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- All Subjects: Biomedical Engineering
Description
DNA methylation (DNAm) is an epigenetic mark with a critical role in regulating gene expression. Altered clinical states, including toxin exposure and viral infections, can cause aberrant DNA methylation in cells, which may persist during cell division. Current methods to study genome-wide methylome profiles of the cells require a long processing time and are expensive. Here, a novel technique called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which is amenable to automation. Up to 15 different samples can be combined into the same run of Mx-MeDIP-Seq, using only 25 ng of DNA per sample. Mx-MeDIP-Seq was used to study DNAm profiles of peripheral blood mononuclear cells (PBMCs) in two biologically distinct RNA viral infections with different modes of transmission, symptoms, and interaction with the host immune system: human immunodeficiency virus1 (HIV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Analysis of 90 hospitalized patients with SARS-CoV-2 and 57 healthy controls revealed that SARS-CoV-2 infection led to alterations in 920 methylated regions in PBMCs, resulting in a change in transcription that affects host immune response and cell survival. Analysis of publicly available RNA-Sequencing data in COVID-19 correlated with DNAm in several key pathways. These findings provide a mechanistic view toward further understanding of viral infections. Genome-wide DNAm changes post HIV-1-infection from 37 chronically ill patients compared to 17 controls revealed dysregulation of the actin cytoskeleton, which could contribute to the establishment of latency in HIV-1 infections. Longitudinal DNAm analysis identified several potentially protective and harmful genes that could contribute to disease suppression or progression.
ContributorsRidha, Inam (Author) / LaBaer, Joshua (Thesis advisor) / Murugan, Vel (Thesis advisor) / Plaisier, Christopher (Committee member) / Nikkhah, Mehdi (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2022
Description
According to the World Health Organization, cancer is one of the leading causes of death around the world. Although early diagnostics using biomarkers and improved treatments with targeted therapy have reduced the rate of cancer related mortalities, there remain many unknowns regarding the contributions of the tumor microenvironment to cancer progression and therapeutic resistance. The tumor microenvironment plays a significant role by manipulating the progression of cancer cells through biochemical and biophysical signals from the surrounding stromal cells along with the extracellular matrix. As such, there is a critical need to understand how the tumor microenvironment influences the molecular mechanisms underlying cancer metastasis to facilitate the discovery of better therapies. This thesis described the development of microfluidic technologies to study the interplay of cancer cells with their surrounding microenvironment. The microfluidic model was used to assess how exposure to chemoattractant, epidermal growth factor (EGF), impacted 3D breast cancer cell invasion and enhanced cell motility speed was noted in the presence of EGF validating physiological cell behavior. Additionally, breast cancer and patient-derived cancer-associated fibroblast (CAF) cells were co-cultured to study cell-cell crosstalk and how it affected cancer invasion. GPNMB was identified as a novel gene of interest and it was shown that CAFs enhanced breast cancer invasion by up-regulating the expression of GPNMB on breast cancer cells resulting in increased migration speed. Lastly, this thesis described the design, biological validation, and use of this microfluidic platform as a new in vitro 3D organotypic model to study mechanisms of glioma stem cell (GSC) invasion in the context of a vascular niche. It was confirmed that CXCL12-CXCR4 signaling is involved in promoting GSC invasion in a 3D vascular microenvironment, while also demonstrating the effectiveness of the microfluidic as a drug screening assay. Taken together, the broader impacts of the microfluidic model developed in this dissertation include, a possible alternative platform to animal testing that is focused on mimicking human physiology, a potential ex vivo platform using patient-derived cells for studying the interplay of cancer cells with its surrounding microenvironment, and development of future therapeutic strategies tailored toward disrupting key molecular pathways involved in regulatory mechanisms of cancer invasion.
ContributorsTruong, Danh, Ph.D (Author) / Nikkhah, Mehdi (Thesis advisor) / LaBaer, Joshua (Committee member) / Smith, Barbara (Committee member) / Mouneimne, Ghassan (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
Breast cancer is the most common cancer and currently the second leading cause of death among women in the United States. Patients’ five-year relative survival rate decreases from 99% to 25% when breast cancer is diagnosed late. Immune checkpoint blockage has shown to be a promising therapy to improve patients’ outcome in many other cancers. However, due to the lack of early diagnosis, the treatment is normally given in the later stages. An early diagnosis system for breast cancer could potentially revolutionize current treatment strategies, improve patients’ outcomes and even eradicate the disease. The current breast cancer diagnostic methods cannot meet this demand. A simple, effective, noninvasive and inexpensive early diagnostic technology is needed. Immunosignature technology leverages the power of the immune system to find cancer early. Antibodies targeting tumor antigens in the blood are probed on a high-throughput random peptide array and generate a specific binding pattern called the immunosignature.
