Assessing and monitoring intratumor heterogeneity in glioblastoma: how far has multimodal imaging come?

Glioblastoma demonstrates imaging features of intratumor heterogeneity that result from underlying heterogeneous biological properties. This stems from variations in cellular behavior that result from genetic mutations that either drive, or are driven by, heterogeneous microenvironment conditions. Among all imaging methods available, only T1-weighted contrast-enhancing and T2-weighted fluid-attenuated inversion recovery are used in standard clinical glioblastoma assessment and monitoring. Advanced imaging modalities are still considered emerging techniques as appropriate end points and robust methodologies are missing from clinical trials. Discovering how these images specifically relate to the underlying tumor biology may aid in improving quality of clinical trials and understanding the factors involved in regional responses to treatment, including variable drug uptake and effect of radiotherapy. Upon validation and standardization of emerging MR techniques, providing information based on the underlying tumor biology, these images may allow for clinical decision-making that is tailored to an individual's response to treatment.Stephen Price is funded by a Clinician Scientist Award from the National Institute for Health Research.This is the author accepted manuscript. The final version is available from Future Medicine via http://dx.doi.org/10.2217/cns.15.2

Probing brain microstructure with multidimensional diffusion MRI: Encoding, interpretation, and the role of exchange

Diffusion MRI (dMRI) is a non-invasive probe of human brain microstructure. It is a long-standing promise to use dMRI for ‘in vivo histology’ and estimate tissue quantities. However, this faces several challenges. First, the microstructure models used for dMRI data are based on assumptions that may cause erroneous interpretations. Also, probing neurites in gray matter assumes high microscopic diffusion anisotropy in both axons and dendrites, which is not supported by evidence. Furthermore, dMRI data analysis typically ignores diffusional exchange between microscopic environments. This thesis investigates and addresses these challenges using ‘multidimensional’ dMRI techniques that vary additional sequence encoding parameters to obtain new information on the tissue. In Paper I, we optimized an acquisition protocol for filter exchange imaging (FEXI). We found slow rates of diffusional exchange in normal brain tissue. In patients with gliomas and meningiomas, faster exchange was tentatively associated with higher tumor grade. In Paper II, we used tensor-valued diffusion encoding to test the NODDI microstructure model. The NODDI assumptions were contradicted by independent data and parameter estimates were found to be biased in normal brain and in gliomas. The CODIVIDE model combined data acquired with different b-tensor shapes to remove NODDI assumptions and reduce the susceptibility to bias. In Paper III, we used tensor-valued diffusion encoding with multiple echo times to investigate challenges in estimating neurite density. We found that microscopic anisotropy in the brain reflected axons but not dendrites. We could not separate the densities and T2 values of a two-component model in normal brain, but we did detect different component T2 values in white matter lesions. Microstructure models ranked regions from normal brain and white matter lesions inconsistently with respect to neurite density. In Paper IV, we optimized an acquisition protocol for tensor-valued diffusion encoding with multiple echo times. The data allowed removing all assumptions on diffusion and T2 relaxation from a two-component model. This increased the measurable parameters from two to six and reduced their susceptibility to bias. Data from the normal brain showed different component T2 values and contradicted common model assumptions. In Paper V, we used tensor-valued diffusion encoding in malformations of cortical development. Lesions that appeared gray matter-like in T1- and T2-weighted contrasts featured white matter-like regions with high microscopic diffusion anisotropy. We interpreted these regions as myelin-poor white matter with a high axonal content. By primarily reflecting axons and not dendrites or myelin, microscopic anisotropy may differentiate tissue where alterations to myelin confound conventional MRI contrasts. In Paper VI, we used SDE with multiple diffusion times in patients with acute ischemic stroke. Subacute lesions exhibited elevated diffusional exchange that predicted later infarction. MD reduction was partially reversible and did not predict infarction. Diffusional exchange may improve definition of ischemic core and identify additional patients for late revascularization

Diffusionâ weighted imaging outside the brain: Consensus statement from an ISMRMâ sponsored workshop

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134160/1/jmri25196_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134160/2/jmri25196.pd

STED Nanoscopy to Illuminate New Avenues in Cancer Research – From Live Cell Staining and Direct Imaging to Decisive Preclinical Insights for Diagnosis and Therapy

