Integrated Study of Liver Fibrosis: Modeling and Clinical Detection

The liver is a vital organ that carries out over 500 essential tasks, including fat metabolism, blood filtering, bile production, and some protein production. Although the structure of the liver and the role of each type of cells in the liver are well known, the biomedical and mechanical interplays within liver tissues remain unclear. Chronic liver diseases are a significant public health challenge. All chronic liver diseases lead to liver fibrosis due to excessive fiber accumulation, resulting in cirrhosis and loss of liver function. Only early stage liver fibrosis is reversible. However, early-stage liver fibrosis is difficult to diagnose. How the progression of fibrosis changes the mechanical properties of the liver tissue and altering the dynamics of blood flow is still not well understood. The objective of this dissertation is to integrate the understanding of liver diseases and mechanical modeling to develop several models relating liver fibrosis to blood flow. In collaboration with clinicians specialized in hepatic fibrosis, we integrated computational modeling and clinicopathologic image analysis and proposed a new technology for early stage fibrosis detection. The key results of this research include: (1) A mathematical model of liver fibrosis progression connecting the cellular and molecular mechanisms of fibrosis to tissue rigidity; (2) A novel machine learning-based algorithm to automatically stage liver fibrosis based on pathology images; (3) A physics model to illustrate how the liver stiffness affects the blood flow pattern, predicting a direct relationship between fibrosis stage and ultrasound Doppler measurement of liver blood flow; (4) Statistical analysis of clinical ultrasound Doppler data from fibrosis patients confirming our model prediction. These results lead to a novel noninvasive technology for detecting early stages of liver fibrosis with high accuracy

Micro Soft Tissues Visualization Based on X-Ray Phase-Contrast Imaging

The current imaging methods have a limited ability to visualize microstructures of biological soft tissues. Small lesions cannot be detected at the early stage of the disease. Phase contrast imaging (PCI) is a novel non-invasive imaging technique that can provide high contrast images of soft tissues by the use of X-ray phase shift. It is a new choice in terms of non-invasively revealing soft tissue details. In this study, the lung and hepatic fibrosis models of mice and rats were used to investigate the ability of PCI in microstructures observation of soft tissues. Our results demonstrated that different liver fibrosis stages could be distinguished non-invasively by PCI. The three-dimensional morphology of a segment of blood vessel was constructed. Noteworthy, the blood clot inside the vessel was visualized in three dimensions which provided a precise description of vessel stenosis. Furthermore, the whole lung airways including the alveoli were obtained. We had specifically highlighted its use in the visualization and assessment of the alveoli. To our knowledge, this was the first time for non-invasive alveoli imaging using PCI. This finding may offer a new perspective on the diagnosis of respiratory disease. All the results confirmed that PCI will be a valuable tool in biological soft tissues imaging

Investigations of the Cavitation and Damage Thresholds of Histotripsy and Applications in Targeted Tissue Ablation.

Histotripsy is a noninvasive ultrasound therapy that controls acoustic cavitation to mechanically fractionate soft tissue. This dissertation investigates the physical thresholds to initiate cavitation and produce tissue damage in histotripsy and factors affecting these thresholds in order to develop novel strategies for targeted tissue ablation. In the first part of this dissertation, the effects of tissue properties on histotripsy cavitation thresholds and damage thresholds were investigated. Results demonstrated that the histotripsy shock scattering threshold using multi-cycle pulses increases in stiffer tissues, while the histotripsy intrinsic threshold using single-cycle pulses is independent of tissue stiffness. Further, the intrinsic threshold slightly decreases with lower frequencies and significantly decreases with increasing temperature. The effects of tissue properties on the susceptibility to histotripsy-induced tissue damage were also investigated, demonstrating that stiffer tissues are more resistant to histotripsy. In the second part of this dissertation, the feasibility of using histotripsy for targeted liver ablation was investigated in an intact in vivo porcine model, with results demonstrating that histotripsy was capable of non-invasively creating precise lesions throughout the entire liver. Additionally, a tissue selective ablation approach was developed, where histotripsy completely fractionated the liver tissue surrounding the major hepatic vessels and gallbladder while being self-limited at the boundaries of these critical structures. In the final part of this dissertation, a novel ablation method combining histotripsy with acoustically sensitive nanodroplets was developed for targeted cancer cell ablation, demonstrating the potential of using nanodroplet-mediated histotripsy (NMH) for targeted, multi-focal ablation. Studies demonstrated that lower frequency and higher boiling point perfluorocarbon droplets can improve NMH therapy. The role of positive and negative pressure on cavitation nucleation in NMH was also investigated, showing that NMH cavitation nucleation is caused directly from the peak negative pressure of the incident wave, similar to histotripsy bubbles generated above the intrinsic threshold. Overall, the results of this dissertation provide significant insight into the physical mechanisms underlying histotripsy tissue ablation and will help to guide the future development of histotripsy for clinical applications such as the treatment of liver cancer.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113591/1/evlaisav_1.pd

