Prostate cancer is the second most often diagnosed cancer and internationally the sixth foremost cause of cancer death in males, as of 2011. Within the United States it is the most common form of cancer in men with 186,000 new cases and with an overall 28,600 deaths in 2008, and it is the second leading kind of cancer-related death in men. The widespread threat that prostate cancer poses against men across the globe cannot be understated, and its initiation and progression must be understood in order to truly comprehend its implicated risks and possible forms of treatment. As its name implies, prostate cancer is a form of cancer that develops in the prostate gland located in the male reproductive system. Its progress starts when standard semen-secreting prostate gland cells mutate into cancer cells. Although its developments may start at the prostate gland, cancer cells may metastasize to other parts of the body through circulation systems such as the lymph nodes. The main sites of metastasis for prostate cancer include the adrenal gland, the bones, the liver and the lungs. Although there are treatments available for prostate cancer, there is no definitive cure. The primary goal of this project was to find an alternative form of treatment, which is what will be necessary to combat this cancer.

<p>Breast cancer remains a major cause of cancer-related deaths among women despite several advances in the field due to metastasis with a 5-year survival rate of less than 30% for metastatic breast cancer. Dissemination of tumor cells to metastatic sites begins as early as the primary tumor is diagnosed at just 4mm in size. These cells remain dormant for extended periods of time evading immune surveillance and later turn into therapy resistant metastases resulting in the poor prognosis in breast cancer patients. Hence, there is a <b>critical need </b>to improve our understanding of the metastatic programs in breast cancer and its contributors to develop better therapy options.</p><p>One such contributor is alcohol which is listed as a carcinogen by the National Toxicology Program. Alcohol consumption is a risk factor for several cancers and increases the risk of breast cancer incidence in a dose dependent manner. We have observed in preliminary studies, that alcohol consumption causes increased neutrophil extracellular trap (NET) formation in the lungs and outgrowth of previously dormant cancer cells in mice. Further, NETs increase cancer cell seeding and play a role in metastasis. Hence, we hypothesized that alcohol consumption breaks cancer cell dormancy by activating neutrophils.</p><p>In this study, we have broken cancer cell dormancy and generated a novel cell line, Alcohol-D2.OR, by inducing outgrowth of the dormant D2.OR cells in mice through alcohol consumption. Reinjection of the Alcohol-D2.OR cells, into alcohol-naïve mice results in aggressive outgrowth of the cells suggesting these cells are modified on a genetic level. Indeed, RNA sequencing analysis of the gene expression in the cells showed that these cells have significantly modified gene expression as well as modified morphology and surface protein expression than the parental D2.OR cells. Importantly, from our analysis we have identified a tumor suppressor, SPINK5 which was significantly downregulated in the alcohol line. Further, SPINK5 expression in cancer cells suppressed neutrophil activity in-vitro. Knockdown of SPINK5 in the parental D2.OR line resulted in outgrowth of the cells in-vivo with increased lung NETs highlighting the importance of this gene for maintenance of dormancy by suppression of neutrophil activity.</p><p>Hence, we have successfully identified a gene responsible for dormancy maintenance, SPINK5 which will aid in not only therapeutic intervention but also in identification of breast cancer patients likely to progress to metastasis. Further, the newly established Alcohol-D2.OR cells provide a novel tool to study other initiators of metastasis in breast cancer.</p><p>A common side-effect of most chemotherapeutic treatments is neutropenia, reduced neutrophils in circulation increasing susceptibility to infections. Hence, GM-CSF is often administered to patients to mobilize bone marrow neutrophils. However, neutrophils have been increasingly shown to promote distant metastases. Circulating disseminated cancer cells (DCCs), which are present as early as primary diagnosis, have been shown to activate neutrophils resulting in the release of neutrophil extracellular traps (NETs). These NETs alter the lung architecture providing a suitable environment for the seeding and growth of DCCs promoting lung metastases. One key player in neutrophil activation is spleen tyrosine kinase (SYK), an intracellular non-receptor kinase which is activated by the engagement of b-integrin on the neutrophil surface.</p><p>Using a chemical genetics approach we are able to specifically inhibit SYK in the murine host. Using our transgenic model of specific SYK inhibition as well as the FDA approved SYK inhibitor, fostamatinib, we see similar results of decreased lung metastases compared to controls. We also observed decreased neutrophil viability in-vitro in the presence of fibronectin, an effect that was not seen on plastic highlighting the importance of integrin mediated activity of SYK. We also observe decreased neutrophil and macrophage infiltration into the lungs upon host-specific SYK inhibition. Overall, these findings suggest a paracrine effect of SYK in stromal cells that promotes favorable tumor microenvironment (TME) and its inhibition may be a useful therapeutic option to combat DCCs from forming metastases.</p><p>Hence, through this work we address two mechanisms of neutrophil-mediated breast cancer metastasis and that therapeutic intervention by rescuing SPINK5 expression in cancer cells or inhibition of SYK in the tumor microenvironment can suppress pulmonary metastasis in breast cancer.</p>