Molecular imaging is established as an indispensable tool in various areas of cancer research, ranging from basic cancer biology and preclinical research to clinical trials and medical practice. In particular, the field of fluorescence imaging has experienced exceptional progress during the last three decades with the development of various in vivo technologies. Within this field, fluorescence microscopy is primarily of experimental use since it is especially qualified for addressing the fundamental questions of molecular oncology. As stimulated emission depletion (STED) nanoscopy combines the highest spatial and temporal resolutions with live specimen compatibility, it is best-suited for real-time investigations of the differences in the molecular machineries of malignant and normal cells to eventually translate the acquired knowledge into increased diagnostic and therapeutic efficacy. This thesis presents the application of STED nanoscopy to two acute topics in cancer research of direct or indirect clinical interest. The first project has investigated the structure of telomeres, the ends of the linear eukaryotic chromosomes, in intact human cells at the nanoscale. To protect genome integrity, a telomere can mask the chromosome end by folding back and sequestering its single-stranded 3’-overhang in an upstream part of the double-stranded DNA repeat region. The formed t-loop structure has so far only been visualized by electron microscopy and fluorescence nanoscopy with cross-linked mammalian telomeric DNA after disruption of cell nuclei and spreading. For the first time, this work demonstrates the existence of t-loops within their endogenous nuclear environment in intact human cells. The identification of further telomere conformations has laid the groundwork for distinguishing cancerous cells that use different telomere maintenance mechanisms based on their individual telomere populations by a combined STED nanoscopy and deep learning approach. The population difference was essentially attributed to the promyelocytic leukemia (PML) protein that significantly perturbs the organization of a subpopulation of telomeres towards an open conformation in cancer cells that employ a telomerase-independent, alternative telomere lengthening mechanism. Elucidating the nanoscale topology of telomeres and associated proteins within the nucleus has provided new insight into telomere structure-function relationships relevant for understanding the deregulation of telomere maintenance in cancer cells. After understanding the molecular foundations, this newly gained knowledge can be exploited to develop novel or refined diagnostic and treatment strategies. The second project has characterized the intracellular distribution of recently developed prostate cancer tracers. These novel prostate-specific membrane antigen (PSMA) inhibitors have revolutionized the treatment regimen of prostate cancer by enabling targeted imaging and therapy approaches. However, the exact internalization mechanism and the subcellular fate of these tracers have remained elusive. By combining STED nanoscopy with a newly developed non-standard live cell staining protocol, this work confirmed cell surface clustering of the targeted membrane antigen upon PSMA inhibitor binding, subsequent clathrin-dependent endocytosis and endosomal trafficking of the antigen-inhibitor complex. PSMA inhibitors accumulate in prostate cancer cells at clinically relevant time points, but strikingly and in contrast to the targeted antigen itself, they eventually distribute homogenously in the cytosol. This project has revealed the subcellular fate of PSMA/PSMA inhibitor complexes for the first time and provides crucial knowledge for the future application of these tracers including the development of new strategies in the field of prostate cancer diagnostics and therapeutics. Relying on the photostability and biocompatibility of the applied fluorophores, the performance of live cell STED nanoscopy in the field of cancer research is boosted by the development of improved fluorophores. The third project in this thesis introduces a biocompatible, small molecule near-infrared dye suitable for live cell STED imaging. By the application of a halogen dance rearrangement, a dihalogenated fluorinatable pyridinyl rhodamine could be synthesized at high yield. The option of subsequent radiolabeling combined with excellent optical properties and a non-toxic profile renders this dye an appropriate candidate for medical and bioimaging applications. Providing an intrinsic and highly specific mitochondrial targeting ability, the radiolabeled analogue is suggested as a vehicle for multimodal (positron emission tomography and optical imaging) medical imaging of mitochondria for cancer diagnosis and therapeutic approaches in patients and biopsy tissue. The absence of cytotoxicity is not only a crucial prerequisite for clinically used fluorophores. To guarantee the generation of meaningful data mirroring biological reality, the absence of cytotoxicity is likewise a decisive property of dyes applied in live cell STED nanoscopy. The fourth project in this thesis proposes a universal approach for cytotoxicity testing based on characterizing the influence of the compound of interest on the proliferation behavior of human cell lines using digital holographic cytometry. By applying this approach to recently developed live cell STED compatible dyes, pronounced cytotoxic effects could be excluded. Looking more closely, some of the tested dyes slightly altered cell proliferation, so this project provides guidance on the right choice of dye for the least invasive live cell STED experiments. Ultimately, live cell STED data should be exploited to extract as much biological information as possible. However, some information might be partially hidden by image degradation due the dynamics of living samples and the deliberate choice of rather conservative imaging parameters in order to preserve sample viability. The fifth project in this thesis presents a novel image restoration method in a Bayesian framework that simultaneously performs deconvolution, denoising as well as super-resolution, to restore images suffering from noise with mixed Poisson-Gaussian statistics. Established deconvolution or denoising methods that consider only one type of noise generally do not perform well on images degraded significantly by mixed noise. The newly introduced method was validated with live cell STED telomere data proving that the method can compete with state-of-the-art approaches. Taken together, this thesis demonstrates the value of an integrated approach for STED nanoscopy imaging studies. A coordinated workflow including sample preparation, image acquisition and data analysis provided a reliable platform for deriving meaningful conclusions for current questions in the field of cancer research. Moreover, this thesis emphasizes the strength of iteratively adapting the individual components in the operational chain and it particularly points towards those components that, if further improved, optimize the significance of the final results rendering live cell STED nanoscopy even more powerful