Liver Tumors

This book is oriented towards clinicians and scientists in the field of the management of patients with liver tumors. As many unresolved problems regarding primary and metastatic liver cancer still await investigation, I hope this book can serve as a tiny step on a long way that we need to run on the battlefield of liver tumors

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro

Dynamic Contrast Enhanced Computed Tomography Measurement of Perfusion in Hepatic Cancer

ABSTRACT In recent years, the incidence and mortality rate for hepatocellular carcinoma (HCC) have increased due to the emergence of hepatitis B, C and other diseases that cause cirrhosis. The progression from cirrhosis to HCC is characterized by abnormal vascularization and by a shift from a venous to an arterial blood supply. A knowledge of HCC vascularity which is manifested as alterations in liver blood flow may distinguish among different stages of liver disease and can be used to monitor response to treatment. Unfortunately, conventional diagnostic imaging techniques lack the ability to accurately quantify HCC vascularity. The purpose of this thesis was to validate and assess the diagnostic capabilities of dynamic contrast enhanced computed tomography (DCE-CT) and perfusion software designed to measure hepatic perfusion. Chapter 2 described a study designed to evaluate the accuracy and precision of hepatic perfusion measurement. The results showed a strong correlation between hepatic artery blood flow measurement with DCE-CT and radioactive microspheres under steady state in a rabbit model for HCC (VX2 carcinoma). Using repeated measurements and Monte Carlo simulations, DCE-CT perfusion measurements were found to be precise; with the highest precision in the tumor rim. In Chapter 3, we used fluorine-18 fluoro-2-deoxy-D-glucose (FDG) positron emission tomography and DCE-CT perfusion to determined an inverse correlation between glucose utilization and tumor blood flow; with an R of 0.727 (P \u3c 0.05). This suggests a limited supply of oxygen (possibly hypoxia) and that the tumor cells were surviving via anaerobic glycolysis. in In Chapter 4, hepatic perfusion data showed that thalidomide caused a reduction of tumor perfusion in the responder group during the first 8 days after therapy, P \u3c 0.05; while perfusion in the partial responder and control group remained unchanged, P \u3e 0.05. These changes were attributed to vascular remodeling and maturation resulting in a more functional network of endothelial tubes lined with pericytes. The results of this thesis demonstrate the accuracy and precision of DCE-CT hepatic perfusion measurements. It also showed that DCE-CT perfusion has the potential to enhance the functional imaging ability of hybrid PET/CT scanners and evaluate the efficacy of anti-angiogenesis therapy

