Browsing by Author "Schmelz, Eva M."
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- The AFM Study of Ovarian Cell Structural Mechanics in the Progression of CancerKetene, Alperen Nurullah (Virginia Tech, 2011-05-06)According to the American Cancer Society, Cancer is the second most common cause of death in the United States, only exceeded by heart disease. Over the past decade, deciphering the complex structure of individual cells and understanding the symptoms of cancer disease has been a highly emphasized research area. The exact cause of Cancer and the genetic heterogeneity that determines the severity of the disease and its response to treatment has been a great challenge. Researchers from the engineering discipline have increasingly made use of recent technological innovations, namely the Atomic Force Microscope (AFM), to better understand cell physics and provide a means for cell biomechanical profiling. The presented work's research objective is to establish a fundamental framework for the development of novel biosensors for cell separation and disease diagnosis. By using AFM nanoindentation, several studies were conducted to identify key distinctions in the trends of cell viscoelasticity between healthy, nontumorigenic cells and their malignant, highly tumorigenic counterparts. The possibility of identifying useful 'biomarkers' was also investigated. Due to the lack of an available human ovarian cell line, experiments were done on a recently developed mouse ovarian surface epithelial (MOSE) cell line, which resembles to human cell characteristics and represents early, intermediate, and late stages of the ovarian cancer. Material properties were extracted via Hertz model contact theory. The experimental results illustrate that the elasticity of late stage MOSE cells were 50% less than that of the early stage. Cell viscosity also decreased by 65% from early to late stage, indicating that the increase in cell deformability directly correlates with increasing levels of malignancy. Various cancer treatment and component-specific drugs were used to identify the causes for the changes in cell biomechanical behavior, depicting that the decrease in the concentration levels of cell structural components, predominantly the actin filament framework, is directly associated with the changes in cell biomechanical property. The investigation of MOSE cells being subject to multiple mechanical loads illustrated that healthy cells react to shear forces by stiffening up to 25% of their original state. On the other hand, cancerous cells are void of such response and at times show signs of decreasing rigidity. Finally, deformation studies on MOSE cancer stem cells have shown that these cells carry a unique elasticity profile among other cell stage phenotypes that could allow for their detection. The results herein carry great potential into contributing to cell separation methods and analysis, furthering the understanding of cell mechanism dynamics. While prior literature emphasizes on the elastic modulus of cells, the study of cell viscosity and other key material properties holds a critical place in the realistic modeling of these complex microstructures. A comprehensive study of individual cells holds a great amount of promise in the development of effective clinical research in the fight against cancer.
- The Biological and Immunological Effects of Irreversible Electroporation and Combination Therapy Options for the Improving the Treatment of Pancreatic CancerBrock, Rebecca Michaela (Virginia Tech, 2020-06-05)While cancer treatments have advanced for multiple cancers, pancreatic cancer remains a lethal cancer with few therapy options available. This is due to the inaccessibility of the tumor by surgical and thermal ablative means, high potential for chemoresistance and metastasis, and strongly immunosuppressive tumor microenvironment that makes new treatment measures ineffective in clinic. Irreversible electroporation (IRE) utilizes short, high voltage electrical pulses to form microlesions in cell membranes and induce cell death. While IRE has had significant impact in pancreatic cancer treatment in clinical trials, little is known on the biological and immunological effects of IRE on pancreatic cancer. By studying the effects of IRE on pancreatic tumor biology and the host immune system, I hypothesize I can identify potential combination therapy targets for IRE. I utilized in vitro, ex vivo, and in vivo animal models of both human and murine cancer to study the effects of IRE on pancreatic cancer progression and its potential for immunomodulation. My findings have shown that IRE can significantly delay cancer progression by inducing necroptosis in the tumor and altering the tumor microenvironment by increasing inflammatory signaling. IRE can also produce viable antigens for presentation to induce local and systemic immunosurveillance. However, these effects are limited by countering expression of programmed-cell death ligand 1 (PD-L1), a checkpoint protein that inhibits cytotoxic lymphocyte activity and allows the tumors to recur. The effects of IRE can therefore be expanded by multiple combination therapy approaches, such as chemotherapeutic application (potentially with nanoparticle packaging), PD-1/PD-L1 antibody immunotherapies, and small molecule inhibitors directed at tumor growth signaling that previously showed limited efficacy in clinic.
