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- Creators: Barrett, The Honors College
Description
After more than 40 years since the signing of the National Cancer Act in 1970, cancer remains a formidable challenge. Cancer is currently the second most common cause of death in the United States, and worldwide cancer cases are projected to rise 50% between 2012 and 2030 [1-2]. While researchers have dramatically expanded our understanding of the biology of cancer, they have also revealed the staggering complexity and difficulty of developing successful treatments for the disease. More complex assays involving three dimensional cell culture offer the potential to model complex interactions, such as those involving the extracellular matrix (ECM), chemical concentration gradients, and the impact of vascularization of a tissue mass. Modern cancer assays thus promise to be both more accurate and more complex than previous models. One promising newly developed type of assay is microfluidics. Microfluidic devices consist of a silicone polymer stamp bonded to a glass slide. The stamp is patterned to produce a network of channels for cell culture. These devices allow manipulation of liquids on a sub-millimeter level, allowing researchers to produce a tightly controlled 3D microenvironment for cell culture. Our lab previously developed a microfluidic device to measure cancer cell invasion in response to a variety of signals and conditions. The small volume associated with microfluidics offers a number of advantages, but simultaneously make it impractical to use certain traditional cell analysis procedures, such as Western Blotting. As a result, measuring protein expression of cells in the microfluidic device was a continuing challenge. In order to expand the utility of microfluidic devices, it was therefore very enticing to develop a means of measuring protein expression inside the device. One possible solution was identified in the technique of In-Cell-Western blotting (ICW). ICW consists of using infrared-fluorescently stained antibodies to stain a protein of interest. This signal is measured using an infrared laser scanner, producing images that can be analyzed to quantitatively measure protein expression. ICW has been well validated in traditional 2D plate culture conditions, but has not been applied in conjunction with microfluidic devices. This project worked to evaluate In-Cell-Western blotting for use in microfluidic devices as a method of quantifying protein expression in situ.
ContributorsKratz, Alexander Franz (Author) / Nikkhah, Mehdi (Thesis director) / Truong, Danh (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The p53 gene functions as a tumor suppressor that inhibits proliferation, regulates apoptosis, DNA repair, and normal cell cycle arrest. Mutation of the p53 gene is linked to be prevalent in 50% of all human cancers. In this paper, we are exploring triple negative breast cancer and the effects of simvastatin on tumor growth and survival. Simvastatin is a drug that is primarily used to treat high cholesterol and heart disease. Simvastatin is unique because it is able to inhibit protein prenylation through regulation of the mevalonate pathway. This makes it a potential targeted drug for therapy against p53 mutant cancer. The mechanism behind this is hypothesized to be correlated to aberrant activation of the Ras pathway. The Ras subfamily functions to transcriptionally regulate cell growth and survival, and will therefore allow for a tumor to thrive if the pathway is continually and abnormally activated. The Ras protein has to be prenylated in order for activation of this pathway to occur, making statin drug treatment a viable option as a cancer treatment. This is because it acts as a regulator of the mevalonate pathway which is upstream of protein prenylation. It is thus vital to understand these pathways at both the gene and protein level in different p53 mutants to further understand if simvastatin is indeed a drug with anti-cancer properties and can be used to target cancers with p53 mutation. The goal of this project is to study the biochemistry behind the mutation of p53's sensitivity to statin. With this information we can create a possible signature for those who could benefit from Simvastatin drug treatment as a possible targeted treatment for p53 mutant cancers.
ContributorsGrewal, Harneet (Co-author) / Loo, Yi Jia Valerie (Co-author) / Anderson, Karen (Thesis director) / Blattman, Joseph (Committee member) / Ferdosi, Shayesteh (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The anthracycline drug Doxorubicin (DOX) is a highly effective treatment for breast cancer, but its clinical utility is limited by dose-dependent cardiovascular toxicity. The toxic effects are partly attributed to DOX-induced generation of reactive oxygen species, which may impair nitric oxide-mediated vasodilation. Exercise training activates antioxidant defense mechanisms and is thus hypothesized to counteract oxidative stress when initiated prior to DOX administration. Adult 8-week old, ovariectomized female Sprague-Dawley rats were divided into 4 groups: sedentary + vehicle (Sed+Veh); Sed+DOX; exercise + veh (Ex+Veh); and Ex+DOX. Rats in the exercise groups were preconditioned with high intensity interval training consisting of 4x4 minute bouts of exercise at 85-95% of VO2peak separated by 2 minutes of active recovery performed 5 days per week. Exercise was implemented one week prior to the first injection and continued throughout the study. Animals received either DOX (4mg/kg) or veh (saline) intraperitoneal injections bi-weekly for a cumulative dose of 12 mg/kg per animal. Five days following the final injection, animals were anesthetized with isoflurane, decapitated and aortas and perivascular adipose tissue (PVAT) were removed for western blot analyses. No significant differences in aortic protein expression were detected for inducible nitric oxide synthase (iNOS) or the upstream activator of endothelial nitric oxide synthase (eNOS), Akt, across groups (p>0.05), whereas eNOS protein expression was significantly downregulated in Sed+DOX (p=0.003). In contrast, eNOS expression was not altered in Ex+DOX treated animals. Protein expression of iNOS in PVAT was upregulated with exercise in the DOX-treated groups (p=0.039). These findings suggest that exercise preconditioning may help mitigate vascular effects of DOX by preventing downregulation of eNOS in the aorta.
