Three-Dimensional Microfluidic Based Tumor-Vascular Model to Study Cancer Cell Invasion and Intravasation

abstract: Breast cancer is the second leading cause of disease related death in women, contributing over 40,000 fatalities annually. The severe impact of breast cancer can be attributed to a poor understanding of the mechanisms underlying cancer metastasis. A primary aspect of cancer metastasis includes the invasion and intravasation that results in cancer cells disseminating from the primary tumor and colonizing distant organs. However, the integrated study of invasion and intravasation has proven to be challenging due to the difficulties in establishing a combined tumor and vascular microenvironments. Compared to traditional in vitro assays, microfluidic models enable spatial organization of 3D cell-laden and/or acellular matrices to better mimic human physiology. Thus, microfluidics can be leveraged to model complex steps of metastasis. The fundamental aim of this thesis was to develop a three-dimensional microfluidic model to study the mechanism through which breast cancer cells invade the surrounding stroma and intravasate into outerlying blood vessels, with a primary focus on evaluating cancer cell motility and vascular function in response to biochemical cues. A novel concentric three-layer microfluidic device was developed, which allowed for simultaneous observation of tumor formation, vascular network maturation, and cancer cell invasion/intravasation. Initially, MDA-MB-231 disseminated from the primary tumor and invaded the acellular collagen present in the adjacent second layer. The presence of an endothelial network in the third layer of the device drastically increased cancer cell invasion. Furthermore, by day 6 of culture, cancer cells could be visually observed intravasating into the vascular network. Additionally, the effect of tumor cells on the formation of the surrounding microvascular network within the vascular layer was evaluated. Results indicated that the presence of the tumor significantly reduced vessel diameter and increased permeability, which correlates with prior in vivo data. The novel three-layer platform mimicked the in vivo spatial organization of the tumor and its surrounding vasculature, which enabled investigations of cell-cell interactions during cancer invasion and intravasation. This approach will provide insight into the cascade of events leading up to intravasation, which could provide a basis for developing more effective therapeutics.Dissertation/ThesisMasters Thesis Biomedical Engineering 201

CD8 T-cell induction against vascular endothelial growth factor receptor 2 by Salmonella for vaccination purposes against a murine melanoma.

The Salmonella type III secretion system (T3SS) efficiently translocates heterologous proteins into the cytosol of eukaryotic cells. This leads to an antigen-specific CD8 T-cell induction in mice orally immunized with recombinant Salmonella. Recently, we have used Salmonella's T3SS as a prophylactic and therapeutic intervention against a murine fibrosarcoma. In this study, we constructed a recombinant Salmonella strain translocating the immunogenic H-2D(b)-specific CD8 T-cell epitope VILTNPISM (KDR2) from the murine vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a member of the tyrosine protein kinase family and is upregulated on proliferating endothelial cells of the tumor vasculature. After single orogastric vaccination, we detected significant numbers of KDR2-tetramer-positive CD8 T cells in the spleens of immunized mice. The efficacy of these cytotoxic T cells was evaluated in a prophylactic setting to protect mice from challenges with B16F10 melanoma cells in a flank tumor model, and to reduce dissemination of spontaneous pulmonary melanoma metastases. Vaccinated mice revealed a reduction of angiogenesis by 62% in the solid tumor and consequently a significant decrease of tumor growth as compared to non-immunized mice. Moreover, in the lung metastasis model, immunization with recombinant Salmonella resulted in a reduction of the metastatic melanoma burden by approximately 60%

Lipid-based nanoparticles for magnetic resonance molecular imaging : design, characterization, and application