- Cell Phenotype Analyzer: Automated Techniques for Cell Phenotyping using Contactless DielectrophoresisBala, Divya Chandrakant (Virginia Tech, 2016-06-23)Cancer is among the leading causes of death worldwide. In 2012, there were 14 million new cases and 8.2 million cancer-related deaths worldwide. The number of new cancer cases is expected rise to 22 million within the next two decades. Most chronic cancers cannot be cured. However, if the precise cancer cell type is diagnosed at an earlier, less aggressive stage then the chance of curing the disease increases with accurate drug delivery. This work is a humble contribution to the advancement of cancer research. This work delves into biological cell phenotyping under a dielectrophoresis setup using computer vision. Dielectrophoresis is a well-known phenomenon in which dielectric particles are subjected to a non-homogeneous electric field. This work is an analytical part of a larger proposed system replete with hardware, software and microfluidics integration to achieve cancer cell characterization, separation and enrichment using contactless dielectrophoresis. To analyze the cell morphology, various detection and tracking algorithms have been implemented and tested on a diverse dataset comprising cell-separation video sequences. Other related applications like cell-counting and cell-proximity detection have also been implemented. Performances were evaluated against ground truth using metrics like precision, recall and RMS cell-count error. A detection approach using difference of Gaussian and super-pixel algorithm gave the highest average F-measure of 0.745. A nearest neighbor tracker and Kalman tracking method gave the best overall tracking performance with an average F-measure of 0.95. This combination of detection and tracking methods proved to be best suited for this dataset. A graphical user interface to automate the experimentation process of the proposed system was also designed.
- Changes in Gene Expression and Cellular Architecture in an Ovarian Cancer Progression ModelCreekmore, Amy L.; Silkworth, William T.; Cimini, Daniela; Jensen, Roderick V.; Roberts, Paul C.; Schmelz, Eva M. (PLOS, 2011-03-03)Background Ovarian cancer is the fifth leading cause of cancer deaths among women. Early stage disease often remains undetected due the lack of symptoms and reliable biomarkers. The identification of early genetic changes could provide insights into novel signaling pathways that may be exploited for early detection and treatment. Methodology/Principal Findings Mouse ovarian surface epithelial (MOSE) cells were used to identify stage-dependent changes in gene expression levels and signal transduction pathways by mouse whole genome microarray analyses and gene ontology. These cells have undergone spontaneous transformation in cell culture and transitioned from non-tumorigenic to intermediate and aggressive, malignant phenotypes. Significantly changed genes were overrepresented in a number of pathways, most notably the cytoskeleton functional category. Concurrent with gene expression changes, the cytoskeletal architecture became progressively disorganized, resulting in aberrant expression or subcellular distribution of key cytoskeletal regulatory proteins (focal adhesion kinase, α-actinin, and vinculin). The cytoskeletal disorganization was accompanied by altered patterns of serine and tyrosine phosphorylation as well as changed expression and subcellular localization of integral signaling intermediates APC and PKCβII. Conclusions/Significance Our studies have identified genes that are aberrantly expressed during MOSE cell neoplastic progression. We show that early stage dysregulation of actin microfilaments is followed by progressive disorganization of microtubules and intermediate filaments at later stages. These stage-specific, step-wise changes provide further insights into the time and spatial sequence of events that lead to the fully transformed state since these changes are also observed in aggressive human ovarian cancer cell lines independent of their histological type. Moreover, our studies support a link between aberrant cytoskeleton organization and regulation of important downstream signaling events that may be involved in cancer progression. Thus, our MOSE-derived cell model represents a unique model for in depth mechanistic studies of ovarian cancer progression.
- Characterization of Influenza:Streptococcus pneumoniae synergistic disease and potential for disease alleviation via sphingolipid therapyGasser, Amanda Lynn (Virginia Tech, 2013-09-06)Influenza A virus (IAV) is generally associated with the seasonal malady that causes brief respiratory illness during the winter months, known simply as "the flu." Most otherwise healthy individuals will suffer from mild fever, congestion, headaches and myalgia that are resolved within 5-7 days of onset. However, there are nearly 500,000 influenza-related deaths that occur world-wide every year. Many of these casualties and patients hospitalized with influenza also test positive for bacterial pneumonia, the most common agent being Streptococcus pneumoniae. Although all individuals are subject to this viral:bacterial synergistic disease, the young, elderly, and immunocompromised are the most susceptible. Previous studies have shown that viral infection creates a prolonged hyper-responsive pro-inflammatory state in the lungs, which increases susceptibility to secondary bacterial infection. Lethality is due to detrimental pulmonary damage from a dysregulated host inflammatory response, known as the "cytokine storm." However, the nature of dual infection has not been well-studied in the elderly demographic. Therefore, we aim to better define this disease synergy in an aged mouse model and explore potential therapeutic alternatives that could be beneficial for the aged and other vulnerable populations. Sphingolipid modulation has emerged as a potential target to ameliorate the excessive inflammation (cytokine storm) elicited by highly pathogenic influenza. There is particular emphasis on sphingosine 1-phosphate (S1P) signaling, as well as control of intracellular S1P levels via sphingosine kinases (SK). Sphingolipids are involved in a multitude of cellular processes, and are tightly regulated by their metabolizing enzymes. We hypothesize that manipulation of sphingolipid signaling and alteration of the internal sphingolipid milieu will diminish the inflammatory response elicited by IAV infection. Using fluorescence-activated cell sorting (FACS), real-time PCR and cytometric bead array (CBA) analysis, we evaluated the immunomodulatory effects of systemic sphingosine analog treatment within the lung microenvironment under homeostatic and influenza-infected conditions. FTY720 treatment caused transient, but significant lymphopenia, influx of neutrophils and efflux of macrophages in the lungs, which was enhanced during a mild influenza infectionGene expression in the lungs was generally unaltered, but protein levels showed increases in specific influenza-induced cytokines, suggesting these treatments may have post-transcriptional effects on cytokine expression. To evaluate sphingolipid modulation in specific pulmonary cell types, we next observed the effects of these compounds and sphingosine kinase (SK) inhibitors in epithelial and alveolar macrophage-like cell lines. SK inhibitors and Enigmol demonstrated anti-viral effects in A549 cells, decreasing viral loads by up to 1.5 logs. Real-time PCR and CBA analysis further demonstrated that these effects were associated with alterations in key cytokine expression, including CCL2, CCL5, CXCL10, IL-6, and IL-8. Collectively, these findings indicate that therapeutic sphingolipid modulation has the potential for creating a protective microenvironment in the lungs that could alleviate or even prevent viral:bacterial synergistic disease.
