Metaboloptics: In Vivo Optical Imaging to Enable Simultaneous Measurement of Glucose Uptake, Mitochondrial Membrane Potential, and Vascular Features in Cancer
Altered metabolism is a hallmark of almost all cancers. A tumor’s metabolic phenotype can drastically change its ability to proliferate and to survive stressors such as hypoxia or therapy. Metabolism can be used as a diagnostic marker, by differentiating neoplastic and normal tissue, and as a prognostic marker, by providing information about tumor metastatic potential. Metabolism can further be used to guide treatment selection and monitoring, as cancer treatments can influence metabolism directly by targeting a specific metabolic dysfunction or indirectly by altering upstream signaling pathways. Repeated measurement of metabolic changes during the course of treatment can therefore indicate a tumor’s response or resistance. Recently, well-supported theories indicate that the ability to modulate metabolic phenotype underpins some cancer cells’ ability to remain dormant for decades and recur with an aggressive phenotype. It follows that accurate identification and repeated monitoring of a tumor’s metabolic phenotype can bolster understanding and prediction of a tumor’s behavior from diagnosis, through treatment, and (sadly) sometimes back again.
The two primary axes of metabolism are glycolysis and mitochondrial metabolism (OXPHOS), and alteration of either can promote unwanted outcomes in cancer. In particular, increased glucose uptake independent of oxygenation is a well-known mark of aggressive cancers that are more likely to metastasize and evade certain therapies. Lately, mitochondria are also gaining recognition as key contributors in tumor metabolism, and mitochondrial metabolism has been shown to promote metastasis in a variety of cell types. Most tumor types rely on a combination of both aerobic glycolysis and mitochondrial metabolism, but the two axes’ relative contributions to ATP production can vary wildly. Knowledge of both glycolytic and mitochondrial endpoints is required for actionable, systems-level understanding of tumor metabolic preference.
Several technologies exist that can measure endpoints informing on glycolytic and/or mitochondrial metabolism. However, these technologies suffer from a combination of prohibitive cost, low resolution, and lack of repeatability due to destructive sample treatments.
There is a critical need to bridge the gap in pre-clinical studies between single-endpoint whole body imaging and destructive ex vivo assays that provide multiple metabolic properties, neither of which can provide adequate spatiotemporal information for repeated tumor monitoring. Optical technologies are well-suited to non-destructive, high resolution imaging of tumor metabolism. A carefully chosen set of endpoints can be measured across a variety of length scales and resolutions to obtain a complete picture of a tumor’s metabolic state. First, the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) can be used to report on glucose uptake. The fluorophore tetramethylrhodamine, ethyl ester (TMRE) reports on mitochondrial membrane potential, which provides information regarding capacity for oxidative phosphorylation. Vascular oxygenation (SO2) and morphological features, which are critical for interpretation of 2-NBDG and TMRE uptake, can be obtained using only endogenous contrast from hemoglobin.
Three specific aims were proposed toward the ultimate goal of developing an optical imaging toolbox that utilizes exogenous fluorescence and endogenous absorption contrast to characterize cancer metabolic phenotype in vivo.
In Aim 1, we optimized the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) to report on glycolytic demand in vivo. Our primary goal was to demonstrate that correcting 2-NBDG uptake (NBDG60) by the rate of delivery (RD) showed improved contrast between distinct tumor phenotypes. We showed that the ratio 2-NBDG60/RD served as a delivery-corrected measure of glucose uptake in the dorsal skin flap window chamber models containing normal tissues and tumors. Delivery correction was able to minimize the effects of a large change in the injected 2-NBDG dose. Further, the endpoint showed a significant inverse correlation with blood glucose levels. Since glucose has been shown to competitively inhibit 2-NBDG transport into cells, this finding indicating that we were indeed reporting on glucose uptake. Importantly, the ratio was able to distinguish specific uptake of 2-NBDG from accumulation of a fluorescent control, 2-NBDLG, which is identical to 2-NBDG in molecular weight and fluorescent spectrum, but is unable to undergo active transport into the cell.
The ratio 2-NBDG60/RD was then leveraged to compare different tumor phenotypes and to characterize the dependence of glucose uptake on vascular oxygenation within these tumors. Our results showed that 2-NBDG60/RD was an effective endpoint for comparing in vivo glucose uptake of metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. Further, the addition of vascular information revealed metabolic heterogeneity within the tumors. The primary conclusion of Aim 1 was that delivery-corrected 2-NBDG uptake (2-NBDG60/RD) is an appropriate indicator of glucose demand in both normal and tumor tissues.
In Aim 2, we optimized fluorescent tetramethyl rhodamine, ethyl ester (TMRE) for measurement of mitochondrial membrane potential (MMP). We then leveraged the relationships between MMP, glucose uptake, and vascular endpoints to characterize the in vivo metabolic landscapes of three distinct and extensively studied murine breast cancer lines: metastatic 4T1 and non-metastatic 67NR and 4T07.
Using two-photon microscopy, we confirmed that TMRE localizes to mitochondrial-sized features in the window chamber when delivered via tail vein. The kinetics of TMRE uptake were robust across both normal and tumor tissues, with a stable temporal window for measurement from 40-75 minutes after injection. We saw that TMRE uptake decreased as expected in response to hypoxia in non-tumor tissue, and in response to chemical inhibition with a mitochondrial uncoupler in both non-tumor and 4T1 tissue. MMP was increased in all tumor types relative to non-tumor (p<0.05), giving further confirmation that TMRE was reporting on mitochondrial activity.
