Franny Jongbloed

70 CHAPTER 3 cells were harvested and after centrifugation, single-cell suspensions were prepared in phosphate buffered saline (PBS) to a final concentration of 2.5 x 10 5 cells/100 µL. Cell viability was determined by trypan blue staining, and was always ≥90%. Experimental setup Mice (n=36) were anesthetized (isoflurane inhalation, 5% isoflurane inhalation initially and then 2% isoflurane with a 1:1 air:oxygen mixture for maintenance of anesthesia) (Figure S1). Both flanks were shaved for precise injection. 2.5 x 10 5 C26 cells were injected subcutaneously on both sides in a volume of 100 µL, using a 21G needle. Tumors were allowed to grow for 12 days before start of the experiment. Mice were weighed and tumors were measured daily with digital calipers. The mice were randomly divided into four groups (n=6/group). Two groups were fasted for three days and two groups were fed ad libitum (AL). After the fasting period, mice were fed AL again. One AL fed group, and one group of fasted animals, were treated with a single weight-adjusted dose of 133 mg/kg (±3.3 mg/kg, and ±2.7 mg/kg respectively) of irinotecan intraperitoneally. The control groups received vehicle treatment (sodium chloride 0.9%). Two hours after injection, all groups received unlimited access to food for 10 hours until the moment of sacrifice. The difference between starting amount and remaining amount of food was used to calculate food consumption per mouse for this 10-hour period. Subsequently, 12 hours after irinotecan administration, the mice were sacrificed by exsanguination. Fasting protocol Mice in the AL fed groups were allowed unrestricted access to food, and amount of food eaten per cage was measured daily. Before the start of the fasting period, all mice were transferred to a clean cage and mice in the fasting groups were withheld food for three days starting at 4:00 PM on a Friday until 10:00 AM on a Monday. All animals were given continuous access to water. Chemotherapy Irinotecan, HCl-trihydrate 20 mg/mL (Hospira, Benelux) was used for in vivo experiments. Irinotecan was diluted in sodium chloride 0.9% (Braun, Melsungen, Germany) to a final volume of 200 µL per injection, and was given intraperitoneally. Serummeasurements Mice were killed by exsanguination with cardiac puncture under anesthesia (isoflurane inhalation, 5% isoflurane inhalation initially and then 2% isoflurane with a 1:1 air:oxygen mixture for maintenance of anesthesia). After cardiac puncture, ± 900 µL of blood per mouse was transferred directly into 1 mL tubes (MiniCollect, Greiner Bio-one), containing