Browsing by Subject "Fluorescence Imaging"

The American Cancer Society reported an estimated 300,000 new cases of breast cancer and 44,000 new breast-cancer related deaths in 2022 in the United States alone. With each new successfully treated primary tumor, there is a subsequent risk of disease recurrence. Recurrence poses a risk to 10% of patients within the first 5 years post treatment and a lifetime risk of 30% across all patients. While new tools are being developed to better understand and mitigate the risk of recurrence, triple negative breast cancers, which exhibit no targetable surface markers, offer little in the way of recurrence prediction or treatment. It is understood that tumor heterogeneity is a driving force in tumor recurrence. Temporal heterogeneity is associated with therapeutic treatment, where the administration either selects for resistance subpopulations of tumor cells that are able to recur or a de novo resistant phenotype arises that leads to recurrence. Additionally, it has been well documented that tumors vary spatially across a primary tumor. This heterogeneity takes the form of genetic, epigenetic, and phenotypic heterogeneity. One such phenotype of interest is metabolic heterogeneity. Metabolism is classified as a ‘Hallmark of Cancer’ and has been studied as a driver of tumor progression for almost a century since Otto Warburg first described the phenomenon of tumors exhibiting high rates of aerobic glycolysis. Optical imaging is well poised to study metabolic heterogeneity due to its ability to image cellular level features, to multiplex multiple endpoints, and the ability to image longitudinally. Endogenous fluorescence contrast of coenzymes NADH and FAD have been used to report on the redox state of in vivo tissue and distinguish cancerous from benign lesions. The Center for Global Women’s Health Technologies (GWHT) has employed the use of exogenous fluorescent contrast agents to provide substrate-specific metabolic information. Three fluorescent agents have been validated including: 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), a glucose derivative that is able to report on glycolysis; Tetramethylrhodamine ethyl ester (TMRE), a cation that is selectively attracted to the charge gradient generated by the mitochondria during ATP synthesis, making it a reporter of OXPHOS; and Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid (Bodipy FL C16), a long chain saturated fatty acid is taken up by the cell and undergoes beta oxidation similar to native fatty acids. More recently, GWHT has begun combing these fluorescence agents for in vivo use to provide a wholistic understanding of cancer metabolism. The work here sets out to develop a novel optical imaging platform that is capable of imaging multiplexed metabolic endpoints, for quantitative intra-image analysis of metabolic gradients. This technology is built on the use of exogenous fluorescence contrast agents to report on substrate or pathway specific axes of metabolism. By simultaneously introducing multiple contrast agents, it is possible to capture a more wholistic snapshot of tissue metabolism. To encourage the adoption of this technology, a novel low-cost instrument will also be developed. Leveraging a consumer grade CMOS camera and variable focus lens, it is possible to image over multiple length scales, capturing both bulk tumor features and also single cell features. The flexibility offered by this simple innovation will allow for metabolic imaging to be applied over a variety sample type. Three specific aims were proposed to realize this goal by developing methods of multi-parametric exogenous contrast and low-cost instrumentation for multi-scale imaging of tumor metabolic heterogeneity in preclinical models. Aim 1 validated and demonstrated a method for the simultaneous injection and measurement of Bodipy FL C16 and TMRE to report on lipid uptake and mitochondrial activity, two potentially interrelated axes of metabolism. To validate this method, three sets of experiments were performed to establish that the two probes do not exhibit chemical, optical, or biological crosstalk. Chemical compatibility was established using liquid chromatography. Briefly, high molar concentration solutions of each individual probe (Bodipy FL C16, TMRE, and 2-NBDG) were created alongside a solution of all three probes at the same concentration. Chromatograms were collected immediately upon mixing, after 1 hour and after 24 hours. The area under the curve for each probe at each time point displayed an area under the curve (AUC) within 2% of the AUC of the single probe solutions, suggesting no chemical reactions. Optical crosstalk was assessed using optical spectroscopy and tissue mimicking phantoms. Optical phantoms were created with tissue mimicking optical properties and various concentration of Bodipy FL C16, TMRE, polystyrene microspheres (tissue scattering mimic), and hemoglobin (tissue absorption mimic). Leveraging an inverse Monte Carlo algorithm, we demonstrated that accurate values for each fluorescent probe could be measured regardless of the concentration of the other optical probe or level of optical scattering or absorption, indicating optical compatibility. To address biological crosstalk, two sets of 4T1 tumor bearing mice were subject to optical spectroscopy with either 1) Bodipy FL C16 alone, 2) TMRE alone, 3) a dual injection of Bodipy FL C16 and TMRE. Fluorescence spectra were measured 2-, 4-, 6-, 8-, 10-, 20-, 30-, 40-, 50-, and 60-minutes post-injection to establish uptake kinetics. It was found that the uptake kinetics of the dual