ContributorsO'Neill, Liam Martin (Author) / Sweazea, Karen (Thesis director) / Angadi, Siddhartha (Committee member) / Dickinson, Jared (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.
ContributorsPaxton, Rebecca Joanne (Author) / Stephanopoulos, Nicholas (Thesis director) / He, Ximin (Committee member) / Gould, Ian (Committee member) / Materials Science and Engineering Program (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Prostate cancer is the second most common kind of cancer in men. Fortunately, it has a 99% survival rate. To achieve such a survival rate, a variety of aggressive therapies are used to treat prostate cancers that are caught early. Androgen deprivation therapy (ADT) is a therapy that is given in cycles to patients. This study attempted to analyze what factors in a group of 79 patients caused them to stick with or discontinue the treatment. This was done using naïve Bayes classification, a machine-learning algorithm. The usage of this algorithm identified high testosterone as an indicator of a patient persevering with the treatment, but failed to produce statistically significant high rates of prediction.
ContributorsMillea, Timothy Michael (Author) / Kostelich, Eric (Thesis director) / Kuang, Yang (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
As advanced as current cancer therapeutics are, there are still challenges that need to be addressed. One of them is the non-specific killing of normal cells in addition to cancerous cells. Ideal cancer therapeutics should be targeted specifically toward tumor cells. Due to the robust self-assembly and versatile addressability of DNA-nanostructures, a DNA tetrahedron nanostructure was explored as a drug carrier. The nanostructure can be decorated with various molecules to either increase immunogenicity, toxicity, or affinity to a specific cell type. The efficiency of the specific binding and internalization of the chosen molecules was measured via flow cytometry. Using a murine B cell lymphoma as the model system, several targeting molecules have been evaluated for their specific binding and induced internalization of DNA nanostructures, including an anti-Igκ antibody, an idiotype-binding peptide, and a g-quadruplex nucleolin specific aptamer. It was found that adding the anti-Igκ antibody appeared to provide increased binding and facilitated cellular internalization. Also, it was found that the presence of CpG appeared to aid in the binding of nanostructures decorated with other molecules, as compared to nanostructures without CpG. The g-quadruplex aptamer thought to specifically bind cancer cells that overexpress nucleolin was tested and found to have better binding to cells when linked to the nanostructure than when alone. The drug doxorubicin was used to load the DNA-nanostructure and attempt to inhibit cancer cell growth. The DNA-nanostructure has the benefit of being self-assembled and customizable, and it has been shown to bind to and internalize into a cancer cell line. The next steps are to test the toxicity of the nanostructure as well as its specificity for cancerous cells compared to noncancerous cells. Furthermore, once those tests are completed the structure’s drug delivery capacity will be tested in tumor bearing mice. The DNA-nanostructure exhibits potential as a cancer specific therapeutic.
ContributorsGomez, Amber Marie (Author) / Chang, Yung (Thesis director) / Anderson, Karen (Committee member) / Liu, Xiaowei (Committee member) / Sanford School of Social and Family Dynamics (Contributor) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Esophageal adenocarcinoma is one of the largest growing cancer types in the United States and the whole world. One of the only known precursors to EAC is Barrett’s Esophagus, the changing of the normal squamous cells which line the esophagus into intestinal cells, following repeated exposure to gastric acids via gastroesophageal reflux disease. There is limited knowledge of the mutations and drivers that contribute to EAC’s low 5-year survival rates, demonstrating a need to identify new therapeutic targets. Given the development of EAC from chronic inflammation and acidic microenvironment, elevated expression of tumor necrosis factor receptor super family member 12A (TNFRSF12A, FN14) and its corresponding ligand, TWEAK, is correlated with disease progression. The functional role of the TWEAK/FN14 signaling axis is well documented in other cancer types, contributing to tumor invasion, migration, and survival. However, reports have shown the TWEAK/FN14 signaling axis can contribute “pro-cancer” and “anti-cancer” phenotypes in different tumor microenvironments. In this study, we seek to demonstrate the functional role of TWEAK and FN14 in EAC survival and migration. We hypothesized TWEAK/FN14 signaling would promoted EAC cell survival and migration. In this study, we illustrate increased expression of FN14 with disease progression. Following treatment with TWEAK, human EAC cell lines had increased sensitivity to standard chemotherapy treatment in vitro. Treatment with TWEAK also correlated with increased cellular migration, most likely in correlation with NF-κB activation. Finally, we showed that inhibition of FN14 via siRNA significantly reduced EAC survival and increased efficacy of standard of care treatments. This data suggests a diverse functional role of the TWEAK/FN14 signaling axis in EAC, and may be a potential target for novel therapeutics.
