Figure 1.
(A) Timeline of imaging events. Mice that were imaged under two imaging conditions were imaged on subsequent days. The order of imaging was scrambled to minimize order effects. (B) A 6 mM injection of 2-NBDG was given and imaged for at least 60 minutes, and the mean of the tumor region for each image was used to construct a kinetic curve. Images for the endpoints 2-NBDG60 (2-NBDG intensity at 60 minutes) and the rate of delivery of 2-NBDG (RD = 2-NBDG60/Tmax) are shown. (C) Trans-illumination images were collected in 10 nm increments from 500–600 nm and used to calculate hemoglobin saturation (SO2). (D) The table shows the number of mice used in each perturbation group. Each mouse was used for up to two imaging sessions, with 24 hours between sessions. The groups were randomized to minimize bias from imaging order, and an analysis of variance (ANOVA) was performed to test for order effects. No significant imaging order effect was observed for any experiment.
Figure 2.
Delivery-corrected 2-NBDG uptake inversely correlates with blood glucose concentration.
(A) Representative images show the kinetics of 2-NBDG uptake in vivo in non-tumor window chambers. The same mouse was given 6 mM or 10 mM 2-NBDG on subsequent days and imaged for 60 minutes following injection. (B) Averaged 2-NBDG kinetics for a cohort of mice injected with 0.1 mL of either 6 mM or 10 mM 2-NBDG. At 5 minutes post-injection (2-NBDG05), the fluorescence ratio of the dose groups (2-NBDG05,10 mM/2-NBDG05,6 mM) was proportional to molarity (p<0.01). The table shows the expected ratio of 10 mM/6 mM fluorescence, if all differences in fluorescence were due to dose. 2-NBDG05,10 mM/2-NBDG05,6 mM corresponds to the ratio of 10 mM and 6 mM fluorescence intensities at t = 5 min. The ratio RD,10 mM/RD,6 mM corresponds to the rate of 2-NBDG delivery for 10 mM and 6 mM. Each group in panel B contains the same n = 7 subjects. p values are from a student's paired t-test. Error bars show standard error. Values in table are mean ± standard error. (C) RD was strongly correlated with 2-NBDG fluorescence at 5 minutes (p<0.001). RD did not correlate with 2-NBDG60 (not shown). (D) 2-NBDG60/RD was inversely correlated with baseline blood glucose in normal mice (R = −0.61, p = 0.02). 2-NBDG60 was also correlated with blood glucose (R = −0.52, p = 0.05, not shown). For animals that received both 6 mM and 10 mM doses, the average values of the endpoints (2-NBDG05, 2-NBDG60, and 2-NBDG60/RD) for both doses were used in calculating the correlations. These subjects are denoted by “mean” in the legend. n = 15 mice for (C) and (D).
Figure 3.
The rate of 2-NBDG delivery, RD, is strongly correlated with blood velocity.
(A) Representative images of blood velocity and the rate of 2-NBDG delivery (RD) in a normal mouse at baseline and during reoxygenation after 1 hour of hypoxia. (B) Paired data for a set of mice at baseline and after 1 hour of hypoxia. After hypoxia, flow velocity and RD increased significantly (p<0.02 for both). N = 6 mice. (C) The rate of 2-NBDG delivery (RD) is highly correlated with blood velocity (R = 0.87, p<0.05). The trendline corresponds to the trend for post-hypoxia data only.
Figure 4.
The ratio 2-NBDG60/RD reflects stereo-specific uptake in vivo.
(A) Mean kinetics of non-specific control (2-NBDLG) and specific tracer (2-NBDG) uptake imaged in the same cohort of mice on subsequent days. Peak fluorescence is significantly greater after 2-NBDLG administration than after 2-NBDG administration (p<0.01). Fluorescence at 60 minutes is comparable for both tracers (p = N.S.) (B) Results of paired tracer and control imaging in a set of four mice. Neither blood glucose nor 2-NBDG60 was significantly different between the two imaging perturbations (p = N.S.). RD was greater for the control 2-NBDLG than for 2-NBDG (p<0.02). 2-NBDG60/RD identifies specific tracer uptake, as it is significantly greater for 2-NBDG than 2-NBDLG (p<0.02). n = 4 mice. Each p-value represents results of a student's paired t-test.