In this dissertation, I propose a scenario for using immunosignature technology to detect breast cancer early and to implement an early treatment strategy by using the PD-L1 immune checkpoint inhibitor. I develop a methodology to describe the early diagnosis and treatment of breast cancer in a FVB/N neuN breast cancer mouse model. By comparing FVB/N neuN transgenic mice and age-matched wild type controls, I have found and validated specific immunosignatures at multiple time points before tumors are palpable. Immunosignatures change along with tumor development. Using a late-stage immunosignature to predict early samples, or vice versa, cannot achieve high prediction performance. By using the immunosignature of early breast cancer, I show that at the time of diagnosis, early treatment with the checkpoint blockade, anti-PD-L1, inhibits tumor growth in FVB/N neuN transgenic mouse model. The mRNA analysis of the PD-L1 level in mice mammary glands suggests that it is more effective to have treatment early.
Novel discoveries are changing understanding of breast cancer and improving strategies in clinical treatment. Researchers and healthcare professionals are actively working in the early diagnosis and early treatment fields. This dissertation provides a step along the road for better diagnosis and treatment of breast cancer.
In this dissertation, I propose a scenario for using immunosignature technology to detect breast cancer early and to implement an early treatment strategy by using the PD-L1 immune checkpoint inhibitor. I develop a methodology to describe the early diagnosis and treatment of breast cancer in a FVB/N neuN breast cancer mouse model. By comparing FVB/N neuN transgenic mice and age-matched wild type controls, I have found and validated specific immunosignatures at multiple time points before tumors are palpable. Immunosignatures change along with tumor development. Using a late-stage immunosignature to predict early samples, or vice versa, cannot achieve high prediction performance. By using the immunosignature of early breast cancer, I show that at the time of diagnosis, early treatment with the checkpoint blockade, anti-PD-L1, inhibits tumor growth in FVB/N neuN transgenic mouse model. The mRNA analysis of the PD-L1 level in mice mammary glands suggests that it is more effective to have treatment early.
Novel discoveries are changing understanding of breast cancer and improving strategies in clinical treatment. Researchers and healthcare professionals are actively working in the early diagnosis and early treatment fields. This dissertation provides a step along the road for better diagnosis and treatment of breast cancer.
ContributorsDuan, Hu (Author) / Johnston, Stephen Albert (Thesis advisor) / Hartwell, Leland Harrison (Committee member) / Dinu, Valentin (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2015
Description
Detection of molecular interactions is critical for understanding many biological processes, for detecting disease biomarkers, and for screening drug candidates. Fluorescence-based approach can be problematic, especially when applied to the detection of small molecules. Various label-free techniques, such as surface plasmon resonance technique are sensitive to mass, making it extremely challenging to detect small molecules. In this thesis, novel detection methods for molecular interactions are described.
First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.
Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.
Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.
Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.
First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.
Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.
Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.
Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.
ContributorsGuan, Yan (Author) / Tao, Nongjian (Thesis advisor) / LaBaer, Joshua (Committee member) / Goryll, Michael (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2015
Description
Though DNA nanostructures (DNs) have become interesting subjects of drug delivery, in vivo imaging and biosensor research, however, for real biological applications, they should be ‘long circulating’ in blood. One of the crucial requirements for DN stability is high salt concentration (like ~5–20 mM Mg2+) that is unavailable in a cell culture medium or in blood. Hence DNs denature promptly when injected into living systems. Another important factor is the presence of nucleases that cause fast degradation of unprotected DNs. The third factor is ‘opsonization’ which is the immune process by which phagocytes target foreign particles introduced into the bloodstream. The primary aim of this thesis is to design strategies that can improve the in vivo stability of DNs, thus improving their pharmacodynamics and biodistribution.
Several strategies were investigated to address the three previously mentioned limitations. The first attempt was to study the effect length and conformation of polyethylene glycol (PEG) on DN stability. DNs were also coated with PEG-lipid and human serum albumin (HSA) and their stealth efficiencies were compared. The findings reveal that both PEGylation and albumin coating enhance low salt stability, increase resistance towards nuclease action and reduce uptake of DNs by macrophages. Any protective coating around a DN increases its hydrodynamic radius, which is a crucial parameter influencing their clearance. Keeping this in mind, intrinsically stable DNs that can survive low salt concentration without any polymer coating were built. Several DNA compaction agents and DNA binders were screened to stabilize DNs in low magnesium conditions. Among them arginine, lysine, bis-lysine and hexamine cobalt showed the potential to enhance DN stability.
This thesis also presents a sensitive assay, the Proximity Ligation Assay (PLA), for the estimation of DN stability with time. It requires very simple modifications on the DNs and it can yield precise results from a very small amount of sample. The applicability of PLA was successfully tested on several DNs ranging from a simple wireframe tetrahedron to a 3D origami and the protocol to collect in vivo samples, isolate the DNs and measure their stability was developed.
