Browsing by Subject "Molecular 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.

Over the past 200 years, the Earth's atmospheric carbon dioxide (CO₂) concentration has increased by more than 35%, and climate experts predict that CO₂ levels may double by the end of this century. Understanding the mechanisms of resource management in plants is fundamental for predicting how plants will respond to the increase in atmospheric CO₂. Plant productivity sustains life on Earth and is a principal component of the planet's system that regulates atmospheric CO₂ concentration. As such, one of the central goals of plant science is to understand the regulatory mechanisms of plant growth in a changing environment. Short-lived positron-emitting radiotracer techniques provide time-dependent data that are critical for developing models of metabolite transport and resource distribution in plants and their microenvironments. To better understand the effects of environmental changes on resource transport and allocation in plants, we have developed a system for real-time measurements of metabolite transport in plants using short-lived positron-emitting radiotracers. This thesis project includes the design, construction, and demonstration of the capabilities of this system for performing real-time measurements of metabolite transport in plants.

The short-lived radiotracer system described in this dissertation takes advantage of the combined capabilities and close proximity of two research facilities at Duke University: the Triangle Universities Nuclear Laboratory (TUNL) and the Duke University Phytotron, which are separated by approximately 100 meters. The short-lived positron-emitting radioisotopes are generated using the 10-MV tandem Van de Graaff accelerator located in the main TUNL building, which provides the capability of producing short-lived positron-emitting isotopes such as carbon-11 (¹¹C; 20 minute half-life), nitrogen-13 (¹³N; 10 minute half-life), fluorine-18 (¹⁸F; 110 minute half-life), and oxygen-15 (¹⁵O; 2 minute half-life). The radioisotopes may be introduced to plants as biologically active molecules such as ¹¹CO₂, ¹³NO₃^-, ¹⁸F^--[H₂O], and H₂^{15<\sup>O. Plants for these studies are grown in controlled-environment chambers at the Phytotron. The chambers offer an array of control for temperature, humidity, atmospheric CO₂ concentration, and light intensity. Additionally, the Phytotron houses one large reach-in growth chamber that is dedicated to this project for radioisotope labeling measurements.}

There are several important properties of short-lived positron-emitting radiotracers that make them well suited for use in investigating metabolite transport in plants. First, because the molecular mass of a radioisotope-tagged compound is only minutely different from the corresponding stable compound, radiotracer substances should be metabolized and transported in plants the same as their non-radioactive counterparts. Second, because the relatively high energy gamma rays emitted from electron-positron annihilation are attenuated very little by plant tissue, the real-time distribution of a radiotracer can be measured in vivo in plants. Finally, the short radioactive half-lives of these isotopes allow for repeat measurements on the same plant in a short period of time. For example, in studies of short-term environmental changes on plant metabolite dynamics, a single plant can be labeled multiple times to measure its responses to different environmental conditions. Also, different short-lived radiotracers can be applied to the same plant over a short period of time to investigate the transport and allocation of various metabolites.

This newly developed system provides the capabilities for production of ¹¹CO₂ at TUNL, transfer of the ¹¹CO₂ gas from the target area at TUNL to a radiation-shielded cryogenic trap at the Phytotron, labeling of photoassimilates with ¹¹C, and in vivo gamma-ray detection for real-time measurements of the radiotracer distribution in small plants. The experimental techniques and instrumentation that enabled the quantitative biological studies reported in this thesis were developed through a series of experiments made at TUNL and the Phytotron. Collimated single detectors and coincidence counting techniques were used to monitor the radiotracer distribution on a coarse spatial scale. Additionally, a prototype Versatile Imager for Positron Emitting Radiotracers (VIPER) was built to provide the capability of measuring radiotracer distributions in plants with high spatial resolution (~2.5 mm). This device enables detailed quantification of real-time metabolite dynamics on fine spatial scales.

The full capabilities of this radiotracer system were utilized in an investigation of the effects of elevated atmospheric CO₂ concentration and root nutrient availability on the transport and allocation of recently fixed carbon, including that released from the roots via exudation or respiration, in two grass species. The ¹¹CO₂ gas was introduced to a leaf on the plants grown at either ambient or elevated atmospheric CO₂. Two sequential measurements were performed per day on each plant: a control nutrient solution labeling immediately followed by labeling with a 10-fold increase or decrease in nutrient concentration. The real-time distribution of ¹¹C-labeled photoassimilate was measured in vivo throughout the plant and root environment. This measurement resulted in the first observation of a rapid plant response to short-term changes in nutrient availability via correlated changes in the photoassimilate allocation to root exudates. Our data indicated that root exudation was consistently enhanced at lower nutrient concentrations. Also, we found that elevated atmospheric CO₂ increased the velocity of photoassimilate transport throughout the plant, enhanced root exudation in an annual crop grass, and reduced root exudation in a perennial native grass.

X-ray computed tomography (CT) is one of the most useful diagnostic tools for clinicians, with widespread availability, fast scan times, and low cost. CT imaging can reveal a patient’s anatomy in exquisite detail and is extremely useful in the diagnosis of a wide variety of diseases. However, CT is currently limited to anatomical imaging due to the lack of appropriate contrast agents and imaging protocols that would allow for molecular imaging, so clinicians must instead rely on other modalities which are more expensive and less readily available. Dual energy CT, a relatively new technique in which two x-ray energies are used for a single scan, can provide valuable information about tissue material composition. This information can potentially be used for molecular imaging if it is coupled with appropriately-designed contrast agents.

