Unmixing dynamic PET images with variable specific binding kinetics

To analyze dynamic positron emission tomography (PET) images, various generic multivariate data analysis techniques have been considered in the literature, such as principal component analysis (PCA), independent component analysis (ICA), factor analysis and nonnegative matrix factorization (NMF). Nevertheless, these conventional approaches neglect any possible nonlinear variations in the time activity curves describing the kinetic behavior of tissues with specific binding, which limits their ability to recover a reliable, understandable and interpretable description of the data. This paper proposes an alternative analysis paradigm that accounts for spatial fluctuations in the exchange rate of the tracer between a free compartment and a specifically bound ligand compartment. The method relies on the concept of linear unmixing, usually applied on the hyperspectral domain, which combines NMF with a sum-to-one constraint that ensures an exhaustive description of the mixtures. The spatial variability of the signature corresponding to the specific binding tissue is explicitly modeled through a perturbed component. The performance of the method is assessed on both synthetic and real data and is shown to compete favorably when compared to other conventional analysis methods

Factor analysis of dynamic PET images

Thanks to its ability to evaluate metabolic functions in tissues from the temporal evolution of a previously injected radiotracer, dynamic positron emission tomography (PET) has become an ubiquitous analysis tool to quantify biological processes. Several quantification techniques from the PET imaging literature require a previous estimation of global time-activity curves (TACs) (herein called \textit{factors}) representing the concentration of tracer in a reference tissue or blood over time. To this end, factor analysis has often appeared as an unsupervised learning solution for the extraction of factors and their respective fractions in each voxel. Inspired by the hyperspectral unmixing literature, this manuscript addresses two main drawbacks of general factor analysis techniques applied to dynamic PET. The first one is the assumption that the elementary response of each tissue to tracer distribution is spatially homogeneous. Even though this homogeneity assumption has proven its effectiveness in several factor analysis studies, it may not always provide a sufficient description of the underlying data, in particular when abnormalities are present. To tackle this limitation, the models herein proposed introduce an additional degree of freedom to the factors related to specific binding. To this end, a spatially-variant perturbation affects a nominal and common TAC representative of the high-uptake tissue. This variation is spatially indexed and constrained with a dictionary that is either previously learned or explicitly modelled with convolutional nonlinearities affecting non-specific binding tissues. The second drawback is related to the noise distribution in PET images. Even though the positron decay process can be described by a Poisson distribution, the actual noise in reconstructed PET images is not expected to be simply described by Poisson or Gaussian distributions. Therefore, we propose to consider a popular and quite general loss function, called the $\beta$-divergence, that is able to generalize conventional loss functions such as the least-square distance, Kullback-Leibler and Itakura-Saito divergences, respectively corresponding to Gaussian, Poisson and Gamma distributions. This loss function is applied to three factor analysis models in order to evaluate its impact on dynamic PET images with different reconstruction characteristics

Development of Novel Molecular Imaging Contrast Agents for Detection of Oxidative Stress

An early and precise diagnosis of disease is a crucial requirement for fast and targeted therapy in the era of precision medicine. Most diseases possess molecular alterations in their early stages, which precede noticeable morphological changes. One important molecular change that contributes to dysfunction in a wide range of pathologies, including cancer and neurological disorders, is an increase in levels of reactive oxygen species (ROS) and associated oxidative damage. Our understanding of how ROS influence disease biology is currently limited by our inability to perform sensitive and specific assessment of ROS levels with high spatial and temporal resolution in living systems. The goal of the research described in this thesis was to overcome the challenge of assessing ROS during disease development in cancer and neurodegenerative disease through the design, synthesis and validation of two classes of novel bifunctional, ROS-sensitive contrast agents. To shed light on the complex redox biology in cancer, the new method of photoacoustic imaging was exploited. A novel activatable, targeted near infrared cyanine dye is reported that enables specific detection of pathological levels of hydrogen peroxide, a major and abundant ROS in living organisms. This approach uses photoacoustic and fluorescence imaging in cancerous tissue to evaluate the performance of the new probe under in vitro and in vivo conditions. In neurodegeneration, there exists a bidirectional interaction between oxidative stress and protein aggregates. To scrutinise this relationship, both bulk and single-molecule fluorescence imaging methods were used to assess the capability of novel bifunctional fluorescence dyes to localise the presence of the two putative disease-causing species, ROS and protein aggregates, simultaneously under in vitro conditions. The data shown here provides a promising foundation for the systematic design of contrast agents based on small molecule dyes, that possess ideal optical and biological characteristics to study oxidative stress in a broad range of pathological applications with high temporal and spatial resolution.Cancer Research U

Oxygen-Enhanced and Dynamic Contrast-Enhanced Optoacoustic Tomography Provide Surrogate Biomarkers of Tumor Vascular Function, Hypoxia, and Necrosis.

