Figure 1.
A. Flowchart describing study design. B. Sample size for the study. C. Illustration of hypoxia protocol. D. 2-NBDG fluorescence images are acquired continuously for a period of 75 minutes to construct a (x,y,λ) data cube. B) At each (x,y) pixel location, a time course of 2-NBDG uptake can be obtained. Based on the time course, three metabolic parameters can be calculated: the initial rate of delivery (RD), rate of clearance (RC), and glucose uptake (2-NBDG60). C) Trans-illumination image cube of hemoglobin absorption is obtained from 520–620 nm. Total hemoglobin content is calculated pixel-wise by fitting a Beer-Lambert equation to the hyperspectral dataset. Knowledge of the wavelength-dependent extinction coefficients of hemoglobin allows calculation of oxy-hemoglobin and deoxy-hemoglobin concentrations. The ratio of oxy-hemoglobin to total hemoglobin concentration is the vascular oxygenation (SO2).
Figure 2.
In vitro uptake of 2-NBDG in 4T1 and 4T07 murine breast cancer cells.
A. Representative images from 4T1 and 4T07 cells incubated with 100 µM 2-NBDG for a period of 1–75 minutes. Cell plates were washed after each time point and imaged. B. Mean 2-NBDG fluorescence at each time point calculated from the images. Fluorescence values at each time point are normalized to the fluorescence measured at 1 minute. Error bars represent standard error of the mean. Incubation experiments were repeated 3 times for each cell line.
Figure 3.
A. In vivo uptake of 2-NBDG in 4T1 dorsal window chamber tumors. Confocal fluorescence images of a dorsal skin flap window chamber with a 4T1 tumor. Red fluorescence is due to constitutive expression of RFP in 4T1 cells. 2-NBDG images show cellular accumulation and co-registration with RFP at and beyond 40 minutes. Note that first four columns (before 2-NBDG injection, 5, 15, and 30 minutes after injection) are at lower resolution (scale bar is 250 µm) and the third and fourth columns are at higher resolution (scale bar is 100 µm). These images are from the dotted ROI shown in column 2. B. 2-NBDG fluorescence images acquired from a 4T1 tumor at lower resolution. The entire tumor is visible in these images. An image of constitutive tumor RFP illustrates the extent of the tumor. Images are acquired at the same time points indicated for 3A. Fluorescence images are not background-subtracted.
Figure 4.
Effect of breathing hypoxic gas on vascular oxygenation of 4T1 and 4T07 tumors.
A. Representative intravital images of vascular oxygenation (SO2) from 4T1 and 4T07 tumors at baseline and after breathing hypoxic gas (10% O2, rest N2). The effect of hypoxia is compared on tumors that showed low and high SO2 values at baseline. White dotted line in each image represents the tumor. B. After breathing hypoxic gas, there was a statistically significant increase in SO2 of the 4T1 tumors (n = 5; p = 0.03). There was no statistically significant increase in the 4T07 group (n = 5; p = 0.06). Error bars represent standard error of the mean. *indicates statistical significance at alpha = 0.05. C. The change in SO2 of 4T1 and 4T07 tumors after breathing hypoxic gas is inversely correlated with SO2 value at baseline (r = −0.74, p = 0.01). D. The change in SO2 of 4T1 and 4T07 tumors after hypoxia is strongly correlated with a change in oxy-hemoglobin concentration (r = 0.87; p = 0.001). E. 24 hours after breathing hypoxic gas, there was a significant decrease in SO2 of the 4T1 tumors (n = 4; p = 0.04), which had returned to a mean value close to pre-hypoxia baseline. 1 animal had to be censored before the 24 hours post-hypoxia measurement. Statistical significance in B and D were assessed using Student’s paired t-test.
Figure 5.
Effect of breathing hypoxic gas on 2-NBDG uptake by 4T1 and 4T07 tumors.