Die extrazelluläre Mikroumgebung spielt eine grundlegende Rolle bei der Entwicklung von Metastasen und hat einen großen Einfluss auf die Wahl der Migrationsstrategien, die von Karzinomzellen während der Invasion angewandt werden. In-vitro Anordnungen sind hilfreiche Instrumente für die Untersuchung von Zellmigration und -invasion, da sie grundlegende Merkmale von In-vivo-Geweben reproduzieren können. Ziel dieser Forschungsarbeit ist es, die Fähigkeit von mesenchymalen und epithelialen Brusttumorzellen zu untersuchen, sich zu verflüssigen und durch enge und starre Mikrostrukturen zu navigieren. Wir verwendeten eine mikrofluidische Vorrichtung mit trichterförmigen Mikroverengungen und verglichen das Verhalten von fünf verschiedenen menschlichen Brustkrebszelllinien in der Mikrovorrichtung bei Stimulation durch Chemoattraktoren. Wir fanden heraus, dass grundsätzlich verschiedene Zelllinien das gleiche invasive Potenzial haben, da normale Epithelzellen in der Lage waren, durch die stark komprimierenden Trichter zu wandern, ähnlich wie die invasiveren mesenchymalen Zellen. Wir fanden auch heraus, dass die Migration der normalen Epithelzellen auch ohne einen chemo-attraktiven Stimulus stattfindet. Wir konzentrierten unsere Beobachtungen auf die Rolle des Aktin- und Intermediärfilament-Zytoskeletts während der eingeschränkten Migration und zeigten, dass das Aktin-Zytoskelett eine starke und langanhaltende Reorganisation erfährt, damit die Zellen durch die engen Verengungen kriechen können. Wir sahen keinen Hinweis darauf, dass das Keratin- und Vimentin-Zwischenfilament- Zytoskelett während der Invasion in die Mikroverengungen eine aktive mechanische Rolle spielte. Insbesondere die Expression des Vimentin-Zwischenfilamentproteins korrelierte in unserem Versuchsaufbau nicht mit der Invasionsfähigkeit einzelner Zellen. Unter diesen Voraussetzungen wurden die passiven (Elastizität und Viskosität) und aktiven (Kontraktilität) viskoelastischen Eigenschaften der Zellen weiter untersucht und quantifiziert. Wir fanden keinen signifikanten Unterschied in der passiven viskoelastischen Reaktion der Zellen, nachdem sie oszillierenden Druckkräften mittels AFM-Sondierung ausgesetzt waren, was darauf hindeutet, dass Elastizität und Viskosität nicht zur Unterscheidung zwischen invasiven und nicht-invasiven Zellen verwendet werden können. Es wurde kein Hinweis darauf gefunden, dass die Kompressionsversteifung die Invasion durch die Mikroverengungen entweder behindert oder fördert. Schließlich haben wir bei der Betrachtung aktiver viskoelastischer Parameter die kontraktile Reaktion unserer Zelllinien verglichen, wenn sie mit dem mikrofluidischen optischen Strecker Laser-Streckkräften ausgesetzt wurden. Hier fanden wir eine klare Korrelation zwischen den Zelllinien, die ein invasives Verhalten in den Mikroverengungen zeigten, und denjenigen, die eine aktive (substratunabhängige) kontraktile Reaktion in der optischen Streckvorrichtung zeigten. Wir kommen zu dem Schluss, dass ein entscheidender Faktor für eine erfolgreiche Migration durch hohe räumliche Enge die Fähigkeit der Zellen ist, aktiv Aktin-Stressfasern zu erzeugen und abzubauen, was sich in der Fähigkeit manifestiert, von einer substratabhängigen und stressfaserbasierten Kontraktilität zu einer substratunabhängigen kortikalen Kontraktilität zu wechseln.

