Doctor of Philosophy
Scholl, Timothy J.
Ronald, John A.
Introduction. The ability to track cells in living organisms with sensitivity, accuracy and high spatial resolution would revolutionize the way we study disease. Reporter genes are valuable tools as they encode detectible products, allowing for quantitative “reporting” of cells that express them. Previously, a gene encoding Organic anion-transporting polypeptide 1a1 (Oatp1a1) was established as a magnetic resonance imaging (MRI) reporter based on its ability to take up the paramagnetic contrast agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). Our objective was to assess, characterize and further develop this system for whole-body tracking of cells in vivo. Methods. Cancer cells were engineered to synthetically express Oatp1a1, or Oatp1b3, a closely related human transporter protein. In our first study, T1-weighted images of Oatp1a1-expressing primary tumours in preclinical animals were acquired before and after administration of 0.1-mmol/kg Gd-EOB-DTPA at 3-Tesla. At endpoint, heterogenous contrast enhancement patterns within the primary tumour architecture were compared to whole-tumour fluorescent histology. In the next study, T1-weighted images of Oatp1b3-expressing primary tumours, and their spontaneous metastases to the lymph nodes and lungs, were acquired before and after administration of 1-mmol/kg Gd-EOB-DTPA at 3-Tesla. In the final study, the feasibility of Oatp1b3 as a photoacoustic reporter gene was assessed by acquiring full-spectrum near infrared photoacoustic images of primary tumours in preclinical animals before and after administration of 8-mg/kg indocyanine green. Results. We were able to demonstrate the feasibility of imaging cancer cells with Oatp1a1 at 3-Tesla and 0.1 mmol/kg Gd-EOB-DTPA. Importantly, as primary tumours grew over time, heterogeneous contrast enhancement patterns that emerged near-endpoint strongly correlated to viable cell distributions on whole-tumour histology. Oatp1b3 was also shown to operate as a MRI reporter gene at 3-Tesla, based on the same principle as Oatp1a1. Impressively, single lymph node metastases and the formation of micrometastases in the lungs of preclinical animals were detected with Oatp1b3-MRI. Finally, we also demonstrated the ability of Oatp1b3 to operate as a photoacoustic reporter gene based on its ability to take up indocyanine green. Conclusion. The Oatp1 reporter gene system is a versatile imaging tool for longitudinal tracking of engineered cells in vivo with sensitivity, high resolution, and 3-dimensional spatial information.
Summary for Lay Audience
The ability to effectively detect a specific cell type, such as cancer cells, in the context of a larger, living biological organism would be useful for the study of medicine and, more broadly, for the life sciences. Though methods do exist to track cells non-invasively, the imaging is limited to superficial surfaces and/or sub-optimal resolution. Our work here tackles cellular imaging by focusing on magnetic resonance imaging (MRI) as a platform, allowing us to overcome several of these limitations. Unlike other imaging methods, MRI scanners produce highly detailed anatomical images and do not lose signal with increasing tissue depth. To image specific cells on MRI, we have developed a nanotechnology in the form of a protein that embeds itself on the surface of the cells we are interested in studying, and allows those cells to “pack” themselves with an MR imaging dye. Meanwhile, other cells do not have this capability. We test the effectiveness of this system for tracking cancer cells in preclinical animal models, and remarkably, it has allowed us to visualize the changing architecture of a primary tumour over time, as well as track the spread of cancer cells from the primary tumour to other parts of the body with high sensitivity and high resolution. This molecular technology can, in principle, be applied to any cell type, and paves the path for imaging various biological processes to better understand their mechanisms and develop new treatments for disease.
Nasri, Nivin N., "A Genetically-Encoded Reporter for In Vivo Imaging in Deep Tissues" (2020). Electronic Thesis and Dissertation Repository. 7130.
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Available for download on Tuesday, December 01, 2020