- Characterization of Metabolic Differences in Benign, Slow Developing and Tumor Initiating Ovarian CancersAnderson, Angela S. (Virginia Tech, 2013-05-14)Ovarian cancer is known as the "silent killer," due to its late diagnosis and frequent recurrence after initial treatment. Finding a new way to diagnose and treat ovarian cancer in conjunction with current therapies is paramount. By capitalizing on metabolic changes that occur during cancer progression, interventions can be developed. The Nobel laureate Otto Warburg is credited with discovering an altered metabolic state within cancer cells known as the Warburg effect. In the Warburg effect, cancer cells participate in an increased rate of aerobic glycolysis with an excess secretion of lactate, allowing for carbon flux into biosynthetic pathways. Exactly which metabolic pathways are altered in ovarian cancer and at which stage in the progression of ovarian cancer they are occurring was unknown. Therefore using the recently established mouse ovarian surface epithelial (MOSE) progression model, we were able to measure metabolic changes in varying states of disease and levels of aggressiveness. As cells progressed from a benign early stage (MOSE-E), through a transitional intermediate stage (MOSE-I), to an aggressive late stage (MOSE-L), the MOSE cells became more glycolytic and lipogenic, establishing the MOSE model as a valuable model for studying ovarian cancer metabolism. Treating the MOSE cells with the naturally occurring chemotherapeutic agent sphingosine decreased p-AKT protein levels in the cell, decreased the glycolytic rate and decreased de novo cholesterol synthesis. Cancer stem cells are known to be resistant to chemotherapy treatments and targeting their metabolism may be promising for combinatorial treatments. Therefore, the metabolism of highly aggressive tumor-initiating cells (TIC), harvested from ascites of C57Bl/6 mice injected with MOSE-L cells were characterized. Although the basal metabolism of the TICs was similar to the MOSE-L cells, TICs were more resistant to cell death as a consequence of external stresses and substrate depletion. The TICs could also up-regulate oxygen consumption rate (OCR) when uncoupled and increase glycolysis when ATP Synthase was inhibited, highlighting their resiliency. Taken together, we have identified targets for treatment strategies that could suppress the growth of primary tumors and may be effective against TICs, thereby suppressing tumor recurrence and possibly prolonging the life of women with ovarian cancer.
- Characterization of sequentially-staged cancer cells using electrorotationTrainito, Claudia I.; Sweeney, Daniel C.; Čemazăr, Jaka; Schmelz, Eva M.; Français; Le Pioufle, Bruno; Davalos, Rafael V. (PLOS, 2019-09-19)The identification and separation of cells from heterogeneous populations is critical to the diagnosis of diseases. Label-free methodologies in particular have been developed to manipulate individual cells using properties such as density and morphology. The electrical properties of malignant cells, including the membrane capacitance and cytoplasmic conductivity, have been demonstrated to be altered compared to non-malignant cells of similar origin. Here, we exploit these changes to characterize individual cells in a sequentially-staged in vitro cancer model using electrorotation (EROT)—the rotation of a cell induced by a rotating electric field. Using a microfabricated device, a dielectrophoretic force to suspend cells while measuring their angular velocity resulting from an EROT force applied at frequencies between 3 kHz to 10 MHz. We experimentally determine the EROT response for cells at three stages of malignancy and analyze the resultant spectra by considering models that include the effect of the cell membrane alone (single-shell model) and the combined effect of the cell membrane and nucleus (double-shell model). We find that the cell membrane is largely responsible for a given cell’s EROT response between 3 kHz and 10 MHz. Our results also indicate that membrane capacitance, membrane conductance, and cytoplasmic conductivity increase with an increasingly malignant phenotype. Our results demonstrate the potential of using electrorotation as a means making of non-invasive measurements to characterize the dielectric properties of cancer cells.