No description available.

Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumor. Even after patients undergo maximum and safe surgical resection followed by adjuvant chemotherapy and radiation therapy, residual GBM cells form secondary tumors which lead to poor survival times and prognoses for patients. This tumor recurrence can be attributed to the inherent GBM heterogeneity that makes it difficult to eradicate the therapy-resistant and tumorigenic subpopulation of GBM cells with stem cell-like properties, referred to as glioma stem cells (GSCs). Additionally, the migratory nature of GBM/GSCs enable them to invade into the healthy brain parenchyma beyond the resection cavity to generate new tumors. In an effort to address these challenges of GBM recurrence, this research aimed to develop a biomaterials-based approach to attract, capture, and eradicate GBM cells and GSCs with chemical and physical stimuli. Specifically, it is proposed that after surgical removal of the primary GBM tumor mass, an injectable hydrogel can be dispensed into the resection cavity for crosslinking in situ. A combination of chemical and physical cues can then induce the migration of the residual GBM/GSCs into the injectable hydrogel to localize and concentrate the malignant cells prior to non-invasively abating them. In order to develop this proposed treatment, this dissertation focused on 1) characterizing and optimizing the thiol-Michael addition injectable hydrogel, 2) attracting and entrapping GBM/GSCs into the hydrogel with CXCL12-mediated chemotaxis, and 3) assessing the feasibility of utilizing histotripsy to mechanically and non-invasively ablate cells entrapped in the hydrogel. The results revealed that hydrogel formulations comprising 0.175 M NaHCO3(aq) and 50 wt% water content were the most optimal for physical, chemical, and biological compatibility with the GBM microenvironment on the basis of their swelling characteristics, sufficiently crosslinked polymer networks, degradation rates, viscoelastic properties, and interactions with normal human astrocytes. Loading the hydrogel with 5 µg/mL of CXCL12 was optimal for the slow, sustained release of the chemokine payload. A dual layer hydrogel platform demonstrated in vitro that the resulting chemotactic gradient induced the invasion of GBM cells and GSCs from the extracellular matrix and into the synthetic hydrogel with ameboid migration and myosin IIA activation. This injectable hydrogel also demonstrated direct therapeutic benefits by passively eradicating entrapped GBM cells through matrix diffusion limitations as well as decreasing the GBM malignancy and GSC stemness upon cancer cell-hydrogel interactions. Research findings revealed the hydrogels can be synthesized under clinically relevant conditions mimicking GBM resection in vitro, and hydrogels were distinguishable with ultrasound imaging. Furthermore, the synthetic hydrogel was acoustically active to generate a stable cavitation bubble cloud with histotripsy treatment for ablation of entrapped red blood cells with well-defined, uniform lesion areas. Overall, the results from this research demonstrate this injectable hydrogel is a promising platform to attract and entrap malignant GBM/GSCs for subsequent eradication with chemical and physical stimuli. Further development of this platform, such as by integrating electric cues for electrotaxis-directed cell migration, may help to improve the cancer cell trapping capabilities and thereby mitigate GBM tumor recurrences in patients. / Doctor of Philosophy / Glioblastoma multiforme (GBM) is the deadliest type of primary brain cancer. Upon GBM diagnosis, patients first undergo surgery to remove the tumor from the brain. After waiting several weeks for the wound healing process due to surgery, patients are administered chemotherapy with drugs and radiation therapy to eradicate any remaining GBM cells. Even after undergoing these combinatorial treatments, the cancer returns and leads to median survival times of only 15 months in 90% of patients. Complete GBM eradication is difficult, since the cancer cells can migrate into healthy brain tissue beyond the original tumor site. Additionally, GBM is highly heterogenous and composed of different cell types that can resist chemotherapy and radiation therapy, which lead to secondary tumors and cancer relapse. To address these challenges, this dissertation aimed to develop a polymer-based material (specifically a hydrogel) that can attract, entrap, and localize the GBM cells into the material to subsequently eradicate them with chemical and physical signals. This hydrogel platform would have important clinical implications, as it can potentially be dispensed into the empty cavity after surgical removal of the tumor in the brain. The hydrogel can then be harnessed to attract residual GBM cells for directed migration into the hydrogel to concentrate and localize the cancer cells for their subsequent destruction with a non-invasive technology. In order to develop this proposed treatment, this dissertation investigated the following three aims: 1) to study and optimize the injectable hydrogel for chemical, physical, and biological compatibility with the GBM therapy; 2) to utilize chemical signals to attract and entrap the GBM cells into the hydrogel; and 3) to apply focused ultrasound with high amplitude, short duration negative pressure pulses to mechanically fractionate and destroy the cells entrapped in the hydrogel. The results revealed that the hydrogel comprising 0.175 M NaHCO3(aq) and 50 wt% water content was the most optimal formulation. CXCL12 chemokine proteins loaded into the hydrogel at 5 µg/mL released slowly from the hydrogel to generate a chemical gradient and thereby attract GBM cells to promote their invasion into the hydrogel matrix. The hydrogel was demonstrated to respond well to focused ultrasound treatment, which was capable of mechanically fractionating and destroying red blood cells in the hydrogel uniformly. Overall, the results from this research provide support that this hydrogel platform can attract, entrap, and eradicate GBM cells with chemical and physical stimuli. Hence, further improvement of this platform and implementation of this novel GBM treatment may in the future help minimize GBM cancer relapse in patients who undergo conventional therapies, thereby extending their survival times.