The role of tumour vasculature in fluid flow and drug transport in solid tumours

The aberrance of the vasculature in tumours has been linked to increased aggressiveness and poor drug delivery in tumours. Complexities in the microarchitecture of tumour vasculature occurring on microscopic scales can affect fluid flow and drug transport making it difficult to predict tumour response to treatment. Given this, mathematical models can play an important role in understanding the various aspects of the tumour vasculature that can promote invasiveness and limit drug delivery. In this work, computational models are developed to investigate the effect of tumour vasculature on fluid flow and drug distribution and novel imaging methods are assessed for their ability to characterise the tumour vasculature in whole human tumours. A mathematical angiogenesis model is used to generate microscopic details including individual vessel properties on a whole vascular network scale which are coupled with a fluid flow and drug transport model. The interstitial fluid pressure (IFP) in the tumour model was found to be elevated with increased heterogeneity caused by the presence of a necrotic core and heterogenous vessel permeability. Subtle changes to the network on a microscopic scale significantly influenced fluid flow in the tumour vessels and tissue. Delivery of doxorubicin to tumours was found to be highly dependent on the properties of tumour vasculature and blood flow, where regions with excessive branching and vessel tortuosity had reduced drug concentrations due to poor blood flow. Hence, the vascular density was not found to be the main factor in the accumulation of the drug within the tissue space and it’s uptake by cancer cells. An interplay between treatment strategy including dose and administration mode and properties of the vasculature was found by evaluating the spatial intracellular concentration. The fluid flow and drug transport models showed the significant effect of incorporating the microscopic properties of the tumour vasculature which can influence fluid flow and drug distribution on a macroscopic scale. The imaging methods assessed in this work shows that Optical projection tomography combined with fluorescent Immunohistochemistry labelling methods can be used to extract angiogenesis related parameters in whole human tumours. Additionally, the method was able to extract clean network topologies that show promise in application to understanding fluid flow and drug transport in real tumours.Open Acces

The role of ligand-gated chloride channels in behavioural alterations at elevated CO2 in a cephalopod

Projected future carbon dioxide (CO2) levels in the ocean can alter marine animal behaviours. Disrupted functioning of γ-aminobutyric acid type A (GABAA) receptors (ligand-gated chloride channels) is suggested to underlie CO2-induced behavioural changes in fish. However, the mechanisms underlying behavioural changes in marine invertebrates are poorly understood. We pharmacologically tested the role of GABA-, glutamate-, acetylcholine- and dopamine-gated chloride channels in CO2-induced behavioural changes in a cephalopod, the two-toned pygmy squid (Idiosepius pygmaeus). We exposed squid to ambient (~450 μatm) or elevated (~1000 μatm) CO2 for 7 days. Squid were treated with sham, the GABAA receptor antagonist gabazine or the non-specific GABAA receptor antagonist picrotoxin, before measurement of conspecific-directed behaviours and activity levels upon mirror exposure. Elevated CO2 increased conspecific-directed attraction and aggression, as well as activity levels. For some CO2-affected behaviours, both gabazine and picrotoxin had a different effect at elevated compared with ambient CO2, providing robust support for the GABA hypothesis within cephalopods. In another behavioural trait, picrotoxin but not gabazine had a different effect in elevated compared with ambient CO2, providing the first pharmacological evidence, in fish and marine invertebrates, for altered functioning of ligand-gated chloride channels, other than the GABAAR, underlying CO2-induced behavioural changes. For some other behaviours, both gabazine and picrotoxin had a similar effect in elevated and ambient CO2, suggesting altered function of ligand-gated chloride channels was not responsible for these CO2-induced changes. Multiple mechanisms may be involved, which could explain the variability in the CO2 and drug treatment effects across behaviours