- A Computer-Aided Framework for Cell Phenotype Identification, Analysis and ClassificationPradeep, Subramanian (Virginia Tech, 2017-09-11)Cancer is arguably one of the most dangerous diseases and the major causes of death in the modern day. It becomes increasingly harder to treat and cure the disease as it makes progress. Detecting cancer at an early stage can help in preventing it from affecting an organism. However, it is very hard to detect at an early stage. The best possible way to tackle this disease is to first study it at a cellular level. This study aims at identifying various phenotypic traits of these cells in the Dielectrophoresis (DEP) based microfluidic device experimental setup and subsequently classifying the cells from the rest. A general framework for automatic labeling, identifying and classifying the malignant from the dead cells is developed in this work. The framework shows a top-down approach starting from static background subtraction, tracking, automatic labeling, feature extraction and finally classification. The data used in this work are videos of live and dead human prostate cancer (PC-3) cells flowing through the microfluidic device. Previous studies have shown that there are significant differences in morphological attributes between cancerous and non-cancerous cells. We focus mainly on shape, texture and geometry as the prominent attribute in our work and subsequently use them for classification. In this work we obtain good tracking results through optical flow as compared to previous work. For classification, linear classifiers such as logistic regression and linear Support Vector Machine (SVM) showed decent results. The machine learning algorithms use Histogram of Oriented Gradient (HOG) features plus the elliptical features as a combined feature vector. The elliptic features branch out this study to another direction that is useful in calculation of physical properties such as the cell elasticity through video processing and we propose a model for the same for the given setup. Currently, the elasticity of a single cell is calculated using expensive and time consuming procedures such as the atomic force microscopy (AFM). Using our framework, we can potentially obtain elasticity for a batch of cells in much less time. Also, our cell classification algorithm procedure is suitable for real time applications and can be a proposed futuristic concept for selective killing of cells.
- Contactless Dielectrophoresis towards Drug Screening and Microdevice Development for Cell SortingElvington, Elizabeth Ashcraft Savage (Virginia Tech, 2013-07-08)Firstly, this work demonstrates that contactless dielectrophoresis (cDEP) was useful to detect a reversal in the electrical phenotype of late-stage ovarian cancer cells to a profile similar to that of slow-growing early-stage ovarian epithelial cells after treatment with a non-toxic bioactive metabolite, sphingosine. Current chemotherapeutics are highly toxic to patients and can cause severe adverse side effects, so non-toxic treatments that could slow or reverse cancer growth would be advantageous. This is the first instance of cDEP for detecting induced changes in cell structure, showing its potential as a rapid, non-biomarker-based drug screening platform. Specifically, low frequency contactless dielectrophoresis devices previously designed by Sano et al were used to extract the crossover frequency and specific membrane capacitance of early and late stage mouse ovarian surface epithelial (MOSE-E and MOSE-L) cells when untreated, treated with the anti-cancer sphingosine (So) metabolite and with a generally cancer-supporting sphingosine-1-phosphate (S1P) metabolite. The specific membrane capacitance of MOSE-L cells treated with So decreased and the normalized crossover frequency increased to levels matching MOSE-E cells. Secondly, a new multilayer cDEP device featuring curved interdigitated electrode channels overlaying a straight sample channel for the purpose of cell sorting was designed, computationally modeled, fabricated, and tested. The goal of this design was to achieve continuous multi-stream sorting of cells, and preliminary testing demonstrated that prostate cancer PC3 cells were continuously deflected toward the top of the channel under an electric field, as predicted by the numerical model.
- Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer TreatmentZou, Peng; Liu, Longhua; Zheng, Louise D.; Payne, Kyle K.; Manjili, Masoud H.; Idowu, Michael O.; Zhang, Jinfeng; Schmelz, Eva M.; Cheng, Zhiyong (Hindawi, 2016-09-26)Overactive mitochondrial fission was shown to promote cell transformation and tumor growth. It remains elusive how mitochondrial quality is regulated in such conditions. Here, we show that upregulation of mitochondrial fission protein, dynamin related protein-1 (Drp1), was accompanied with increased mitochondrial biogenesis markers (PGC1α, NRF1, and Tfam) in breast cancer cells. However, mitochondrial number was reduced, which was associated with lower mitochondrial oxidative capacity in breast cancer cells. This contrast might be owing to enhanced mitochondrial turnover through autophagy, because an increased population of autophagic vacuoles engulfing mitochondria was observed in the cancer cells. Consistently, BNIP3 (a mitochondrial autophagy marker) and autophagic flux were significantly upregulated, indicative of augmented mitochondrial autophagy (mitophagy). The upregulation of Drp1 and BNIP3 was also observed in vivo (human breast carcinomas). Importantly, inhibition of Drp1 significantly suppressed mitochondrial autophagy, metabolic reprogramming, and cancer cell viability. Together, this study reveals coordinated increase of mitochondrial biogenesis and mitophagy in which Drp1 plays a central role regulating breast cancer cell metabolism and survival. Given the emerging evidence of PGC1α contributing to tumor growth, it will be of critical importance to target both mitochondrial biogenesis and mitophagy for effective cancer therapeutics.