probe group were not statistically different from the single probe group, indicating biological compatibility. With no observable crosstalk between Bodipy FL C16 and TMRE, the two probes method was applied to characterize murine mammary gland and two tumor of differing metastatic potential (4T1 and 67NR). In addition, to Bodipy FL C16 and TMRE, oxygen saturation and total hemoglobin were extracted from estimates of optical absorption, and these 4 endpoints were used to attempt to cluster groups of tumor and normal tissue. Difficulty clustering tumor groups of varying metastatic potential suggest a need for imaging technology. In Aim 2 a low-cost fluorescence microscope was developed capable of performing quantitative fluorescence imaging over a variety of samples. The goal of this work was to design a system that could be adapted to image a number of different sample types include core-needle tissue biopsies, preclinical window chambers, and in vitro organoids. To accomplish this a low-cost CMOS detector was used with a variable magnification lens allowing for imaging at multiple length scales. Uniform illumination was a necessary criterion for quantitative imaging. To generate uniform illumination that could be scaled across multiple length scales, an LED coupled 1:4 fanout optical fiber was employed alongside a computational model to determine the positioning of each fiber. To automate the design of illumination, a computational model was employed where each optical fiber was modeled as a Lambertian emitter in a spherical coordinate system. To determine the ideal placement of each fiber such that the individual illumination contributions of all fibers summed to a uniform distribution, a global optimizer was employed. A genetic pattern search allowed for the selection of coordinates to produce uniform illumination that could be feasibly employed at the benchtop. This integrated system is referred to as the CapCell microscope. Using this computational approach, two uniform illumination profiles were designed, one with a high aspect ratio (length ≫ width) and one with a low aspect ratio (length = width). To demonstrate the utility of optimized illumination, core needle biopsies from 4T1 tumors were stained with a tumor-specific fluorescent contrast agent, HS-27 and imaged with either optimized or unoptimized gaussian illumination. The repeatability of intra-image features was compared for the two illumination scenarios, and it was found that uniform illumination repeatedly revealed the same fluorescent features across the sample. These features were further confirmed with standard histology. Window chamber imaging demonstrated the importance of designing application specific illumination. 4T1 mammary tumors were grown orthotopically before a window chamber was surgically implanted. Animals were injected with either Bodipy FL C16, 2-NBDG, or HS-27 and imaged with both the high AR and low AR illumination platforms. As expected, the low AR, designed for window chambers, had a higher power density at the sample site and thus increased contrast compared to the low AR images. With a method and a system in place, the goal of Aim 3 was to apply the optical imaging platform to observe spatiotemporal metabolic heterogeneity. To achieve this, the CapCell microscope was upgraded to enhance contrast and improve resolution for the visualization of capillaries and single cells. This was demonstrated using 4T1 window chamber models stained with acridine orange, a nucleus specific stain, and green light reflectance to highlight hemoglobin absorption in microvessels. Given the interplay between metabolism and vasculature it was desirable to employ a vessel segmentation approach to describe vascular features within an image. A Gabor filter and Djikstra segmentation approach was employed on metabolic images to enable metabolic and vascular comparisons across an image field of view. To test the improved CapCell system, 4T1 tumors were treated with combretastatin A-1, a vascular disrupting agent. Across the course of treatment, the CapCell was able to observe bulk changes in metabolism and vascular density. Additionally, by employing high resolution imaging, it was possible to observe relationships between each metabolic probe and vessel tortuosity. This analysis allowed for the identification of metabolically unique regions within each group of animals, demonstrating the ability of this technology to parse metabolically distinct regions of tumor. In total, the work outlined here describes the development of a novel optical imaging platform capable of quantifying intratumor metabolic heterogeneity of multiple metabolic endpoints over multiple length scales. The system expands on previous work developing methods for simultaneous measurement of exogenous fluorescent contrast agents to report on lipid uptake and mitochondrial activity. The system also introduced a novel computational approach to design uniform illumination for a low-cost microscope capable of imaging across multiple sample types. Together these technologies were used to observe metabolic heterogeneity in preclinical window chamber models following chemical perturbation. The technology introduced here, is primed for future exploration. First, it would be desirable to integrate all three exogenous contrast agents for simultaneous imaging of three axes of metabolism in vivo. Once accomplished, the sample technology could be applied to study metabolic and vascular changes associated with residual disease and tumors that are entering recurrence.

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.