu/Nu mice (Janvier, France) were inoculated in both flanks with 2 × 10⁶ 22Rv1 cells in 100:100 µL medium/Matrigel (VWR, Radnor, PA, USA). Once tumors reached a volume of 150 mm³, mice were treated by intraperitoneal injection with solvent (9:1 saline/EtOH), metformin (250 mg/kg, every day), GANT61 (50 mg/kg every other day) or the combination of both drugs for 7 weeks. At day 5 of drug treatment, tumors were irradiated with a single dose of 6 Gy. During the entire treatment period, tumor growth was followed by 2-weekly caliper measurements and tumor volumes were calculated (V = (length × width × height) × π/6). In addition, the body weight of the mice was monitored to assess potential treatment toxicity. Mice were euthanized at the end of drug treatment or when tumors reached the maximum ethically permitted volume of 2 × 10³ mm³. Thirty min before euthanasia, pimonidazole was intraperitoneally injected. Afterwards, tumors were excised and half of the tumor was fixed in formalin and embedded in paraffin for immunohistochemical analysis and the other half was snap-frozen for protein analysis.

Ovarian cancer cells OVCAR-5 were sorted for CXCR4 and CD133 as previously described29 (link). For tumorigenicity assays, serial dilutions of single-cells re-suspended in Matrigel^TM (BD Bioscience, Heidelberg, Germany) were subcutaneously injected into female nude NMRI nu/nu mice (Janvier, Le Genest-Saint-Isle, France).

All animal experiments
were conducted in accordance with the German Animal Welfare Law and
were approved by local authorities. Female NMRI nu/nu mice (Janvier,
France) were dosed once at 100 mg/kg orally (p.o., in PEG400/ethanol 9:1) (n = 3 per group), intraperitoneally
(i.p., in solutol/ethanol/water 4:1:5), or subcutaneously
(s.c., in castoroil/benzylbenzoate 9:1). After 0.5,
3, 6, 14, 24, and 48 h, mice were sacrificed by decapitation and blood
sampled in potassium-EDTA tubes (Sarstedt, Germany). 100 μL
of plasma was used for analysis. Samples were precipitated by the
immediate administration of ice cold acetonitrile in a 1:5 dilution.
Samples were frozen at −20 °C overnight and subsequently
centrifuged at 3000 rpm for 15 min. The supernatant was analyzed with
an Agilent 1200 HPLC system with MS/MS detection (AB Sciex, Framingham,
MA). Obtained exposures were corrected for plasma–protein binding
and reported in relation to the antiproliferative IC₅₀u.

All animal experiments were conducted according to the European and Belgian legislation and with approval of the Animal Ethics Committee of the faculty of Biomedical Sciences of the KU Leuven (project license 221/2015). SWISS, NMRI nu/nu mice (Janvier labs) and B6.Cg-Tg(ACTb-eGFP) mice were used in these studies. For timed matings, male and female SWISS mice were placed together and left overnight. The next day (0.5 dpc) the success of the mating was assessed by the presence of a vaginal plug. At 14.5 dpc pregnant females were sacrificed by cervical dislocation.

Nine NMRI nu/nu mice (Janvier Laboratories, Le Genest-St-Isle, France) aged 8 weeks were used as recipients for the xenografts. They were housed in cages under filtered hoods (MicroIsolator, Uno) in rooms maintained at an ambient temperature between 22 and 24°C with a day/night cycle of 12 h. All housing material and food were autoclaved before use. The mice were fed ad libitum on laboratory chow (complete food for rats and mice; Pavan Carfil) and acidified water. All experiments in this study were approved by the Ethics Review Board and the Committee on Animal Research of the Catholic University of Louvain.