In this thesis research is described which was aimed to develop lipidic nanoparticles for the investigation and visualization of atherosclerosis and angiogenesis with both magnetic resonance molecular imaging and optical techniques. The underlying rationale for this is that conventional MR imaging techniques are only capable of visualizing physiological and morphological changes, while magnetic resonance molecular imaging aims to depict cellular and molecular processes that are associated with or lie at the basis of pathological processes. This may lead to earlier detection, and improved diagnosis and prognosis of disease processes. Furthermore this technique may be very useful for the evaluation of a given therapy. The introduction of MRI as a molecular imaging modality is hampered by its low sensitivity compared to nuclear methods like PET and SPECT. With recent developments in chemistry and the synthesis of powerful, innovative, specific, and multimodal contrast agents, e.g. by introducing fluorescent properties as well, MRI is becoming increasingly important for molecular imaging. Therefore, the first aim of the research described in this thesis was to develop biocompatible nanoparticles that can be made target specific and can be detected by both MRI and optical techniques to allow the investigation of disease processes with two highly complementary imaging methods. Chapter 1 gives a general introduction in magnetic resonance molecular imaging and its potential use for the investigation of several pathological processes. Furthermore, contrast enhanced MRI based on differences in T1 and T2 relaxation times is explained. Lastly, different classes of contrast agents and their contrast generating properties are described. Amphphilic molecules are widely applied to serve as building blocks for nanoparticles in biomedical applications. In the field of drug targeting for example, liposomes comprised of amphiphilic molecules hold great promise and have been used extensively the last several decades. Furthermore, micelles, microemulsions, and other amphiphilic aggregates are also under investigation to serve as drug carriers. A relatively new application of lipidic nanoparticles is their use as contrast generating materials for MRI. In Chapter 2 the properties of amphiphilic molecules and their assembly in a wide range of aggregated structures are described. This is followed by an overview of different strategies that are employed to conjugate targeting ligand to such lipid based nanoparticles. The emphasis of this chapter is a literature overview of what has been realized in this research field thus far. Chapter 3 describes the physical characterization of novel liposomal contrast agents. The morphology of different formulations was investigated with electron microscopy, which revealed the necessity of incorporating cholesterol in the liposomal bilayer. Furthermore the relaxation properties of these contrast agent were measured as a function of temperature and magnetic field strength. In Chapter 4 a liposomal contrast agent with both fluorescent and magnetic properties is described. The liposomes were made target specific by conjugating multiple E-selectin specific antibodies to the surface of the nanoparticle. Its feasibility to serve as molecular imaging contrast agent for the detection of the inflammation marker E-selectin is demonstrated in vitro. The specific uptake of the liposomes by human endothelial cells stimulated to express E-selectin was visualized by MRI and fluorescence microscopy. Chapter 5 describes a superparamagnetic nanoparticle encapsulated in a micellular shell. Fluorescent properties were introduced to this contrast agent by the incorporation of fluorescent lipids in the lipid layer. The contrast agent has a very high r2/r1 ratio and therefore is especially suitable to be used for T2 (*) enhanced MRI. The nanoparticle can be made target specific by covalently linking targeting ligands distally to the PEG chains of lipids incorporated in the micellular shell via maleimide-sulfhydryl coupling. Specificity for apoptotic cells was realized by conjugating multiple Annexin A5 proteins. The feasibility to use this contrast agent for molecular imaging purposes was demonstrated in vitro on apoptotic Jurkat cells. In Chapter 6 the synthesis and characterization of a novel bimodal nanoparticle based on semiconductor nanocrystals encapsulated within the corona of paramagnetic micelles is described. The CdSe nanoparticle, also referred to as quantum dot, serves as the contrast generating material for fluorescence imaging, while the paramagnetic micellular coating is employed for contrast enhanced MRI. The in vitro association of this nanoparticle with isolated cells by either conjugating multiple avß3-integrin specific RGDpeptides or multiple phosphatidyl serine specific Annexin A5 proteins was demonstrated with both fluorescence microscopy and MRI. The second aim of the research described in this thesis was to apply the novel nanoparticles for the investigation of atherosclerosis and tumor angiogenesis in mouse models with magnetic resonance molecular imaging. Chapter 7 describes the application of non targeted paramagnetic liposomes for the improved and sustained visualization of neointimal lesions induced after placing a constrictive collar around the right carotid artery of apoE-KO mice. Commercially available Gd-DTPA (Magnevist) showed little potential for the detection of such lesion. In Chapter 8 pegylated micelles conjugated with macrophage scavenger receptor (MSR) specific antibodies were employed for improved atherosclerotic plaque detection and characterization. Existing nanoparticulate agents that are used to detect macrophages, such as USPIO or lipophilic micelles, show little specificity. The micelles used for this study have a hydrophilic PEG coating, and therefore show minimal non-specific interaction with plaque, which results in negligible background signal. In case of the MSR micelles a pronounced enhancement of atherosclerotic plaque was observed. Furthermore, the micelles exhibit fluorescent properties by the incorporation of either quantum dots or fluorescent lipids. This allowed the detection of macrophages with optical techniques as well. Chapter 9 and Chapter 10 describe the application of avß3 targeted bimodal liposomes for the visualization of angiogenically activated tumor blood vessels with both MRI in vivo and fluorescence microscopy ex vivo. The specificity of the contrast agent was demonstrated with an MRI competition experiment, while the exclusive association with endothelial cells was demonstrated with fluorescence microscopy. The follow-up study demonstrates the usefulness of contrast enhanced MRI after applying this contrast agent for the evaluation of angiostatic therapies, i.e. using endostatin and anginex, at two time points after onset of therapy. Most importantly, the in vivo MRI data show very good correlation with ex vivo microvessel density determinations. In the last experimental Chapter 11 of this thesis a sophisticated method for the parallel visualization of angiogenic tumor blood vessels with both intravital microscopy (IVM) and MRI is described. The nanoparticulate contrast agent conjugated with avß3-specific RGDpeptides described in Chapter 6 was administrated to tumor bearing mice. IVM allowed the investigation of the disease process at the cellular level, while MRI was used to investigate angiogenesis at the anatomical level. The contrast agent possesses excellent contrast generating properties for these complementary imaging techniques. Widespread angiogenic activity within the rim of the tumor, and up to 1 cm from the tumor boundary could be observed by using both techniques