Figure 5.
Delivery-corrected glucose uptake reveals distinct glycolytic phenotypes in metastatic (4T1) and non-metastatic (4T07) mammary tumors.
(A) Representative images of vascular oxygen saturation (SO2) and delivery-corrected 2-NBDG (2-NBDG60/RD) for a 4T1 tumor and a 4T07 tumor, in vivo. (B) 2-NBDG60/RD showed contrast in glucose uptake between metastatic 4T1 and non-metastatic 4T07 tumors in vivo (p<0.01). A Seahorse Glycolysis Stress Test also revealed that the glycolytic capacity, defined as extracellular acidification rate (ECAR) after blockade of respiration by oligomycin, was significantly greater for 4T1 than for 4T07 (p<0.01). (C) Mean vascular oxygen saturation (SO2) was comparable for 4T07 and 4T1 tumors in window chambers (p = N.S.). Vascular density was indistinguishable between tumor lines (p = N.S.). A Seahorse Glycolysis Stress Test showed that oxygen consumption rate (OCR) is comparable for 4T1 and 4T07 tumors (p = N.S.). Number of mice per group indicated by group name on axis. For Seahorse results, n = 12 cell samples from 3 distinct assays. Midline of box plots show median, box edges correspond to 25th and 75th percentiles, and scatter points show all data values.
Figure 6.
The ratio 2-NBDG/RD facilitates assessment of glucose demand in heterogeneous regions of metastatic mammary tumors.
(A) Representative images of vascular oxygenation (SO2) and delivery-corrected 2-NBDG (2-NBDG60/RD) for a 4T1 tumor with low mean SO2, a 4T1 tumor with intermediate mean SO2, and a 4T07 with high mean SO2. Adapted from Rajaram, et al. 2013. (B) Survival curves (1-cumulative distributions) show 2-NBDG60, RD, and 2-NBDG60/RD for regions of distinct SO2 (%) in 4T07 and 4T1 tumors. For 4T1, 2-NBDG60 is lower for 0<SO2,4T1<10 regions than for any other SO2,4T1 (p = N.S.). Significantly lower rates of RD are seen for the 0<SO2,4T1<10 group than for well-oxygenated 4T1 regions (p<0.05 or p<0.01 for 0<SO2,4T1<10 vs. 20<SO2,4T1<40 or 40<SO2,4T1<60, respectively). After correction for low RD, 2-NBDG60/RD increased slightly but significantly in hypoxic regions (p<0.01 for 0<SO2,4T1<10 vs. 40<SO2,4T1<60). For 4T07, 2-NBDG uptake for the highest SO2,4T07 regions decreased compared to the lowest SO2,4T07 (p<0.01 for all 20<SO2,4T07<40 vs. 60<SO2,4T1<80). RD is indistinguishable between SO2,4T07 levels. After correction by RD, 2-NBDG60/RD is lowest for 60<SO2,4T07<80 (p<0.01). Comparison between 4T1 and 4T07 shows that 2-NBDG60 is higher for all SO2,4T1 than all SO2,4T07 (p<0.01). On the other hand, RD for the best oxygenated 4T07 groups (40<SO2,4T07<60 and 60<SO2,4T07<80) is greater than for all 4T1 groups (p<0.01 for all groups except 40<SO2,4T1<60 vs. 60<SO2,4T07<80 where p<0.06). After correction by RD, 2-NBDG60/RD is higher for all SO2,4T1 than all SO2,4T07 (p<0.01 for all SO2,4T1 compared to all SO2,4T07). Number of mice per group indicated by group name in legend.