Optical measures of SO₂ are significantly lower in tumors compared with normal tissue.

<p><b>A</b>. Representative reflectance spectra (open circles) from normal tissue (blue), 4T1 (red) and 4T07 (green) murine mammary tumors, and MC model fits (solid line). These spectra were measured prior to 2-NBDG injection <b>B</b>. Extracted absorption spectra for the reflectance spectra shown in 2A illustrate higher absorption in the 4T1 tumor compared with the normal and 4T07 tumor. <b>C</b>. Baseline oxygenation levels are significantly lower in 4T1 and 4T07 tumors compared with normal tissue. <b>D</b>. The injection of 2-NBDG causes a statistically insignificant increase in SO₂ in normal tissue and 4T1 tumors, and no change in the control and 4T07 groups <b>E</b>. Oxygen consumption rates (OCR) of 4T1 and 4T07 cells are statistically similar. Measurements were made using a Seahorse Glycolysis stress test. Data represent n = 12 cell samples from 3 distinct assays. Error bars represent standard error of the mean.</p

2-NBDG₆₀ is sensitive to a decrease in blood glucose levels.

<p><b>A</b>. Representative 2-NBDG kinetic profiles for 3 different fasting groups of mice after injection of a 6 mM (2mg/ml) dose via the tail-vein. There are no significant differences in maximum 2-NBDG-fluorescence between the different fasting groups, confirming that the delivery of 2-NBDG is similar across all animal groups. <b>B</b>. 2-NBDG₆₀ is significantly higher in mice fasted for 6 hours and 12 hours compared with mice that were not fasted (p = 0.02). There are no significant differences between the 6 and 12-hour fasting groups. <b>C</b>. Blood glucose measurements were performed using a Freestyle Lite monitor by drawing 3 μl of blood from the tail of a separate cohort of mice (n = 5). Fasting for 6 hours led to a significant decrease in blood glucose levels. However, fasting for 12 hours did not lead to any further decrease in blood glucose levels. Statistical analysis was conducted using Wilcoxon rank sum tests.</p

Intrinsic tissue fluorescence corrected for absorption and scattering improves 2-NBDG contrast between 4T1 tumors and normal tissue.

<p><b>A</b>. Measured 2-NBDG₆₀ from the 4T1 and 4T07 tumors is distorted by hemoglobin absorption, and is on par with fluorescence from normal tissue. A normal tissue data-point and representative tumors with similar measured fluorescence values were selected to illustrate the effect of correction. <b>B</b>. Normalized spectra of measured 2-NBDG₆₀ illustrate the distortion in better detail. <b>C</b>. Measured 2-NBDG₆₀ is not significantly different between the different groups. <b>D</b>. Correction with the MC fluorescence model removes hemoglobin-induced distortions and improves contrast between normal and tumor. <b>E</b>. Corrected 2-NBDG₆₀ spectra from normal tissue, a 4T1 tumor, and a 4T07 tumor shown in 2D, normalized to their respective maxima are presented along with a true 2-NBDG fluorescence measurement, illustrating good agreement between the extracted in vivo spectral line shapes and native 2-NBDG. <b>F</b>. Corrected 2-NBDG₆₀ is significantly higher in 4T1 tumors compared with normal tissue (p = 0.02). Although mean 2-NBDG₆₀ in 4T07 tumors is higher compared with normal tissue, this is not statistically significant. <b>G</b>. The extracellular acidification rate (ECAR) of 4T1 and 4T07 cells, as calculated with a Seahorse Glycolysis stress test, is not significantly different. Data represent n = 12 cell samples from 3 distinct assays. Error bars represent standard error of the mean.</p

Flowchart illustrating the working of the MC reflectance and fluorescence models to extract optical properties and distortion-free fluorescence from tissue.

<p>μ_a(λ) and μ_s(λ) refers to the absorption and scattering coefficients, respectively.</p

The ratio 2-NBDG/R_D facilitates assessment of glucose demand in heterogeneous regions of metastatic mammary tumors.