- Development of a Dielectrophoresis-Based Cancer-Cell Analysis ToolDouglas, Temple Anne (Virginia Tech, 2018-10-04)One significant obstacle in cancer treatment is tumor heterogeneity. Different subpopulations within a tumor can respond differently to chemotherapy, resulting in resistance and recurrence. Addressing these differences while choosing a treatment modality could significantly improve chemotherapy outcomes. This work focuses on the development of a new modular device that leverages the unique advantages of a contactless dielectrophoresis, a method that uses applied electric fields in a microfluidic device to separate cells by biophysical phenotype. By optimizing force balancing between the dielectrophoretic force and the drag force on cells in the device, and by using cell-size pillars to maximize electric field gradients per volt applied while reducing cell-cell interactions,we demonstrate that it is possible to separate mouse ovarian surface epithelial (MOSE) cells at different stages while maintaining high viability. We also show other cell types to be separable with this device and develop an algorithm to rapidly analyze cell response to a variety of frequency/voltage/flow rate combinations. We also propose a microfluidic device downstream of the DEP chip that can be used to provide an integrated system for studying the subpopulations separated using dielectrophoresis by moving them into a culture chamber with hydrogel where they can be grown in 3D and characterized for a variety of parameters such as biophysical structure, metastatic capacity, and chemotherapy resistance.
- Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresisSalmanzadeh, Alireza; Kittur, Harsha; Sano, Michael B.; Roberts, Paul C.; Schmelz, Eva M.; Davalos, Rafael V. (American Institute of Physics, 2012-06-01)Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3699973] Actual pdf downloaded from NCBI.
- Do Probiotics Protect Against the Deleterious Effects of a High-Fat Diet?Fundaro, Gabrielle F. (Virginia Tech, 2014-06-27)High-fat diets and obesity have been linked to unfavorable changes in gut bacteria and increased leakage of bacterially-derived lipopolysaccharide (endotoxin) from the intestinal tract into circulation, which is associated with low-grade inflammation, metabolic dysregulation and degradation of tight-junction proteins between intestinal cells. Probiotic supplementation is the practice of ingesting live strains of bacteria that are proposed to have a beneficial effect on the host by enriching the intestine with healthy bacteria. The purpose of this project was to determine if probiotic supplementation would prevent increased inflammatory tone, decreased oxidative capacity, and decreased tight-junction protein expression associated with high-fat feeding and elevated endogenous endotoxin. Male C57BL/6J mice were fed either a control (CD, 10% fat) or high-fat (HFD, 60% fat) diet for 4 weeks while receiving a daily oral gavage of water (C-VSL#3, HF-VSL#3) or probiotics (C+VSL#3, HF+VSL#3) equivalent to 1.2 billion live cultures. Changes in body weight, body composition, respiratory exchange ratio, energy expenditure, and glucose and insulin tolerance were measured in live mice. Markers of metabolic function were measured in whole muscle homgenates and mitochondria isolated from red and white skeletal muscle. Plasma endotoxin was measured in blood collected from fasted mice at the time of euthanization. The large and small intestines were collected and mRNA levels of tight-junction proteins and markers of nutrient sensing were measured. To determine a possible protective effect against endogenous LPS, a second cohort of mice were given an intraperitoneal injection of 0.1µg/kg LPS or saline to induce endotoxemia after four weeks of the aforementioned feeding protocol. Markers of metabolic function and inflammation were measured in mitochondria, skeletal muscle and liver. VSL#3 supplementation improved glucose homeostasis and markers of inflammation while enhancing nutrient sensing in the gut.
- The effect of hypoxia and 3D culture conditions on heterogeneous ovarian cancer spheroidsLiu, Lu (Virginia Tech, 2017-01-10)Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy due to the insufficient accurate screening programs for the early detection of EOC. To improve the accuracy of the early detection, there is a need to deeply understand the mechanism of EOC progression and the interaction between cancer cells with their unique microenvironment. Therefore, this work investigated the metabolic shift in the mouse model for progressive ovarian cancer, and evaluated the effects of hypoxic environment, spheroid formation as well as stromal vascular fractions (SVF) on the metabolic shift, proliferation rate, drug resistance and protein markers in functional categories. The results demonstrated an increasingly glycolytic nature of MOSE cells as they progress from a tumorigenic (MOSE-L) to a highly aggressive phenotype (MOSE-FFL), and also showed changes in metabolism during ovarian cancer spheroid formation with SVF under different oxygen levels. More specifically, the hypoxic environment enhanced glycolytic shift by upregulating the glucose uptake and lactate secretion, and the spheroid formation affected the cellular metabolism by increasing the lactate secretion to acidify local environments, modulating the expression of cell adhesion molecules to enhance cell motility and spheroids disaggregation, and up-regulating invasiveness markers and stemness makers to promote ovarian cancer aggressive potential. Hypoxia and spheroid formation decreased ovarian cancer cells growth but increased the chemoresistance, which leads to the promotion of aggressiveness and metastasis potential of ovarian cancer. SVF co-cultured spheroids further increased the glycolytic shift of the heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer.
