Delivery rate affects uptake of a fluorescent glucose analog in murine metastatic breast cancer.

We demonstrate an optical strategy using intravital microscopy of dorsal skin flap window chamber models to image glucose uptake and vascular oxygenation in vivo. Glucose uptake was imaged using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). SO2 was imaged using the differential absorption properties of oxygenated [HbO2] and deoxygenated hemoglobin [dHb]. This study was carried out on two sibling murine mammary adenocarcinoma lines, 4T1 and 4T07. 2-NBDG uptake in the 4T1 tumors was lowest when rates of delivery and clearance were lowest, indicating perfusion-limited uptake in poorly oxygenated tumor regions. For increasing rates of delivery that were still lower than the glucose consumption rate (as measured in vitro), both 2-NBDG uptake and the clearance rate from the tumor increased. When the rate of delivery of 2-NBDG exceeded the glucose consumption rate, 2-NBDG uptake decreased with any further increase in rate of delivery, but the clearance rate continued to increase. This inflection point was not observed in the 4T07 tumors due to an absence of low delivery rates close to the glucose consumption rate. In the 4T07 tumors, 2-NBDG uptake increased with increasing rates of delivery at low rates of clearance. Our results demonstrate that 2-NBDG uptake in tumors is influenced by the rates of delivery and clearance of the tracer. The rates of delivery and clearance are, in turn, dependent on vascular oxygenation of the tumors. Knowledge of the kinetics of tracer uptake as well as vascular oxygenation is essential to make an informed assessment of glucose demand of a tumor

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

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

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

Outline of methods.

<p>(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 (R_D = 2-NBDG₆₀/T_max) 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.</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