We leveraged the relationships between the now-optimized endpoints of MMP (Aim 2), glucose uptake (Aim 1) and vascular endpoints (Aims 1 and 2) to characterize the in vivo metabolic landscapes of three distinct and extensively studied murine breast cancer lines: metastatic 4T1 and non-metastatic 67NR and 4T07. Imaging the combination of endpoints revealed a classic “Warburg effect” coupled with hyperpolarized mitochondria in 4T1; 4T1 maintained vastly increased glucose uptake and comparable MMP relative to 4T07 or 67NR across all SO2. We also showed that imaging trends were concordant with independent metabolomics analysis, though the lack of spatial and vascular data from metabolomics obscured a more detailed comparison of the technologies.
We observed that vascular features in tumor peritumoral areas (PA) were equally or more aberrant than vessels in the tumor regions that they neighbored. This prompted consideration of the metabolic phenotype of the PA. Regional metabolic cooperation between the tumor region and the PA was seen only in 4T1, where MMP was greater in 4T1 tumors and glucose uptake was greater in 4T1 PAs.
Because of their regional metabolic coupling as well as their demonstrated capacity for glycolysis and mitochondrial activity, we hypothesized that the 4T1 tumors would have an increased ability to maintain robust MMP during hypoxia. 67NR and 4T07 tumors showed expected shifts toward decreased MMP and increased glucose uptake during hypoxia, similar to the trends we observed in normal tissue. Surprisingly, 4T1 tumors appeared to increase mitochondrial metabolism during hypoxia, since MMP increased and SO2 dramatically decreased. Overall, this aim demonstrated two key findings: 1. TMRE is a suitable marker of mitochondrial membrane potential in vivo in normal tissue and tumors, and 2. imaging of multiple metabolic and vascular endpoints is crucial for the appropriate interpretation of a metabolic behavior.
Finally, in Aim 3 we evaluated the feasibility of combined 2-NBDG and TMRE imaging. The primary objective was to enable simultaneous imaging of the two fluorophores by minimizing sources of “cross-talk”: chemical reaction, optical overlap, and confounding biological effects. A secondary objective was to transition our imaging method to a new platform, a reflectance-mode, high-resolution fluorescence imaging system built in our lab, which would expand the use of our technique beyond the dorsal window chamber model. We first used liquid chromatography- mass spectrometry to confirm that the chemical properties of the two fluorophores were compatible for simultaneous use, and indeed saw that the mixing of equimolar 2-NBDG and TMRE did not form any new chemical species.
We also performed a phantom study on the hyperspectral imaging system, used for all animal imaging in Aim 1 and Aim 2, to estimate the range of 2-NBDG and TMRE concentrations that are seen at the tissue level in normal and tumor window chambers. We created a new phantom set that spanned the range of estimated in vivo concentrations, and imaged them with the reflectance-mode fluorescence imaging system. The phantom experiments gave us two important findings. First, we saw that fluorescence intensity increased linearly with fluorophore concentration, allowing for accurate quantification of concentration changes between samples. Most importantly, we found that we could exploit the optical properties of the fluorophores and our system’s spectral detection capability to excite the two fluorophores independently. Specifically, we could excite 2-NBDG with a 488nm laser without detectable emission from TMRE, and could excite TMRE with a 555nm laser without detectable emission from 2-NBDG. With this characterization, the optical properties of the two fluorophores were considered compatible for simultaneous imaging.
Next, we sought to determine whether biological or delivery interactions would affect uptake of the two fluorophores. Surprisingly, both in vitro and in vivo imaging suggested that simultaneous dosing of the 2-NBDG and TMRE caused significant changes in uptake of both probes. Since we previously found that TMRE equilibrates rapidly at the tissue site, we hypothesized that staggering the injections to allow delivery of TMRE to tissue before injecting 2-NBDG would restore the full uptake of both fluorophores. Two sequential injection protocols were used: in the first group, TMRE was injected first followed by injection of 2-NBDG after only 1-5 minutes, and in the second group, TMRE was injected first followed by injection of 2-NBDG after 10-15 minutes. Both sequential injection strategies were sufficient to restore the final fluorescence of both fluorophores to that seen in the separate TMRE or 2-NBDG imaging cohorts; however, the shorter time delay caused changes to the initial delivery kinetics of 2-NBDG. We concluded that sequential imaging of TMRE followed by 2-NBDG with a 10-15 minute delay was therefore the optimal imaging strategy to enable simultaneous quantification of glucose uptake and mitochondrial membrane potential in vivo.
Applying the sequential imaging protocol to 4T1 tumors demonstrated a highly glycolytic phenotype compared to the normal animals, as we had seen in Aim 2. However, mitochondrial membrane potential was comparable for the normal and tumor groups. The next study will test an extended delay between the injections to allow more time for TMRE delivery to tumors prior to 2-NBDG injection. Overall, the key finding of Aim 3 was that a carefully chosen delivery strategy for 2-NBDG and TMRE enabled simultaneous imaging of the two endpoints, since chemical and optical cross-talk were negligible.
The work presented here indicates that an optical toolbox of 2-NBDG, TMRE, and vascular endpoints is well poised to reveal interesting and distinct metabolic phenomena in normal tissue and tumors. Future work will focus on the integration of optical spectroscopy with the microscopy toolbox presented here, to enable long-term studies of the unknown metabolic changes underlying a tumor’s response to therapy, its escape into dormancy, and ultimately, its recurrence.
Mitochondrial membrane potential
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