It is becoming increasingly apparent that cell populations are heterogeneous in their functions, disease states and response to therapy. Tumor heterogeneity is one of the main factors contributing to acquire drug resistance. Substantial interest is now devoted to characterization methods that operate at the single-cell level, as opposed to bulk analyses that can only measure average properties over a given population. Fluorescence methods have long been used to measure molecular processes in single living cells. However, a vast number of small molecules remain invisible to fluorescence probing. These molecules lack inherent fluorescence and cannot be fluorescently labeled without altering their biochemical activity. Precise and sensitive quantitation of small molecules (e.g. in drug pharmacokinetics studies) remains the domain of radionuclide detection methods (scintillation counting, autoradiography, and positron emission tomography) since radiolabeling in most cases can preserve biochemical activity. The recent finding that radionuclide molecules too can be imaged at the cellular level in a microscope represents a radical departure from what was previously known. This new method, called radioluminescence microscopy, can measure the accumulation of a radionuclide molecule in single living cells. While the technique has been demonstrated for a variety of applications, the technology is still in its infancy. In this proposa, we are proposing several radical improvements that will allow us to measure radionuclide probe uptake in up to 1000 individual cells, in a single acquisition. Studies that investigate large cell numbers are necessary for statistical reasons and due to the existence of rare cell subpopulations. Based on encouraging preliminary results, we are proposing a new instrument design called the radioluminescence microarray that can achieve this goal. This new design incorporates several radical improvements that will transform radioluminescence microscopy into a versatile tool for high-throughput studies with many potential applications.
In Aim 1, we will develop a new device called the radioluminescence microarray. This device includes a micrometer-thin scintillator for high-resolution imaging, a microwell array for optimized cell placement, a fluidics platform for repeatable sample preparation, and an improved epifluorescence add-on for multi-modality imaging.
In Aim 2, we will implement software for real-time display and automated analysis of radioluminescence microscopy images. Last, in Aim 3, we will validate the overall approach by investigating the interaction with single living cells of -fluorouracil (5-FU), a small-molecule non-fluorescent chemotherapeutic agent. Using the radioactive form of the drug ([18F] 5-FU), we will determine how 5FU distributes in heterogeneous cancer cell populations. Fluorescence microscopy will be used to assign cells to different subpopulations according to factors such as position in the cell cycle or stem-cell status. In summary, this project will develop new a new instrument with unmatched capabilities, which will be applied to deepening our understanding of drug resistance in heterogeneous cancer cell populations.
This project proposal aims to develop a new imaging device that can characterize how small molecules interact with single cells. Once built, the imaging device will be used to measure the differential uptake of drugs by single cancer cells and visualize how heterogeneous cancer cell populations acquire resistance to drugs. Such single-cell studies will help develop more effective therapies that overcome drug resistance.
| Kiru, Louise; Kim, Tae Jin; Shen, Bin et al. (2018) Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB. Mol Imaging Biol 20:378-387 |
| Jung, Kyung Oh; Jo, Hunho; Yu, Jung Ho et al. (2018) Development and MPI tracking of novel hypoxia-targeted theranostic exosomes. Biomaterials 177:139-148 |
| Wang, Qian; Sengupta, Debanti; Kim, Tae Jin et al. (2018) In silico optimization of radioluminescence microscopy. J Biophotonics 11: |
| Türkcan, Silvan; Kiru, Louise; Naczynski, Dominik J et al. (2018) Lactic acid accumulation in the tumor microenvironment suppresses 18F-FDG uptake. Cancer Res : |
| Sengupta, Debanti; Kim, Tae Jin; Almasi, Sepideh et al. (2018) Development and characterization of a scintillating cell imaging dish for radioluminescence microscopy. Analyst 143:1862-1869 |
| Kim, Tae Jin; Türkcan, Silvan; Pratx, Guillem (2017) Modular low-light microscope for imaging cellular bioluminescence and radioluminescence. Nat Protoc 12:1055-1076 |
| Wang, Qian; Sengupta, Debanti; Kim, Tae Jin et al. (2017) Performance evaluation of 18 F radioluminescence microscopy using computational simulation. Med Phys 44:1782-1795 |
| Gallina, Maria Elena; Kim, Tae Jin; Shelor, Mark et al. (2017) Toward a Droplet-Based Single-Cell Radiometric Assay. Anal Chem 89:6472-6481 |
| Türkcan, Silvan; Naczynski, Dominik J; Nolley, Rosalie et al. (2016) Endoscopic detection of cancer with lensless radioluminescence imaging and machine vision. Sci Rep 6:30737 |
| Sengupta, Debanti; Pratx, Guillem (2016) Imaging metabolic heterogeneity in cancer. Mol Cancer 15:4 |
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