published_or_final_version / Biochemistry / Master / Master of Medical Sciences

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Pancreatic cancer remains a major unsolved health problem, with conventional cancer treatments having little impact on disease course. The objective of this thesis is to create innovative tools to better understand and improve chemotherapeutic treatment of pancreatic cancer. Towards this end, nanosystems were designed with a dual purpose: to carry chemotherapeutic drugs and act as photoacoustic imaging contrast agents. The overarching hypothesis is that these nanosystems can provide enhanced therapy for pancreatic cancer and enable visualization of drug delivery. Demonstrated in this dissertation is the design, synthesis, and characterization of two such nanosystems built to carry the chemotherapeutic agent gemcitabine while acting as a photoacoustic imaging contrast agent. The nanosystems were also shown to be multifunctional with possible application as photothermal therapy agents and cellular functional sensors. Although future research is required to fully investigate the clinical potential of these systems for pancreatic cancer, the work presented in this dissertation is a step towards creation of multifunctional nanosystems that will enable non-invasive, in vivo photoacoustic imaging of drug delivery. / text

Obesity is an established risk and progression factor for many types of cancer, including pancreatic and colon cancer, and is characterized by abnormal metabolic hormone production and a chronic low-grade state of inflammation. However, the links between obesity, hormones, inflammation and tumorigenesis in colon and pancreatic tissue are poorly understood. Calorie restriction (CR), an anti-obesity dietary regimen with potent anticancer effects, reduces serum metabolic hormones and protumorigenic cytokines. Insulin-like growth factor (IGF)-1 is a metabolic hormone that activates NF-[kappa]B, a key regulator of inflammation. NF-[kappa]B is a transcription factor that mediates transcription of many cancer- and inflammation-related genes and is upregulated in both colon and pancreatic cancer. We hypothesized that CR inhibits colon and pancreatic tumor cell growth through modulation of hormone-stimulated NF-[kappa]B activation and protumorigenic gene expression. To test this hypothesis, we used CR and ad libitum feeding to generate a lean and overweight (control) phenotype, respectively; in C57BL/6 mice transplanted with MC38 colon cancer cells or Panc 02 pancreatic cancer cells, and analyzed the effect of diet on circulating hormone levels, markers of inflammation, and tumor growth. We also investigated the in vitro effects of IGF-1 on NF-[kappa]B activation and downstream protumorigenic gene expression in MC38 and Panc 02 cells. CR, relative to control diet, reduced body weight, circulating IGF-1 levels, and transplanted MC38 and Panc 02 tumor growth, as well as protumorigenic gene expression in the MC38 and Panc 02 tumor microenvironment. IGF-1 increased cell viability, NF-[kappa]B nuclear translocation and DNA binding, transcriptional activation, and downstream gene expression of inflammation and other protumorigenic genes in MC38 colon cancer cells and Panc 02 pancreatic cancer cells in vitro. Knockdown studies of NF-[kappa]B in Panc 02 cells using si-RNA established that the IGF-1-induced increase in protumorigenic gene expression is mediated, at least partially, through an NF-[kappa]B-dependent mechanism. In conclusion, these findings in models of pancreatic and colon cancer help clarify the links between obesity, IGF-1, NF-[kappa]B-mediated inflammation, and cancer. This work provides the underpinnings for several new molecular targets and strategies to test in model systems and translational studies for preventing or controlling obesity-related cancer. / text

