Figure 7: Nutrient regulation of CendR uptake in physiological context.

(a) CendR uptake in live tumour slices responds to nutrient availability. Live tumour tissue slices (PPC1 xenografts; 4T1 mouse mammary tumours) were obtained and incubated under the indicated conditions (y axis) for 6 h. The slices were then suspended in the same kind of media containing 15 pM R-Ag and incubated for another 2 h at 37 °C with gentle shaking on a rocker before etching and fixation. The fluorescence intensity of R-Ag per cell was quantified using ImageJ, normalized to slices cultured in complete media and shown as relative uptake (x axis). **P<0.01 and ***P<0.001 (Student’s t-test) in comparison with complete media conditions of the corresponding tumour types. Error bars indicate s.e.m. (4–7 replicates). (b) Representative images of R-Ag internalization into PPC1 tumour slices. R-Ag, red; nuclei, blue. Scale bar, 50 μm. (c) Representative image of glucose uptake into 4T1 tumours after GLUT IV inhibitor treatment. Mice bearing 4T1 tumours were treated with intratumoral injections of vehicle only or GLUT IV inhibitor as described in Methods. IRDye 800CW 2-deoxy-D-glucose (2-DG) in 10 nmol in 100 μl PBS was then injected intravenously into a tail vein and 24 h later, the fluorescence intensity of 2-DG was captured using Xenogen IVIS 200 imager (PerkinElmer Inc.). The experiment was conducted twice with at least three mice per group. (d) iRGD accumulation in tumours responds to glucose deprivation. FAM (fluorescein)-labelled iRGD (200 μg in 100 μl PBS) was intravenously injected into mice bearing 4T1 tumours treated with vehicle alone or with the GLUT IV inhibitor. After 2 h of circulation, the mice were perfused with PBS containing 1% BSA to remove peptide remaining in the circulation. The tumours were excised, fixed and sectioned. FAM-iRGD in the tumours was detected with rabbit anti-FITC antibody. The average signal intensity per cell was normalized to vehicle-treated tumours as relative uptake. The experiment was conducted twice with at least three mice per group, and error bars indicate s.e.m. *P<0.05 (Student’s t-test).