- Effects of low linolenic soy oil on pre-malignant human mammary epithelial cell progressionMcCall, Elaine Teresa (Virginia Tech, 2008-12-10)Beginning January 1, 2006 the U.S. Food and Drug Administration mandated that the amount of trans fats per serving be listed on the Nutrition Facts panel. Consequently new soybean breeds that would no longer be subject to the hydrogenation process, thus reducing trans fats, were developed. By traditional plant breeding techniques, plant breeders have developed a low linolenic soybean with 83.36% less alpha-linolenic acid (ALA; omega-3) than conventional soybean. A number of studies have demonstrated that the influence of dietary fats on cancer depends on the quantity as well as the type of lipids and diets with a disproportionately high omega-6 (n-6)/omega-3 (n-3) ratio are thought to contribute to cardiovascular disease, inflammation and cancer. Conventional soybean oil (SO) has an n-6/n-3 ratio of 8/1 while the new low linolenic soy bean oil (LLSO) has an n-6/n-3 ratio of 56/1. In this study, we evaluated the effects of dietary LLSO, SO and lard on the progression of breast cancer (BC). Thirty-five, 6-wk old, ovariectomized, athymic mice received human pre-malignant breast cells (MCF-10AT1 1 x 105 cells/40μl/ Matrigel/spot, 4 spots/mouse). Mice were divided into three groups and then fed isocaloric and isonitrogenous diets with disparate fat sources: LLSO (20% of total energy intake), SO (20%) and lard (20%). The dietary treatment lasted 24 weeks upon which the study was terminated and tumors, tissues and blood samples were analyzed. Average tumor surface area at termination for the LLSO group was 45.11 ± 4.46 mm2, 40.08 ± 4.2 mm2 for lard and 56.63 ± 5.42 mm2 for SO. Messenger RNA (mRNA) expression of HER2/neu, epidermal growth factor receptor (EGFR), H-ras, Bcl-2, cyclooxygenase-2 (COX-2), vascular epidermal growth factor (VEGF), and fatty acid synthase (FAS) in tumors were analyzed using quantitative real time-polymerase chain reaction (qRT-PCR). We found that dietary LLSO supplementation significantly (p < 0.05, Tukey's test) increased tumor expression of oncogenes HER2/neu, EGFR, FAS, and H-ras, but not in the SO or lard supplemented groups. Relative mRNA expression was also significantly increased in both LLSO and SO groups, however, there was no marked difference in mRNA expression for Bcl-2 and COX-2. Removed tumors were evaluated microscopically for histologic lesion progression corresponding to human breast cancer progression. Tumors from the LLSO group showed more advanced lesions (grade 2) (p < 0.05, Chi Square test) with areas of four or more layers of epithelial cells and irregularly shaped lumens. These data suggest that dietary intake of LLSO may accelerate mammary tumor progression at a faster rate than conventional SO or lard.
- The effects of Low α-Linolenic fatty acid Soybean Oil and Mid Oleic acid Soybean Oil on the growth of Her-2/neu and Fatty acid synthase over-expressing human breast cancer (SK-Br3) cellsBark, Jee Hyun (Virginia Tech, 2010-09-09)A variety of soybean oils (SOs) were developed with improved functional properties. Some of the modified SOs contain altered fatty acid (FA) composition by selective breeding methods. Currently, low α- linolenic acid soybean oil (LLSO) and low α- linolenic acid and mid oleic acid soybean oil (LLMOSO) are available FA modified SOs in the market. The consumption of FA modified SOs has been increased because the United States Food and Drug Administration required listing trans fat content in food products sold in U.S. as an effort to reduce possible health risks caused by trans fat beginning 2006. However, the effects of these FA modified SOs on human chronic diseases including breast cancer (BC) have not been studied. BC has become the most frequently diagnosed cancer and is the second leading cause of cancer death among American women. The type of dietary fat, FA composition, and n-6/n-3 ratio are known to influence BC development. Therefore, it is possible that the changed FA composition and n-6/n-3 ratio in the FA modified SOs may affect BC progression, and its critical health concern needs to be investigated. Increased human epithelial growth factor receptor 2 (Her-2/neu) and fatty acid synthase (FAS) are associated with BC progression. In fact, FAS activity and expression are affected by dietary FA composition and FA metabolism. Hypothesis of this research is that LLSO and LLMOSO may affect Her-2/neu and FAS expressing human BC (SK-Br3) cell growth in vitro and in vivo. To test our hypothesis, we investigated the potential adverse or beneficial effects of LLSO and LLMOSO in comparison with conventional SO and lard on human BC cells and then examined the possible mechanisms of action by evaluating the expression level of genes