Perfusion computed tomography of the liver

Background: Perfusion CT (P-CT) is a relatively new imaging technique that permits the visual and quantitative assessment of the micro- and macrocirculation of a target organ and focal lesions. P-CT has shown promising results in the evaluation of hyper-vascular tumors such as hepatocellular carcinoma (HCC). HCC is the sixth most common cancer globally and it has a poor prognosis when discovered at a late tumor stage. Any improvement in HCC detection would be directly beneficial for patient care. This thesis aims to investigate the strengths and limitations of whole liver P-CT and to evaluate if PCT can improve the detection of hyper-vascular liver lesions in patients with chronic liver disease. Methods: Study I: Twenty-four patients, who underwent dynamic P-CT for detection of HCC were retrospectively divided into three groups: (1) without portal-venous hypertension (PVH) (n = 8), (2) with PVH (n = 8), (3) with PVH and thrombosis (n = 8). Time to peak splenic- and peak renal enhancement (PSE resp. PRE), as well as arterial liver perfusion (ALP), portal- venous liver perfusion (PLP) and hepatic perfusion-index (HPI) of the liver and HCC derived from PSE- versus PRE- based modelling were compared between the groups. Study II: Group A (n=15) and Group B (n= 38) underwent P-CT using 50 ml contrast medium (CM). Group B underwent an additional standard multiphasic liver CT using 120ml (70-143 ml). Triple-arterial CT image sets were reconstructed from P-CT by fusing three dedicated arterial time points. Triple-arterial CT and single-arterial CT were compared by two readers (R1, R2), who assessed subjective image quality (IQ) and HCC detection rate. A third reader assessed objective IQ.Study III: Fifty study participants (Group A) were scanned with P-CT, a high CM volume protocol and bolus-tracking technique to depict ten arterial phases. Time attenuation curves were created for hyper-vascular liver lesions, liver parenchyma and hepatic vascular structures. 16 participants of Group A with lesions were further analyzed and radiation dose-neutral quadruple arterial phase image sets were created (Group A1). Group A1 was then compared to a Control Group (Group B) consisting of 16 consecutive patients undergoing standard single arterial phase scans. Lesion depiction and quantitative IQ were compared. Results: Study I: Time to PSE was significantly delayed in PVH groups 2 and 3 (P = 0.02), whereas PRE was similar in groups 1, 2 and 3 (P > 0.05). In group 1, liver and HCC perfusion parameters were similar for PSE- and PRE-based modelling (all P > 0.05), while significant differences were seen for PLP and HPI (liver only) in group 2 and ALP in group 3 (all P < 0.05). Study II: The mean CTDIvol of triple-arterial CT and single-arterial CT was equivalent (P=0.73). Triple-arterial CT showed lower image noise and better contrast-to-noise-ratio (P<0.001, P=0.032, respectively), but no significant difference in lesion-to-liver-contrast-ratio (P=0.31). Subjective IQ was good for both protocols. The detection rate of the 65 HCC lesions was 82%/83% (R1/R2) at triple-arterial CT and 80%/77% (R1/R2) at single-arterial CT (P=0.4). Study III: Both Group A1 and B had 33 hyper-enhancing liver lesions each. The mean CTDIvol of quadruple-arterial CT and single-arterial CT was equivalent (P=0.16). The mean time to reach peak lesion-to-liver contrast (LLC) was 20.1s (±4.2s) with a range of 12.5s to 29.1s. Quadruple arterial CT performed significantly better than the Control Group in regards to LLC (P= .009), CNR (P= .002), Image Noise (P<0.001) and hepatic artery enhancement(P<0.001). Conclusions: Study I: PSE is significantly delayed in patients with portal venous hypertension, which results in a miscalculation of P-CT parameters. Maximum-slope based P-CT could be improved by replacing the spleen with the kidney as the reference organ. The difference between time to PSE and time to PRE might serve as a non-invasive biomarker of portal venous hypertension. Study II: Radiation dose-equivalent triple arterial phase imaging is feasible and showed superior image quality and similar HCC detection rate as standard single arterial phase CT despite a substantially smaller CM volume. Study III: The optimal scan delay at single arterial phase CT for depiction of hyper-vascular liver lesions occurs at 20 s, when using a high iodine dose CM protocol and bolus-tracking. Fused quadruple arterial phase CT significantly increases lesion depiction, quantitative IQ and hepatic artery enhancement as compared to standard single arterial phase CT, without elevating the total radiation dose

The Development of Hyaluronan-Based Contract Agents for the Intraoperative Detection of Pancreatic Tumors

Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. Tumor-specific intraoperative fluorescence imaging could improve staging and surgical resection, thereby improving prognosis. In the first study, hyaluronic acid derived NPs with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG accumulated significantly in an orthotopic pancreatic ductal adenocarcinoma model with safety profile both in vitro and in vivo. To maximize tumor signal, while minimizing signal in healthy pancreas and RES capture of macromolecules, in the next study, we describe the rational development of a series of hyaluronic acid (HA) conjugates that vary in molecular weight and are conjugated to near-infrared fluorescent (NIRF) dyes that have differences in hydrophilicity, serum protein binding affinity, and clearance mechanism. We systematically investigated the roles of each of these properties on tumor accumulation, relative biodistribution, and the impact of intraoperative imaging of orthotopic, syngeneic pancreatic cancer. Overall, each HA-NIRF conjugate displayed intra-pancreatic tumor enhancement compared to uninvolved pancreas at 24 and 96 h. Regardless of HA molecular weight, Cy7.5 conjugation directed biodistribution to the liver, spleen, and bowels. Conjugation of IRDye-800 to 5 and 20 kDa HA resulted in low liver and spleen signal, while preserving tumor contrast enhancement up to 14-fold compared to healthy pancreas. When IRDye800 was conjugated to 100 kDa HA, the conjugate preferentially distributed to RES organs. When assessing the imaging efficacy of HA-based conjugates in hepatic metastases, those that accumulated to the liver utmost (HA100k-Cy7.5, HA100k-IRDye800, NanoICG) turned to aid the identification of hepatic malignancy with hypo-contrast. These studies demonstrate that by tuning HA molecular weight and the physicochemical properties of the conjugated moiety, in this case a NIRF probe, peritoneal biodistribution can be substantially altered to achieve optimized delivery to tumors with robust contrast enhancement for intraoperative imaging to abdominal tumors. Aside from assisting the accurate delineation of primary tumor, HA-NIRF conjugates demonstrated potential for identification of occult metastases in the intraoperative setting, as a versatile tool for accurate staging

Development of three-dimensional, ex vivo optical imaging

The ability to analyse tissue in 3-D at the mesoscopic scale (resolution: 2-50 µm) has proven essential in the study of whole specimens and individual organs. Techniques such as ex vivo magnetic resonance imaging (MRI) and X-ray computed tomography (CT) have been successful in a number of applications. Although MRI has been used to image embryo development and gene expression in 3-D, its resolution is not sufficient to discriminate between the small structures in embryos and individual organs. Furthermore, since neither MRI nor X-ray CT are optical imaging techniques, none of them is able to make use of common staining techniques. 3-D images can be generated with confocal microscopy by focusing a laser beam to a point within the sample and collecting the fluorescent light coming from that specific plane, eliminating therefore out-of-focus light. However, the main drawback of this microscopy technique is the limited depth penetration of light (~1 mm). Tomographic techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (also known as single plane illumination microscopy, SPIM) are novel methods that fulfil a requirement for imaging of specimens which are too large for confocal imaging and too small for conventional MRI. To allow sufficient depth penetration, these approaches require specimens to be rendered transparent via a process known as optical clearing, which can be achieved using a number of techniques. The aim of the work presented in this thesis was to develop methods for threedimensional, ex vivo optical imaging. This required, in first instance, sample preparation to clear (render transparent) biological tissue. In this project several optical clearing techniques have been tested in order to find the optimal method per each kind of tissue, focusing on tumour tissue. Indeed, depending on its structure and composition (e.g. amount of lipids or pigments within the tissue) every tissue clears at a different degree. Though there is currently no literature reporting quantification of the degree of optical clearing. Hence a novel, spectroscopic technique for measuring the light attenuation in optically cleared samples is described in this thesis and evaluated on mouse brain. 5 Optical clearing was applied to the study of cancer. The main cancer model investigated in this report is colorectal carcinoma. Fluorescently labelled proteins were used to analyse the vascular network of colorectal xenograft tumours and to prove the effect of vascular disrupting agents on the vascular tumour network. Furthermore, optical clearing and fluorescent compounds were used for ex vivo analysis of perfusion of a human colorectal liver metastasis model