Measuring the functional status of tumor vasculature, including blood flow fluctuations and changes in oxygenation, is important in cancer staging and therapy monitoring. Current clinically approved imaging modalities suffer long procedure times and limited spatiotemporal resolution. Optoacoustic tomography (OT) is an emerging clinical imaging modality that may overcome these challenges. By acquiring data at multiple wavelengths, OT can interrogate hemoglobin concentration and oxygenation directly and resolve contributions from injected contrast agents. In this study, we tested whether two dynamic OT techniques, oxygen-enhanced (OE) and dynamic contrast-enhanced (DCE)-OT, could provide surrogate biomarkers of tumor vascular function, hypoxia, and necrosis. We found that vascular maturity led to changes in vascular function that affected tumor perfusion, modulating the DCE-OT signal. Perfusion in turn regulated oxygen availability, driving the OE-OT signal. In particular, we demonstrate for the first time a strong per-tumor and spatial correlation between imaging biomarkers derived from these in vivo techniques and tumor hypoxia quantified ex vivo Our findings indicate that OT may offer a significant advantage for localized imaging of tumor response to vascular-targeted therapies when compared with existing clinical DCE methods.Significance: Imaging biomarkers derived from optoacoustic tomography can be used as surrogate measures of tumor perfusion and hypoxia, potentially yielding rapid, multiparametric, and noninvasive cancer staging and therapeutic response monitoring in the clinic.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5980/F1.large.jpg Cancer Res; 78(20); 5980-91. ©2018 AACR

Advancing fluorescent contrast agent recovery methods for surgical guidance applications

Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

Contributions au traitement des images multivariées

Ce mémoire résume mon activité pédagogique et scientifique en vue de l’obtention de l’habilitation à diriger des recherches

Bioanalytical applications of multicolour bioluminescence imaging: new tools for drug discovery and development

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular

Prediction of Proapoptotic Anticancer Therapeutic Response Based on Visualization of Death Ligand-Receptor Interaction and Specific Marker of Cellular Proliferation

Emerging targeted therapeutics hold great promise for the treatment of human cancer. However there are still challenges for selecting patients that most likely will benefit from targeted drugs. One of the major limitations of classical imaging methods is the significant delay to provide quantifiable and objective evidence of response to cancer therapy. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and predicting therapeutic response in both preclinical and clinical settings. The apoptosis pathway triggered by the Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) receptors is a potential target for therapeutic intervention. TRAIL and its proapoptotic receptor agonistic monoclonal antibodies are being developed as targeted therapeutics in the treatment of human cancer. It is our hypothesis that visualization of proapoptotic receptors and binding of their agonists to proapoptotic receptors can noninvasively predict proapoptotic response if the pathway is intact. Hence the objective of this work is to develop efficient multimodality molecular imaging methods to predict proapoptotic anticancer therapy response before or at the very early stage of treatment. Towards this goal, we have labeled proapoptotic receptor agonists (PARAs) with near-infrared (NIR) fluorescent dyes to image PARAs binding to their targets expressed on the cell surface in cultured cells and in human tumor xenografts grown subcutaneously in immunodeficient mice. Both in vitro and in vivo studies demonstrated that imaging PARAs binding to their targets was well correlated with proapoptotic anticancer therapeutic response when TRAIL signaling pathway was intact. To pursue a more general molecular imaging marker that can predict anticancer therapeutic response even when the signaling pathway is impaired, we explored a novel radiotracer for positron emission tomography (PET) imaging [(18)F]-3\u27-fluoro-3\u27-deoxy-L-thymidine ([(18)F]-FLT), an analogue of thymidine and a specific marker of DNA replication and cellular proliferation. Our results suggested that early changes in [(18)F]-PET may not only predict the tumor histological response to anticancer therapeutics but also determine superiority of one treatment regimen over another. In summary our proof-of-concept studies show that multimodality molecular imaging will greatly aid in accelerating anticancer drug approval process and improving survival and response rates in hard-to-treat cancer

Imaging studies of peripheral nerve regeneration induced by porous collagen biomaterials

