A Multiplexed, Multi-scale Optical Imaging Platform to Quantify Tumor Metabolic Heterogeneity

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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.





Deutsch, Riley Joseph (2023). A Multiplexed, Multi-scale Optical Imaging Platform to Quantify Tumor Metabolic Heterogeneity. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/29124.


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