This work aims to extend the use of CT into the molecular imaging realm by developing and testing nanoparticle contrast agents for use with dual energy CT. Several studies were carried out, each of which focused on a different application of using nanoparticle contrast agents together with dual energy CT for molecular imaging.

A commercial blood pool iodine contrast agent for pre-clinical CT (Exia-160) has been shown to accumulate in the myocardium and continue to enhance the myocardium after the contrast agent has been cleared from the bloodstream. It was hypothesized that this agent would not accumulate in infarcted myocardium, which would allow for specific identification of myocardial infarction by CT. Mice were injected with the contrast agent following myocardial infarction, and dual energy CT was used to identify the iodine within the myocardium and separate the iodine from the calcium in the neighboring ribs. Regions of myocardial infarction showed no enhancement on CT, while the healthy myocardium was highly enhanced. Size and position of myocardial infarction determined by dual energy CT were compared with the standard molecular imaging technique for measuring myocardial viability (SPECT). It was found that dual energy CT using this nanoparticle contrast agent reliably agreed with the gold standard molecular imaging method.

Molecular imaging for the improved detection and characterization of lung tumors was also explored through two different studies. The first study used both gold nanoparticles and iodine-containing liposomes together with dual energy CT in order to measure tumor vascular functional parameters, including tumor fractional blood volume and vascular permeability. These dual energy CT measurements were confirmed with ex vivo tissue analysis to demonstrate the validity and accuracy of the in vivo dual energy CT method. The second study used antibody-targeted gold nanoparticles to image EGFR-positive tumors. Two different types of antibodies were tested: a clinically used humanized anti-EGFR antibody, and a small llama-derived single domain anti-EGFR antibody. The single domain antibody showed improved blood half-life and reduced immune clearance compared to the full-sized antibody when attached to gold nanoparticles, but the higher affinity of the full-sized antibody led to much higher overall tumor accumulation. This antibody significantly increased the accumulation of gold nanoparticles in tumors expressing high levels of EGFR. Together, these two studies showed that dual energy CT and nanoparticle contrast agents can be used to measure a wide variety of tumor functional parameters, including tumor vascular density, vascular permeability, and receptor expression. All these parameters can be combined with the anatomical CT imaging to better characterize lung tumors and differentiate between benign and malignant lesions.

The use of dual energy CT for measuring tumor vascular permeability changes after gold nanoparticle-augmented radiation therapy was also demonstrated. Liposomal iodine was injected into mice following radiation therapy in order to measure vascular permeability. Dual energy CT was used to differentiate the signal of the liposomal iodine from the CT signal of the gold nanoparticles already in the tumor. Tumor permeability was measured with CT using multiple combinations of gold nanoparticles and radiation doses to find the optimal conditions for enhancing the effect of radiation therapy on the vasculature. These conditions were then used to increase the delivery of a liposomal chemotherapy agent to tumors. Tumors treated with the gold-augmented radiation therapy and liposomal drug showed significant growth delay compared to the other groups, confirming the predictions made in the dual energy CT imaging.

Finally, a protease-responsive contrast agent was developed for use with dual energy CT imaging. Clusters of gold nanoparticles cross-linked together by protease-sensitive peptides were injected into mice along with liposomal iodine. In the presence of tumor proteases, the clusters degraded and the concentration of gold within the tumor decreased. Clusters without the protease-sensitive peptide did not degrade and did not leave the tumors. The ratio of iodine to gold in each tumor was measured, and it was found that the ratio was significantly higher in mice injected with the degradable gold clusters compared to mice injected with non-degradable control clusters. This demonstrated the ability to use multiple contrast agents with dual energy CT for enzyme-specific ratiometric molecular imaging.

This work confirms that dual energy CT can be used along with multiple nanoparticle contrast agents for molecular imaging applications. With continued contrast agent development and further application of dual energy CT, these methods can potentially be applied clinically to improve the power of CT imaging and improve diagnosis of a wide variety of pathologies.

Hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI) enables non-invasive visualization of metabolism and physiological activities in real-time. Hyperpolarized agents developed to date are primarily 13C-labeled metabolites with short polarization lifetimes of less than a minute, limiting the imaging assay to fast metabolic pathways. To expand on the applications of HP-MRSI, we proposed the use of a versatile 15N-molecular tagging strategy.

This dissertation reports our exploration and application of highly adaptable 15N-molecular handles for development of hyperpolarized molecular imaging probes. Towards this goal, we have investigated 15N2-diazirines and 15N3-azides as biocompatible HP tags with long polarization lifetimes. Several 15N-tagged biological molecules were prepared, including amino acid, glucose, and drug molecules. Hyperpolarization with a d-DNP method demonstrated high signal enhancements (over 400,000-fold) and long 15N relaxation lifetimes (T1) of average 3–4 minutes in aqueous solutions, which warrants a long MR imaging window.

Moreover, we have rationally designed and synthesized novel 15N-labeled reaction-based probes for sensing hydrogen peroxide (H2O2) and nitric oxide (NO) as biomarkers for oxidative stress. We were able to observe the 15N-signal from our 15N-labeled H2O2 sensing probe in vivo using an animal model, which presents exciting progress in the field. Additionally, we explored various molecular probe designs to pursue the most practical 15N-labeled gamma-glutamyl transferase (GGT) sensor. These reaction-based redox and enzyme sensing probes showed favorable hyperpolarization and bioimaging properties. Our work on innovative de novo chemical probes highlights the unprecedented HP-MRSI applications for imaging disease biomarkers.