markers involved in growth factor mediated signal transduction pathway, specifically Her-2/neu PI 3-kinase (phophoinositide 3- kinase)-FAS signal transduction pathway. In vitro study demonstrated that all the tested oils at 0-2 μl/ml level have cytotoxic effects. LLMOSO had less cytotoxic effects on the growth of SK-Br3 cells compared to SO. However, there was no difference in SK-Br3 cell growth between LLSO and SO. The apoptotic protein markers (mutant p53 and caspase-3) analysis revealed that the cell growth inhibition by oil treatments was cytotoxic by triggering apoptosis. Western blot analysis demonstrated that LLSO- and LLMOSO- induced changes on cell growth involve Her-2/neu and FAS signaling transduction pathway and sterol regulatory element binding protein-1 (SREBP-1), mitogen activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI 3-kinase) are possible down-stream effectors of Her-2/neu signaling pathway. We also evaluated the dietary effects of LLSO (20% fat of total calorie), SO (20%), and lard (20%) on the growth of SK-Br3 tumors implanted in athymic mice. Changes in tumor surface area, body weight, and food intake were monitored during the 6 months feeding study. After termination, tumor net weight, Her-2/neu and FAS mRNA expression in tumors, FAS protein expression in liver, lipid composition in diets, abdominal fat, and serum, as well as plasma total cholesterol and triglyceride levels were analyzed. In vivo study showed that there were no statistical differences in tumor size and tumor net weight among SO, LLSO, and lard groups. No differences in FAS mRNA and protein expression levels between the LLSO and SO groups were observed. Tumors from the lard group expressed higher Her-2/neu and FAS mRNA than those from the LLSO and SO group. The lipid analysis demonstrated that LLSO was not significantly distinct from SO in trans fat concentration after metabolism. Serum cholesterol and triglyceride levels were unchanged in LLSO fed compared to SO fed mice. In summary, LLSO which contained modification in αLA concentration showed similar effects on SK-Br3 as SO in both in vitro and in vivo. However, LLMOSO which contained more drastic modifications on FA composition exhibited less cytotoxicity compared to SO in vitro.
- Elucidating the Role of Pattern Recognition Receptors in Understanding, Treating, and Targeting CancerScaia, Veronica Marie (Virginia Tech, 2019-04-23)Pattern Recognition Receptors (PRRs) are a group of evolutionarily conserved and germline-encoded cellular receptors of the innate immune system that are responsible for recognizing and responding to the entirety of the pathogens a host encounters. The ingenuity of the innate immune system is that with a comparatively miniscule pool of receptors, these receptors are capable of responding to a diverse and large array of pathogens and damage signals. Two highly relevant subsets of PRRs include nucleotide binding domain leucine rich repeat containing (NOD-like) receptors (NLRs) and Toll-like receptors (TLRs). Both NLRs and TLRs have been implicated in several diseases, including autoimmune disorders, inflammatory conditions, and cancer. Mice lacking a specific NLR, NLRP1, are more susceptible to chemically induced colitis and colitis-associated tumorigenesis. We investigated whether the absence of NLRP1 in the gastrointestinal tract influenced the composition of the microbiome, and whether it was responsible for the predisposition of these animals to colitis-associated cancer. By carefully controlling for non-genotype influences, we found that in fact maternal and housing factors were greater predictors over genotype of gut flora composition. This study concluded with a clearer understanding of NLRP1. We next investigated the effectiveness of a novel tumor ablation therapy, termed High-Frequency Irreversible Electroporation (H-FIRE) in a murine model of triple negative breast cancer. The chosen 4T1 model closely mimics aggressive human metastatic triple negative breast cancer, and metastasizes to the same organs. After ablation of the primary mammary tumor, we saw significant improvements in disease burden and metastases, both of which were accompanied by PRR activation within the tumor microenvironment, implicating PRRs in the successful treatment outcome following H-FIRE ablation. Lastly, we generated novel CRISPR-Cas9 plasmids to genetically manipulate the Tlr4 gene of wild type C57Bl/6 mice in order to recapitulate the LPS-hyporesponsive TLR4 protein of C3H/HeJ mice. This proof-of-concept study successfully demonstrated that PRRs can be targets for gene editing purposes, and that nanoparticle delivery leads to enhanced and improved delivery. Collectively, this work attempts to better appreciate the role of PRRs in understanding, treating, and targeting cancer.