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.There is urgent need to develop treatments for inducing regeneration in injured organs. Porous collagen-based scaffolds have been utilized clinically to induce regeneration in skin and peripheral nerves, however still there is no complete explanation about the underlying mechanism. This thesis utilizes advanced microscopy to study the expression of contractile cell phenotypes during wound healing, a phenotype believed to affect significantly the final outcome. The first part develops an efficient pipeline for processing challenging spectral fluorescence microscopy images. Images are segmented into regions of objects by refining the outcome of a pixel-wide model selection classifier by an efficient Markov Random Field model. The methods of this part are utilized by the following parts. The second part extends the image informatics methodology in studying signal transduction networks in cells interacting with 3D matrices. The methodology is applied in a pilot study of TGFP signal transduction by the SMAD pathway in fibroblasts seeded in porous collagen scaffolds. Preliminary analysis suggests that the differential effect of TGFP1 and TGFP3 to cells could be attributed to the "non-canonical" SMADI and SMAD5. The third part is an ex vivo imaging study of peripheral nerve regeneration, which focuses on the formation of a capsule of contractile cells around transected rat sciatic nerves grafted with collagen scaffolds, 1 or 2 weeks post-injury. It follows a recent study that highlights an inverse relationship between the quality of the newly formed nerve tissue and the size of the contractile cell capsule 9 weeks post-injury. Results suggest that "active" biomaterials result in significantly thinner capsule already 1 week post-injury. The fourth part describes a novel method for quantifying the surface chemistry of 3D matrices. The method is an in situ binding assay that utilizes fluorescently labeled recombinant proteins that emulate the receptor of , and is applied to quantify the density of ligands for integrins a113, a2p1 on the surface of porous collagen scaffolds. Results provide estimates for the density of ligands on "active" and "inactive" scaffolds and demonstrate that chemical crosslinking can affect the surface chemistry of biomaterials, therefore can affect the way cells sense and respond to the material.by Dimitrios S. Tzeranis.Ph. D

CT-PET guided target delineation in head and neck cancer and implications for improved outcome

Aim: Fifty percent of patients with squamous cell carcinoma of the Head and Neck develop loco-regional recurrence after treatment. Factors leading to this failure are most likely altered intra-tumoural glucose metabolism and increased hypoxia. Tissue glucose utilisation and the degree of hypoxia can be visualised by CTPET imaging with 18FDG and hypoxic radio-nuclides. This thesis has investigated 18FDG CT-PET guided target volume delineation methods and attempted to validate 64Cu-ATSM as a hypoxic radio-nuclide in patients with squamous cell carcinoma of the Head and Neck. Materials and Methods: Eight patients with locally advanced disease underwent 18FDG CT-PET imaging before and during curative radiotherapy or chemo-radiotherapy. Fixed (SUV cut off and percentage threshold of the SUVmax) and adaptive thresholds were investigated. The functional volumes automatically delineated by these methods and SUVmax were compared at each point, and between thresholds. Four patients with locally advanced disease, two to seven days prior to surgery, underwent 3D dynamic CT-PET imaging immediately after injection of 64Cu- ATSM. Two patients were also imaged 18 hours after injection, and two underwent a dynamic contrast-enhanced CT to evaluate intra-tumoural perfusion. All patients received pimonidazole before surgery. The pimonidazole, GLUT1, CAIX, and HIF1a immuno-histochemical hypoxic fractions were defined. Staining was correlated with the retention pattern of 64Cu-ATSM at 3 time points. Hypoxic target volumes were delineated according to tumour to muscle, blood and background ratios. Results: 18FDG primary and lymph node target volumes significantly reduced with radiation dose by the SUV cut off method and correlated with the reduction in the SUVmax within the volume. Volume reduction was also found between thresholds by the same delineation method. The volumes delineated by the other methods were not significantly reduced (except the lymph node functional volume when defined by the adaptive threshold). 64Cu-ATSM correlated with hypoxic immuno-histochemical staining but not with blood flow. Tumour ratios increased with time after injection, which influenced the delineated hypoxic target volume. Conclusion: Dose-escalated image-guided radiotherapy strategies using these CT-PET guided functional volumes have the potential to improve loco-regional control in patients with squamous cell carcinoma of the Head and Neck. CT-PET 18FDG volume delineation is intricately linked to the method and threshold of delineation and the timing of the imaging. 64Cu-ATSM is promising as a hypoxic radio-nuclide and warrants further investigation