ContributorsFornefeld, Lucas Christien (Author) / Stout, Valerie (Thesis director) / Whitsett, Timothy (Committee member) / Carson, Vashti (Committee member) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
While specific resistance mechanisms to targeted inhibitors in BRAF-mutant cutaneous melanoma have been identified, surprisingly little is known about the rate at which resistance develops under different treatment options. There is increasing evidence that resistance arises from pre-existing clones rather than from de novo mutations, but there remains the need for a better understanding of how different drugs affect the fitness of clones within a tumor population and promote or delay the emergence of resistance. To this end, we have developed an assay that defines the in vitro rate of adaptation by analyzing the progressive change in sensitivity of a melanoma cell line to different treatments. We performed a proof-of-theory experiment based on the hypothesis that drugs that cause cell death (cytotoxic) impose a higher selection pressure for drug-resistant clones than drugs that cause cell-cycle arrest (cytostatic drugs), thereby resulting in a faster rate of adaptation. We tested this hypothesis by continuously treating the BRAFV600E melanoma cell line A375 with the cytotoxic MEK inhibitor E6201 and the cytostatic MEK inhibitor trametinib, both of which are known to be effective in the setting of constitutive oncogenic signaling driven by the BRAF mutation. While the identification of confounding factors prevented the direct comparison between E6201-treated and trametinib-treated cells, we observed that E6201-treated cells demonstrate decreased drug sensitivity compared to vehicle-treated cells as early as 18 days after treatment begins. We were able to quantify this rate of divergence at 2.6% per passage by measuring the increase over time in average viability difference between drug-treated and vehicle-treated cells within a DDR analysis. We argue that this value correlates to the rate of adaptation. Furthermore, this study includes efforts to establish a barcoded cell line to allow for individual clonal tracking and efforts to identify synergistic and antagonist drug combinations for use in future experiments. Ultimately, we describe here a novel system capable of quantifying adaptation rate in cancer cells undergoing treatment, and we anticipate that this assay will prove helpful in identifying treatment options that circumvent or delay resistance through future hypothesis-driven experiments.
ContributorsDe Luca, Valerie Jean (Author) / Wilson Sayres, Melissa (Thesis director) / Trent, Jeff (Committee member) / Hendricks, William (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Big Data Network Analysis of Genetic Variation and Gene Expression in Individuals with Breast Cancer
Description
The advent of big data analytics tools and frameworks has allowed for a plethora of new approaches to research and analysis, making data sets that were previously too large or complex more accessible and providing methods to collect, store, and investigate non-traditional data. These tools are starting to be applied in more creative ways, and are being used to improve upon traditional computation methods through distributed computing. Statistical analysis of expression quantitative trait loci (eQTL) data has classically been performed using the open source tool PLINK - which runs on high performance computing (HPC) systems. However, progress has been made in running the statistical analysis in the ecosystem of the big data framework Hadoop, resulting in decreased run time, reduced storage footprint, reduced job micromanagement and increased data accessibility. Now that the data can be more readily manipulated, analyzed and accessed, there are opportunities to use the modularity and power of Hadoop to further process the data. This project focuses on adding a component to the data pipeline that will perform graph analysis on the data. This will provide more insight into the relation between various genetic differences in individuals with breast cancer, and the resulting variation - if any - in gene expression. Further, the investigation will look to see if there is anything to be garnered from a perspective shift; applying tools used in classical networking contexts (such as the Internet) to genetically derived networks.
ContributorsRandall, Jacob Christopher (Author) / Buetow, Kenneth (Thesis director) / Meuth, Ryan (Committee member) / Almalih, Sara (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
PD-L1 blockade has shown recent success in cancer therapy and cancer vaccine regimens. One approach for anti-PD-L1 antibodies has been their application as adjuvants for cancer vaccines. Given the disadvantages of such antibodies, including long half-life and adverse events related to their use, a novel strategy using synbodies in place of antibodies can be tested. Synbodies offer a variety of advantages, including shorter half-life, smaller size, and cheaper cost. Peptides that could bind PD-L1 were identified via peptide arrays and used to construct synbodies. These synbodies were tested with inhibition ELISA assays, SPR, and pull down assays. Additional flow cytometry analysis was done to determine the binding specificity of the synbodies to PD-L1 and the ability of those synbodies to inhibit the PD-L1/PD-1 interaction. Although analysis of permeabilized cells expressing PD-L1 indicated that the synbodies could successfully bind PD-L1, those results were not replicated in non-permeabilized cells. Further assays suggested that the binding of the synbodies was non-specific. Other tests were done to see if the synbodies could inhibit the PD-1/PD-L1 interaction. This assay did not yield any conclusive results and further experimentation is needed to determine the efficacy of the synbodies in inhibiting this interaction.
ContributorsMujahed, Tala (Author) / Johnston, Stephen (Thesis director) / Blattman, Joseph (Committee member) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12