A Genetically-Encoded Reporter for In Vivo Imaging in Deep Tissues

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

Improved outcome prediction in tetralogy of Fallot

Successful advances in cardiac surgery have led to a paradigm shift in the management of an expanding population of repaired tetralogy of Fallot (rTOF) patients. However, late morbidity and mortality have not been abolished, with patients vulnerable to arrhythmia and sudden death. Outcome prediction remains challenging, mandating the identification of novel sensitive and specific non-invasive biomarkers. Cardiac fibrosis in rTOF has been shown to correlate to adverse clinical features, and therefore merits further study, particularly with regards to interstitial fibrosis. Cardiac remodelling following surgical pulmonary valve replacement in patients with rTOF was investigated. Structural reverse remodelling was observed to occur immediately after surgery, followed by gradual biological remodelling. A proactive surgical approach before right ventricular (RV) end-systolic indexed volumes exceed 82ml/m2 confers optimal postoperative RV normalisation. Novel cardiovascular magnetic resonance T1 mapping techniques were developed and tested to improve identification of RV interstitial cardiac fibrosis in rTOF. Multi-echo imaging to separate fat from myocardium, combined with blood signal suppression is promising as a feasible method in saturation-recovery T1 mapping, but requires further technical study prior to clinical application and validation. The genomic signatures of the pathological RV in rTOF were investigated by next generation RNA sequencing. Differential gene expression was evident, and potential molecular determinants of fibrotic and restrictive phenotypes were ascertained. Ubiquitin C may have important functional implications as a ‘network hub’ gene in rTOF. Finally, the longitudinal predictive role of neurohormone expression in patients with rTOF was examined. Neurohormonal activation was confirmed in rTOF, with serum brain natriuretic peptide being prognostic for mortality and sustained arrhythmias during extended follow-up. In conclusion, this work reflects the complex interplay of candidate biomarkers in influencing clinical outcomes. Myocardial fibrosis in rTOF remains a key diagnostic and therapeutic target for improving risk stratification and ameliorating morbidity in the lifelong care of these individuals.Open Acces

Acoustic Angiography: A New Imaging Platform for High Resolution Mapping of Microvasculature and Tumor Assessment

Statistically, one in four Americans will die from cancer. Many new tumor detection and therapeutic approaches have improved patient outcomes, but cancer continues to run rampant in our country; it claimed the lives of 1.6 million Americans in 2012. To put this number of annual deaths in perspective, it is over 500 times the number of people who died in the horrific attacks on September 11, 2001. This dissertation does not offer either an antidote to the disease, nor a detection mechanism appropriate for all tumor types. It does, however, present the description and characterization of a novel dual-frequency ultrasound imaging transducer, capable of operating in a new imaging mode we call `acoustic angiography.' These images offer high resolution and high contrast 3D depictions of the microvasculature; herein we demonstrate its cancer assessment utility by way of multiple imaging studies. Throughout this dissertation, image data from both healthy and diseased tissues are presented. Additionally, acoustic assessments of vasculature within an ex vivo biomatrix scaffold model (a platform for creating of artificial organs) are presented. A vessel mapping algorithm, originally developed for human magnetic resonance angiography images, has been implemented in both in vivo and ex vivo tissue volumes. A novel microvessel phantom generation technique is presented, which allows ground-truth coordinates for vascular networks to be defined and imaged. Finally, the ultrasound pulsing technique, radiation force, was used as a method to improve the diagnostic sensitivity of ultrasound to malignant tumors. Together, the results of these studies suggest that the imaging approach, acoustic angiography, enabled by our new dual-frequency ultrasound transducer, could eventually be used to detect and monitor tumors in a clinical imaging context. This dissertation supports the following three hypotheses: 1) A prototype dual-frequency ultrasound transducer can be used to depict in vivo microvasculature, 2) These microvascular images can be quantitatively assessed as a means to characterize the presence of a tumor, and evaluate tumor response to therapy, and 3) Radiation force can be used as a method to improve ultrasonic diagnostic sensitivity to the presence of a tumor.Doctor of Philosoph

Deep Learning in Medical Image Analysis

The accelerating power of deep learning in diagnosing diseases will empower physicians and speed up decision making in clinical environments. Applications of modern medical instruments and digitalization of medical care have generated enormous amounts of medical images in recent years. In this big data arena, new deep learning methods and computational models for efficient data processing, analysis, and modeling of the generated data are crucially important for clinical applications and understanding the underlying biological process. This book presents and highlights novel algorithms, architectures, techniques, and applications of deep learning for medical image analysis

Preclinical MRI of the Kidney

This Open Access volume provides readers with an open access protocol collection and wide-ranging recommendations for preclinical renal MRI used in translational research. The chapters in this book are interdisciplinary in nature and bridge the gaps between physics, physiology, and medicine. They are designed to enhance training in renal MRI sciences and improve the reproducibility of renal imaging research. Chapters provide guidance for exploring, using and developing small animal renal MRI in your laboratory as a unique tool for advanced in vivo phenotyping, diagnostic imaging, and research into potential new therapies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Preclinical MRI of the Kidney: Methods and Protocols is a valuable resource and will be of importance to anyone interested in the preclinical aspect of renal and cardiorenal diseases in the fields of physiology, nephrology, radiology, and cardiology. This publication is based upon work from COST Action PARENCHIMA, supported by European Cooperation in Science and Technology (COST). COST (www.cost.eu) is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation. PARENCHIMA (renalmri.org) is a community-driven Action in the COST program of the European Union, which unites more than 200 experts in renal MRI from 30 countries with the aim to improve the reproducibility and standardization of renal MRI biomarkers

Preclinical MRI of the kidney : methods and protocols

This Open Access volume provides readers with an open access protocol collection and wide-ranging recommendations for preclinical renal MRI used in translational research. The chapters in this book are interdisciplinary in nature and bridge the gaps between physics, physiology, and medicine. They are designed to enhance training in renal MRI sciences and improve the reproducibility of renal imaging research. Chapters provide guidance for exploring, using and developing small animal renal MRI in your laboratory as a unique tool for advanced in vivo phenotyping, diagnostic imaging, and research into potential new therapies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Preclinical MRI of the Kidney: Methods and Protocols is a valuable resource and will be of importance to anyone interested in the preclinical aspect of renal and cardiorenal diseases in the fields of physiology, nephrology, radiology, and cardiology. This publication is based upon work from COST Action PARENCHIMA, supported by European Cooperation in Science and Technology (COST). COST (www.cost.eu) is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation. PARENCHIMA (renalmri.org) is a community-driven Action in the COST program of the European Union, which unites more than 200 experts in renal MRI from 30 countries with the aim to improve the reproducibility and standardization of renal MRI biomarkers