- Evaluating the Effects of Fluid Shear Stress on Ovarian Cancer Progression and Metastatic PotentialHyler, Alexandra Rochelle (Virginia Tech, 2018-04-06)Most women die of ovarian metastasis rather than the effects of the primary tumor. However, little is known about the factors that support the survival and secondary outgrowth of exfoliated ovarian cancer cells. In addition to genetic and molecular factors, the unique environment of the peritoneal cavity exposes ovarian cells to biophysical forces, particularly fluid shear stress (FSS). These biomechanical forces, only recently identified as a hallmark of cancer, induce rapid signaling events in attached and aggregated cells, a process termed mechanotransduction. The cellular responses to these forces and their impact on tumor initiation, progression, and metastasis are not understood. In order to delineate these phenomena, dynamic and syngeneic cell models are needed that represent the development of the disease and can be used in relevant engineered testing platforms. Thus, in an interdisciplinary approach, this work bridges molecular and cancer biology, device engineering, fluid mechanics, and biophysics strategies. The results demonstrated that even a low level of continual FSS significantly and differentially affected the viability of epithelial ovarian cancer cells of various stages of progression over time, and enhanced their aggregation, adhesion, and cellular architecture, traits of more aggressive disease. Furthermore, benign cells that survived FSS displayed phenotypic and genotypic changes resembling more aggressive stages of the disease, suggesting an impact of FSS on early stages of tumor development. After identifying a biological affect, we designed an in vitro testing platform for controlled FSS investigations, and we modeled the system fluid mechanics to understand the platform's performance capability. A cylindrical platform divided into annular sections with lid-driven flow was selected to allow continuous experiments sustainable for long durations. Tuning of the lid speed or fluid height resulted in a wide range of FSS magnitudes (0- 20 N/m2) as confirmed by analytical and numerical modeling. Further, detailed numerical modeling uncovered that FSS magnitudes experienced by cell aggregates were larger than previously observed, suggesting an even larger role of FSS in ovarian cancer. Finally, we built and engineered the designed platform to investigate changes in benign and cancer cells as a function of time and FSS magnitude. Device precision was balanced with biological consistency needs, and a novel platform was built for controlled FSS investigations. This work provides a foundational understanding of the physical environment and its potential links to ovarian cancer progression and metastatic potential.
- Exploiting Clausius-Mossotti Factor to Isolate Stages of Human Breast Cancer Cells: Theory and ExperimentHenslee, Erin A. (Virginia Tech, 2010-01-19)This work demonstrates the ability of contactless dielectrophoresis (cDEP) for isolation of breast cancer cell stages. The ability to selectively concentrate breast tumor cells from a non-transformed or normal cell population is the key to successfully detecting tumors at an early stage of growth and treating transformed cells before they proliferate. Since all cell types have a unique molecular composition it is expected that their dielectrophoretic properties are also unique. DEP force is dependent on the frequency and magnitude of the applied field, as well as a particle's size and electric properties. Specifically, the Clausius-Mossotti factor in the DEP force equation determines a specific cell type's interaction with the electric field and the DEP force response. Cell properties affecting this parameter were investigated numerically and experimentally. MCF10A, MCF7, and MDA-MB231 human breast cancer cells were used to represent early, intermediate, and late staged breast cancer respectively. Experiments were conducted at 0.02ml/hr with applied voltages of 20Vrms, 25Vrms, 30Vrms, 35Vrms, 40Vrms and 50Vrms (n=8). Frequency measurements were recorded for the initial onset of DEP force and when 90% trapping was obtained. The trapping frequency ranges for each cell were distinct from one another with the least amount of overlap between the MCF10A cells and MDA-MB231cells. The MCF7 cell line had, on average, the smallest trapping region at each applied voltage, and fell in between the normal and late staged cells' trapping frequency ranges. Voltages of 20Vrms to 30Vrms were found the most efficient for cell isolation.
- Fluid shear stress impacts ovarian cancer cell viability, subcellular organization, and promotes genomic instabilityHyler, Alexandra R.; Baudoin, Nicolaas C.; Brown, Megan S.; Stremler, Mark A.; Cimini, Daniela; Davalos, Rafael V.; Schmelz, Eva M. (PLOS, 2018-03-22)Ovarian cancer cells are exposed to physical stress in the peritoneal cavity during both tumor growth and dissemination. Ascites build-up in metastatic ovarian cancer further increases the exposure to fluid shear stress. Here, we used a murine, in vitro ovarian cancer progression model in parallel with immortalized human cells to investigate how ovarian cancer cells of increasing aggressiveness respond to < 1 dyne/cm2 of fluid-induced shear stress. This biophysical stimulus significantly reduced cell viability in all cells exposed, independent of disease stage. Fluid shear stress induced spheroid formation and altered cytoskeleton organization in more tumorigenic cell lines. While benign ovarian cells appeared to survive in higher numbers under the influence of fluid shear stress, they exhibited severe morphological changes and chromosomal instability. These results suggest that exposure of benign cells to low magnitude fluid shear stress can induce phenotypic changes that are associated with transformation and ovarian cancer progression. Moreover, exposure of tumorigenic cells to fluid shear stress enhanced anchorage-independent survival, suggesting a role in promoting invasion and metastasis.
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