Cell-Oriented Modeling of Angiogenesis

Due to its significant involvement in various physiological and pathological conditions, angiogenesis (the development of new blood vessels from an existing vasculature) represents an important area of the actual biological research and a field in which mathematical modeling proved particularly useful in supporting the experimental work. In this paper, we focus on a specific modeling strategy, known as “cell-centered” approach. This type of mathematical models work at a “mesoscopic scale,” assuming the cell as the natural level of abstraction for computational modeling of development. They treat cells phenomenologically, considering their essential behaviors to study how tissue structure and organization emerge from the collective dynamics of multiple cells. The main contributions of the cell-oriented approach to the study of the angiogenic process will be described. From one side, they have generated “basic science understanding” about the process of capillary assembly during development, growth, and pathology. On the other side, models were also developed supporting “applied biomedical research” for the purpose of identifying new therapeutic targets and clinically relevant approaches for either inhibiting or stimulating angiogenesis

In Vitro and In Vivo Anti-Angiogenic Activities of Panduratin A

Targeting angiogenesis has emerged as an attractive and promising strategy in anti-cancer therapeutic development. The present study investigates the anti-angiogenic potential of Panduratin A (PA), a natural chalcone isolated from Boesenbergia rotunda by using both in vitro and in vivo assays.PA exerted selective cytotoxicity on human umbilical vein endothelial cells (HUVECs) with IC(50) value of 6.91 ± 0.85 µM when compared to human normal fibroblast and normal liver epithelial cells. Assessment of the growth kinetics by cell impedance-based Real-Time Cell Analyzer showed that PA induced both cytotoxic and cytostatic effects on HUVECs, depending on the concentration used. Results also showed that PA suppressed VEGF-induced survival and proliferation of HUVECs. Furthermore, endothelial cell migration, invasion, and morphogenesis or tube formation demonstrated significant time- and dose-dependent inhibition by PA. PA also suppressed matrix metalloproteinase-2 (MMP-2) secretion and attenuated its activation to intermediate and active MMP-2. In addition, PA suppressed F-actin stress fiber formation to prevent migration of the endothelial cells. More importantly, anti-angiogenic potential of PA was also evidenced in two in vivo models. PA inhibited neo-vessels formation in murine Matrigel plugs, and angiogenesis in zebrafish embryos.Taken together, our study demonstrated the distinctive anti-angiogenic properties of PA, both in vitro and in vivo. This report thus reveals another biological activity of PA in addition to its reported anti-inflammatory and anti-cancer activities, suggestive of PA's potential for development as an anti-angiogenic agent for cancer therapy