A. Representative intravital images of 2-NBDG60 from 4T1 and 4T07 tumors at baseline and after breathing hypoxic gas (10% O2, rest N2). 2-NBDG60 is the background-subtracted fluorescence image at 60 minutes (I60−I0). For comparison, 2-NBDG60 of tumors with low and high SO2 from 4T1 and 4T07 groups are shown here. B. 2-NBDG60 at baseline is significantly different between the 4T1 and 4T07 tumors. There are no significant changes in mean 2-NBDG60 after hypoxia. Each set of bars in 5B represents data from one animal at baseline and after hypoxia (5 animals per cell line). C. Survival curves of 2-NBDG60 from all animals as a function of SO2 of nearest blood vessel. At baseline, 2-NBDG60 of 4T1 tumors is lowest when SO2<10% (blue line; p<0.01). There are no significant differences in 2-NBDG60 for all other SO2 ranges. In the 4T07 tumors, 2-NBDG60 is lowest when SO2>60%. There are no significant differences in 2-NBDG60 for other SO2 ranges. Only SO2 ranges for which there are at least 3 animals are plotted. After breathing hypoxic gas, there are no significant differences in 2-NBDG60 between the different SO2 ranges in 4T1 and 4T07 tumors, except SO2>60% in the 4T07 tumors. Number of animals in each SO2 range is shown in parantheses in the legend. Error bars represent standard error of the mean. Statistical tests to compare 2-NBDG60 survival curves were conducted using repeated measures ANOVA.
Figure 6.
Effect of breathing hypoxic gas on 2-NBDG kinetics.
A. Kinetic profiles of 2-NBDG uptake in 4T1 and 4T07 tumors for high and low SO2. Blue line corresponds to the initial rate of delivery (RD). Rate of clearance (RC) is calculated from the falling part of the curve. Solid lines indicate baseline data and dashed lines indicate post-hypoxia data. B. Breathing hypoxic gas caused a significant increase in RD of 4T1 tumors (p = 0.002). There was significant increase in RC of 4T1 and 4T07 tumors after breathing hypoxic gas. Each set of bars in 6B represents data from one animal at baseline and after hypoxia (5 animals per cell line). C. Survival curves illustrating the relationship between RD and SO2. In the 4T1 tumors, RD is lowest when SO2<10%, illustrating that low SO2 leads to poor perfusion. After hypoxia, the region of lowest SO2 is no longer present in the 4T1 tumors, indicating an increase in SO2. There is a small, but significant increase in RD with an increase in SO2. In the 4T07 tumors, RD is highest for the highest level of SO2 (>60%). Post hypoxia, there are no significant changes in RD as a function of SO2. Statistical tests to compare 2-NBDG60 survival curves were conducted using repeated measures ANOVA. For each SO2 range, the number of animals is indicated in parentheses. Error bars represent standard error of the mean. *indicates statistical significance at a = 0.05.
Table 1.
Summary of RD and RC values for the representative curves shown in Figure 7A.
Figure 7.
Relationship between SO2, RD, RC and 2-NBDG60 for 4T1 and 4T07 tumors.
A. Illustration of 2-NBDG uptake curve indicating how RD, RC and 2-NBDG60 are calculated. B. Contour plots showing the relationship between delivery, clearance and uptake of 2-NBDG. RD and RC represent the x- and y-axes, respectively, while 2-NBDG60 represents the z-axis projecting out of the x-y plane. For the 4T1 tumors at baseline, 2-NBDG60 increases with RD at low values of RC. At higher values of RC, 2-NBDG60 reaches a maximum at RD = 2.5 s−1, levels off and then declines gradually for increasing RD. After hypoxia, a secondary maximum is seen at very high values of RD (RD>6 s−1) and low RC. In the 4T07 tumors, 2-NBDG60 increases with RD and reaches a maximum at approximately 6 s−1. The same feature is also present after hypoxia. At higher values of RC, 2-NBDG60 was nearly negligible for increasing values of RC.
Table 2.
A summary of possible relationships between NBDG60, RD, RC, and SO2 and final outcome corresponding to each combination.
Figure 8.
Ratio of 2-NBDG uptake to delivery is higher in 4T1 tumors.
The ratio of 2-NBDG60 and RD, which are indicative of glucose uptake and flow, respectively, is significantly higher in the 4T1 group (p = 0.01) compared to the 4T07 group.