Mathematical methods for modeling the microcirculation

The microcirculation plays a major role in maintaining homeostasis in the body. Alterations or dysfunctions of the microcirculation can lead to several types of serious diseases. It is not surprising, then, that the microcirculation has been an object of intense theoretical and experimental study over the past few decades. Mathematical approaches offer a valuable method for quantifying the relationships between various mechanical, hemodynamic, and regulatory factors of the microcirculation and the pathophysiology of numerous diseases. This work provides an overview of several mathematical models that describe and investigate the many different aspects of the microcirculation, including geometry of the vascular bed, blood flow in the vascular networks, solute transport and delivery to the surrounding tissue, and vessel wall mechanics under passive and active stimuli. Representing relevant phenomena across multiple spatial scales remains a major challenge in modeling the microcirculation. Nevertheless, the depth and breadth of mathematical modeling with applications in the microcirculation is demonstrated in this work. A special emphasis is placed on models of the retinal circulation, including models that predict the influence of ocular hemodynamic alterations with the progression of ocular diseases such as glaucoma

Novel approaches to MRI of glioma

Gliomas are extremely heterogeneous, both morphologically and biologically, which contributes to a very poor prognosis. Current imaging of glioma is insufficient for a thorough diagnosis, therapy assessment and prognosis prediction. Moreover, refined and more sophisticated imaging technique could help in furthering our knowledge of gliomas. In order to facilitate proliferation, cancer cells undergo a change in structure and an increase in metabolism that results in distortion and disruption of tissue architecture. Gliomas are characterised by an increase in cells of variable sizes, as well as changes in the tissue microstructure. Diffusion-Weighted Imaging (DWI) and the apparent diffusion coefficient (ADC), have been extensively studied as potential imaging biomarkers for cellularity and tissue architecture. However, several studies have shown partial overlap in the measured values between tumour subtypes. Moreover, ADC is influenced by several factors and does not provide detailed information on the tissue microstructure. The Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumours (VERDICT) is a novel diffusion model that infers tissue microstructure compartment from conventional DWI measurements. This model derives metrics for the intracellular, intravascular and extracellular– extravascular spaces providing a more detailed interpretation of the tissue microstructure. To date, VERDICT has been applied to xenograft models of colorectal cancer, patient studies of prostate cancer and recently its feasibility in glioma has been shown. In this PhD I have applied a shortened version of the VERDICT method to image intratumoral and intertumoral heterogeneity in glioma. The results have also been validated with histology as part of a prospective study. Gliomas also exhibit a significant increase in mitotic activity within the tumour. The increased number of mitosis alters cell density which, in turn, affects the total concentration of tissue sodium as the concentration of tissue sodium is approximately ten-fold higher in the extracellular compared to the intracellular space. In addition, there is a decrease in Na+/K+-ATPase activity in tumours due to ATP depletion, which contributes to disturb sodium homeostasis. Non-invasive detection of 23Na with MRI has the potential to quantify sodium concentration and therefore could be an imaging probe of cell morphology and membrane function within the tumour microenvironment, as well as a method of probing tissue heterogeneity. During my PhD, a novel 23Na-MRI technique has been used to evaluate sodium distribution within glioma and in the surrounding tissue. Metabolic reprogramming is one of the major driving forces for determining glioma growth and invasion. Therefore, the non-invasive characterization of metabolic intratumoral, peritumoral and intertumoral heterogeneity in vivo could help to better stratify patients and to develop novel therapeutic strategies targeting cancer-specific metabolic pathways. 13C magnetic resonance imaging (MRI) using dynamic nuclear polarization (DNP) is a novel technique that allows non-invasive assessment of the metabolism of hyperpolarized (HP) 13C-labelled molecules in vivo, such as the exchange of [1-13C]pyruvate to [1-13C]lactate in tumours (Warburg effect). Part of my PhD has focused on developing and translating HP [1-13C]pyruvate MRI to explore metabolic reprogramming in glioma and the surrounding microenvironment. The overall aim of my PhD has been to develop novel approaches to imaging glioma with MRI to probe both the architectural and metabolic changes of Glioma. The preliminary evidence suggests that these tools can more deeply phenotype tumours than conventional imaging approaches. Although the main focus of this work has been gliomas, the techniques developed and presented here may be applied to study other pathological conditions within the brain, which raises the possibility of other potential clinical applications for this work