<p>  </p> <p>Breast cancer is a significant public health concern being the second leading cause of cancer-related death in women, with a projected 43,250 deaths in the US in 2022. However, cancer progression to metastatic sites is the primary cause of death in breast cancer patients. A hallmark of cancer is the dysregulation of cellular metabolism. Cancer cells have the ability to hijack their metabolism and drive cellular processes supporting cancer progression. As cancer cells continue through the metastatic cascade, they are challenged with various bioenergetic processes that can be supported by the influx of glucose. Thus, altering glycogen accumulation, where glucose is stored in cells, may be beneficial in supporting cancer progression. In this study, we aim to determine what drives glycogen accumulation in metastatic cells and if it is utilized to support cancer progression. We employed the non-metastatic MCF10A-<em>ras</em> and the metastatic MCF10CA1a cells for these studies. Our results demonstrate that metastatic MCF10CA1a have 20-fold accumulation of glycogen compared to the MCF10A-<em>ras</em> cells. Utilizing 13C6-glucose flux analysis, surprisingly, most of the glucose incorporated into glycogen of the MCF10CA1a cells was in the M+5 glucose labeling pattern instead of the expected M+6 pattern which occurs when glucose is directly converted to glycogen. We showed that glycogen was accumulated due to increased gluconeogenesis through cataplerosis (PEPCK) utilizing inhibitors of the enzyme. Additionally, in a pulse-chase experiment using 13C6-glucose flux analysis, there was an approximate 50% reduction in labeled glucose in glycogen, 3 hours after removing the label, suggesting that the MCF10CA1a cells also have a rapid turnover of glycogen. Glucose can be released through two mechanisms, glycogenolysis or glycophagy. Utilizing siRNAs to a rate limiting steps in each pathway, results suggest both glycogenolysis (PYGL) and glycophagy (GAA) are necessary to support cell migration, a critical step in metastasis of the MCF10CA1a cells. Thus, glycogen metabolism is dysregulated in the MCF10CA1a breast cancer cells such that they have increased glycogen accumulation and that glycogen is required to support cell migration. Further understanding the mechanism by which glucose is accumulated and released in a specific cancer and in specific steps or stressors in cancer progression may contribute to potential therapeutic targets to help mitigate metastasis, and potentially breast cancer mortality.</p>