Several strategies were investigated to address the three previously mentioned limitations. The first attempt was to study the effect length and conformation of polyethylene glycol (PEG) on DN stability. DNs were also coated with PEG-lipid and human serum albumin (HSA) and their stealth efficiencies were compared. The findings reveal that both PEGylation and albumin coating enhance low salt stability, increase resistance towards nuclease action and reduce uptake of DNs by macrophages. Any protective coating around a DN increases its hydrodynamic radius, which is a crucial parameter influencing their clearance. Keeping this in mind, intrinsically stable DNs that can survive low salt concentration without any polymer coating were built. Several DNA compaction agents and DNA binders were screened to stabilize DNs in low magnesium conditions. Among them arginine, lysine, bis-lysine and hexamine cobalt showed the potential to enhance DN stability.
This thesis also presents a sensitive assay, the Proximity Ligation Assay (PLA), for the estimation of DN stability with time. It requires very simple modifications on the DNs and it can yield precise results from a very small amount of sample. The applicability of PLA was successfully tested on several DNs ranging from a simple wireframe tetrahedron to a 3D origami and the protocol to collect in vivo samples, isolate the DNs and measure their stability was developed.
ContributorsBanerjee, Saswata (Author) / Yan, Hao (Thesis advisor) / Angell, Austen (Committee member) / Woodbury, Neal (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2018
Description
Solid tumors advance from benign stage to a deadly metastatic state due to the complex interaction between cancer cells and tumor microenvironment (TME) including stromal cells and extracellular matrix (ECM). Multiple studies have demonstrated that ECM dysregulation is one of the critical hallmarks of cancer progression leading to formation of a desmoplastic microenvironment that participates in tumor progression. Cancer associated fibroblasts (CAFs) are the predominant stromal cell type that participates in desmoplasia by depositing matrix proteins and increasing ECM stiffness. Although the influence of matrix stiffness on enhanced tumorigenicity has been well studied, the biological understanding about the dynamic changes in ECM architecture and the role of cancer-stromal cell interaction on ECM remodeling is still limited.
In this dissertation, the primary goal was to develop a comprehensive cellular and molecular level understanding of ECM remodeling due to the interaction of breast tumor cells and CAFs. To that end, a novel three-dimensional (3D) high-density tumor-stroma model was fabricated in which breast tumor cells (MDA-MB-231 and MCF7) were spatially organized surrounded by CAF-embedded collagen-I hydrogel (Aim 1). Further the platform was integrated with atomic force microscopy to assess the dynamic changes in ECM composition and stiffness during active tumor invasion. The results established an essential role of crosstalk between breast tumor cells and CAFs in ECM remodeling. The studies were further extended by dissecting the mode of interaction between tumor cells and CAFs followed by characterization of the role of various tumor secreted factors on ECM remodeling (Aim 2). The results for the first time established a critical role of paracrine signaling between breast tumor cells and CAFs in modulating biophysical properties of ECM. More in-depth analysis highlighted the role of tumor secreted cytokines, specifically PDGF-AA/BB, on CAF-induced desmoplasia. In aim 3, the platform was further utilized to test the synergistic influence of anti-fibrotic drug (tranilast) in conjugation with chemotherapeutic drug (Doxorubicin) on desmoplasia and tumor progression in the presence of CAFs. Overall this dissertation provided an in-depth understanding on the impact of breast cancer-stromal cell interaction in modulating biophysical properties of the ECM and identified the crucial role of tumor secreted cytokines including PDGF-AA/BB on desmoplasia.
In this dissertation, the primary goal was to develop a comprehensive cellular and molecular level understanding of ECM remodeling due to the interaction of breast tumor cells and CAFs. To that end, a novel three-dimensional (3D) high-density tumor-stroma model was fabricated in which breast tumor cells (MDA-MB-231 and MCF7) were spatially organized surrounded by CAF-embedded collagen-I hydrogel (Aim 1). Further the platform was integrated with atomic force microscopy to assess the dynamic changes in ECM composition and stiffness during active tumor invasion. The results established an essential role of crosstalk between breast tumor cells and CAFs in ECM remodeling. The studies were further extended by dissecting the mode of interaction between tumor cells and CAFs followed by characterization of the role of various tumor secreted factors on ECM remodeling (Aim 2). The results for the first time established a critical role of paracrine signaling between breast tumor cells and CAFs in modulating biophysical properties of ECM. More in-depth analysis highlighted the role of tumor secreted cytokines, specifically PDGF-AA/BB, on CAF-induced desmoplasia. In aim 3, the platform was further utilized to test the synergistic influence of anti-fibrotic drug (tranilast) in conjugation with chemotherapeutic drug (Doxorubicin) on desmoplasia and tumor progression in the presence of CAFs. Overall this dissertation provided an in-depth understanding on the impact of breast cancer-stromal cell interaction in modulating biophysical properties of the ECM and identified the crucial role of tumor secreted cytokines including PDGF-AA/BB on desmoplasia.
ContributorsSaini, Harpinder (Author) / Nikkhah, Mehdi (Thesis advisor) / Ros, Robert (Committee member) / LaBaer, Joshua (Committee member) / Kodibagkar, Vikram (Committee member) / Ebrahimkhani, Mohammad (Committee member) / Arizona State University (Publisher)
Created2019