<p>(A) Representative images of vascular oxygenation (SO₂) and delivery-corrected 2-NBDG (2-NBDG₆₀/R_D) 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, R_D, and 2-NBDG₆₀/R_D for regions of distinct SO₂ (%) in 4T07 and 4T1 tumors. For 4T1, 2-NBDG₆₀ is lower for 02,4T1<10 regions than for any other SO_2,4T1 (p = N.S.). Significantly lower rates of R_D are seen for the 02,4T1<10 group than for well-oxygenated 4T1 regions (p<0.05 or p<0.01 for 02,4T1<10 vs. 202,4T1<40 or 402,4T1<60, respectively). After correction for low R_D, 2-NBDG₆₀/R_D increased slightly but significantly in hypoxic regions (p<0.01 for 02,4T1<10 vs. 402,4T1<60). For 4T07, 2-NBDG uptake for the highest SO_2,4T07 regions decreased compared to the lowest SO_2,4T07 (p<0.01 for all 202,4T07<40 vs. 602,4T1<80). R_D is indistinguishable between SO_2,4T07 levels. After correction by R_D, 2-NBDG₆₀/R_D is lowest for 602,4T07<80 (p<0.01). Comparison between 4T1 and 4T07 shows that 2-NBDG₆₀ is higher for all SO_2,4T1 than all SO_2,4T07 (p<0.01). On the other hand, R_D for the best oxygenated 4T07 groups (402,4T07<60 and 602,4T07<80) is greater than for all 4T1 groups (p<0.01 for all groups except 402,4T1<60 vs. 602,4T07<80 where p<0.06). After correction by R_D, 2-NBDG₆₀/R_D is higher for all SO_2,4T1 than all SO_2,4T07 (p<0.01 for all SO_2,4T1 compared to all SO_2,4T07). Number of mice per group indicated by group name in legend.</p

The rate of 2-NBDG delivery, R_D, is strongly correlated with blood velocity.

<p>(A) Representative images of blood velocity and the rate of 2-NBDG delivery (R_D) 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 R_D increased significantly (p<0.02 for both). N = 6 mice. (C) The rate of 2-NBDG delivery (R_D) is highly correlated with blood velocity (R = 0.87, p<0.05). The trendline corresponds to the trend for post-hypoxia data only.</p

Delivery-corrected 2-NBDG uptake inversely correlates with blood glucose concentration.

<p>(A) Representative images show the kinetics of 2-NBDG uptake <i>in vivo</i> 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-NBDG_{05,10 mM}/2-NBDG_{05,6 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-NBDG_{05,10 mM}/2-NBDG_{05,6 mM} corresponds to the ratio of 10 mM and 6 mM fluorescence intensities at t = 5 min. The ratio R_{D,10 mM}/R_{D,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) R_D was strongly correlated with 2-NBDG fluorescence at 5 minutes (p<0.001). R_D did not correlate with 2-NBDG₆₀ (not shown). (D) 2-NBDG₆₀/R_D 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/R_D) 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).</p

Delivery-corrected glucose uptake reveals distinct glycolytic phenotypes in metastatic (4T1) and non-metastatic (4T07) mammary tumors.

<p>(A) Representative images of vascular oxygen saturation (SO₂) and delivery-corrected 2-NBDG (2-NBDG₆₀/R_D) for a 4T1 tumor and a 4T07 tumor, <i>in vivo</i>. (B) 2-NBDG₆₀/R_D showed contrast in glucose uptake between metastatic 4T1 and non-metastatic 4T07 tumors <i>in vivo</i> (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 25^th and 75^th percentiles, and scatter points show all data values.</p

Relationship between SO₂, R_D, R_C and 2-NBDG₆₀ for 4T1 and 4T07 tumors.

<p><b>A.</b> Illustration of 2-NBDG uptake curve indicating how R_D, R_C and 2-NBDG₆₀ are calculated. <b>B.</b> Contour plots showing the relationship between delivery, clearance and uptake of 2-NBDG. R_D and R_C 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 R_D at low values of R_C. At higher values of R_C, 2-NBDG₆₀ reaches a maximum at R_D = 2.5 s⁻¹, levels off and then declines gradually for increasing R_D. After hypoxia, a secondary maximum is seen at very high values of R_D (R_D>6 s⁻¹) and low R_C. In the 4T07 tumors, 2-NBDG₆₀ increases with R_D and reaches a maximum at approximately 6 s⁻¹. The same feature is also present after hypoxia. At higher values of R_C, 2-NBDG₆₀ was nearly negligible for increasing values of R_C.</p

A summary of possible relationships between NBDG₆₀, R_D, R_C, and SO₂ and final outcome corresponding to each combination.

<p>A summary of possible relationships between NBDG₆₀, R_D, R_C, and SO₂ and final outcome corresponding to each combination.</p