Laser-induced fluorescence (LIF) has been shown to be potentially useful for identifying microorganisms in real time. It is a selective and sensitive technique because the excitation is performed at one wavelength while the emission is monitored at longer wavelengths so that background from the excitation source can be eliminated. This specialized optical property of LIF can be applied to development of an optical sensor capable of quickly, non-invasively, and quantitatively probing complex biochemical transformations in microorganisms. Various bio-photonic optical fiber sensors based on laser-induced fluorescence (LIF) spectroscopy were developed as diagnostic tools for microorganisms. In the first phase, the enhancement of the sensitivity and selectivity of the optical sensor system focused on diagnosis of human breast cancer cell lines and Azotobacter vinelandii (an aerobic soil-dwelling organism). Autoluorescence spectra from human breast cancer cell lines and Azotobacter vinelandii corresponding to different growth environments were investigated. Then, the study has expanded to include the use of gold nanoparticles for specific DNA detection. The use of gold nanoparticales opens a door into construction of a compact, highly specific, inexpensive and userriendly optical fiber senor for specific DNA detection. An optical fiber laser-induced fluorescence (LIF) sensor based has been developed to detect single-strand (ss) DNA hybridization at the femtomolar level. Effects of various experimental parameters and configuration were investigated in order to optimize sensor performance and miniaturize sensor size.

No description available.

<p>Prostate cancer is the most frequently diagnosed cancer and the second leading causes of cancer-related death in men in the United States. Despite high overall incidence, the disease is relatively well controlled due to slow progression and early detection. However, surgical resection and external beam radiation therapy, first lines of defense, are the only potentially curative options in the clinic. Radiation resistant and metastatic prostate cancer are treated with androgen signaling inhibition (ASI) therapy to target the major growth/proliferation signaling axis that drives prostate cancer cells, but resistance invariably develops. Further, as ASI therapeutic compounds become more potent and are approved for use as neoadjuvant therapeutic options, up to 25% of prostate cancer patients on ASI therapy develop neuroendocrine prostate cancer (NEPC) that is actually induced by the ASI therapy itself. NEPC is resistant to taxane and platinum-based therapies, has no curative or specific targeting options clinically, results in mean overall survival under 9 months in some patient cohorts, and represents a significant unmet clinical need.</p> <p>Protein arginine methyltransferase 5 (PRMT5) is a methyltransferase with histone (epigenetic) and non-histone (non-epigenetic) substrates. PRMT5 is a critical mediator of stemness-associated genes as well as epigenetic regulation of cell fate determination. Further, PRMT5 is a validated therapeutic target in multiple hematological and solid tumor malignancies with multiple clinical trials ongoing. The Hu lab has recently demonstrated that 1) PRMT5 drives androgen receptor (AR) expression in hormone naïve prostate cancer (HNPC) cells, 2) PRMT5 positively regulates DNA damage response gene expression to confer radiation resistance in prostate cancer cells, 3) PRMT5 cooperates with cofactor pICln to drive AR expression in  castration resistant prostate cancer (CRPC) cells, and 4) targeting PRMT5 inhibits development of radiation-induced NEPC development, and that PRMT5 is a valid therapeutic target for prevention of radiation-induced neuroendocrine differentiation (NED). </p> <p>The research presented in this thesis demonstrates that PRMT5 and MEP50 are required for ASI-induced NED in prostate cancer cells, that the PRMT5:MEP50 protein:protein interaction can be pharmacologically targeted, and that ASI-induced NED occurs in an AR-dependent manner. Further, this work contributes a novel class of PRMT5:MEP50 PPI inhibitors in addition to a single-cell, time-resolved model system for interrogating pharmacological targeting of ASI-induced NED <em>in vitro</em>.</p>

No description available.