A significant challenge in the surgical resection of tumors is accurate identification of tumor margins. Current methods for margin detection are time-intensive and often result in incomplete tumor excision and recurrence of disease. The objective of this project was to develop a near-infrared narrowband imaging (NIR NBI) system to image tumor and its margins in real-time during surgery utilizing the contrast provided by gold nanoparticles (GNPs). NIR NBI images narrow wavelength bands to enhance contrast from plasmonic particles in a widefield, portable and non-contact device that is clinically compatible for real-time tumor margin demarcation. GNPs have recently gained significant traction as nanovectors for combined imaging and photothermal therapy of tumors. Delivered systemically, GNPs preferentially accumulate at the tumor site via the enhanced permeability and retention effect, and when irradiated with NIR light, produce sufficient heat to treat tumor tissue. The NIR NBI system consists of 1) two LED's: green (530 nm) and NIR (780 nm) LED for illuminating the blood vessels and GNP, respectively, 2) a filter wheel for wavelength selection, and 3) a CCD to collect reflected light from the sample. The NIR NBI system acquires and processes images at a rate of at least 6 frames per second. We have developed custom control software with a graphical user interface that handles both image acquisition and processing/display in real-time. We used mice with a subcutaneous tumor xenograft model that received intravenous administration and topical administration of gold nanoshells and gold nanorods. We determined the GNP's distribution and accumulation pattern within tumors using NIR NBI. Ex vivo NIR NBI of tumor xenografts accumulated with GNPs delivered systemically, demonstrated a highly heterogeneous distribution of GNP within the tumor with higher accumulation at the cortex. GNPs were observed in unique patterns surrounding the perivascular region. The GNPs clearly defined the tumor while surrounding normal tissue did not indicate the presence of particles. In addition, we present results from NBI of tumors that received topical delivery of conjugated GNPs. We determined that tumor labeling using topical delivery approach resulted in a more homogenous distribution of GNPs compared to the systemic delivery approach. Finally, we present results from the on-going in vivo tumor margin imaging studies using NIR NBI. Our results demonstrate the feasibility of NIR NBI in demarcating tumor margins during surgical resection and potentially guiding photo-thermal ablation of tumors. / text

Cell-based insulin therapies can potentially improve glycemic regulation in insulin dependent diabetes patients and thus help reduce secondary complications. The long-term goal of our work is to engineer autologous insulin-secreting intestinal endocrine cells as a non-beta cell approach to alleviate donor cell shortage and immune rejection issues associated with islet transplantation. These cells have been chosen for their endogenous similarity to beta cells, but generating cell constructs with sufficient insulin secretion for therapeutic effect has proven challenging. Previous work in our lab showed that a tissue engineered pancreatic substitute (TEPS) based on an engineered insulin-secreting L cell line, GLUTag-INS, was insufficient in affecting blood glucose levels in streptozotocin-induced diabetic mice, but promising since human insulin was detected in the blood. The objective of this project was therefore to fabricate an improved TEPS based on GLUTag-INS cells and evaluate its suitability as a standalone diabetes therapy. To achieve this objective, the following specific aims were (1) to investigate gene incorporation as a strategy to enhance recombinant insulin secretion from GLUTag-INS cells; (2) to develop and characterize a TEPS in vitro based on a microcapsule system containing improved GLUTag-INS cells with bioluminescence monitoring capability; and (3) to assess therapeutic efficacy of the graft in a diabetic, immune-competent mouse model and use bioluminescence monitoring to elucidate in vivo transplant behavior. This thesis therefore reports on the progression of studies from the genetic and molecular levels for improved insulin secretion per-cell, to the tissue level for enhanced secretion per-graft, and lastly to the preclinical level for therapeutic assessment in a diabetic mouse model.

Pancreatic ductal adenocarcinoma is an extremely lethal cancer that is difficult to treat. A better understanding of the biology of pancreatic ductal cancer will help to develop targeted therapies that may improve clinical outcomes. Recently, the lipid signaling molecule sphingosine-1-phosphate (S1P) has emerged as a driver of malignant behavior in many types of cancer. Its role in pancreatic cancer remains unknown. Pancreatic cancer cells express high levels of the S1P receptor known as S1PR1, which is the receptor most important for mediating growth and migration through S1P signaling. In addition, the subcellular expression of the sphingosine kinases is altered in pancreatic cancer cells, which may contribute to their malignant behavior. Exogenous S1P increases pancreatic cancer cell migration, while inhibition of S1P signaling decreases the metabolic activity of pancreatic cancer cells as well as their ability to invade and migrate. Taken together, these results demonstrate the importance of S1P signaling in maintaining malignant behavior in pancreatic cancer cells. In addition, inhibition of S1P signaling represents a potential therapeutic target in pancreatic ductal cancer.

In this research a variety of drug delivery systems were synthesized and characterized. For the most part, these consisted of a matrix of poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and polyvinyl alcohol (PVA) containing encapsulated anticancer drugs as chemotherapy agents. The drug release from biodegradable nanoparticles was analyzed mathematically using new approaches that simultaneously incorporates the three major mechanisms of release: initial burst, nanoparticle degradation-relaxation, and diffusion. The theoretical release studies were corroborated experimentally by evaluating the cytotoxicity effectiveness of PHT-427-loaded nanoparticles over pancreatic cancer cells in vitro. These studies showed that the encapsulated PHT-427 drug in the nanoparticles is more accessible and thus more effective when compared with the drug alone. Also, the PHT-427-loaded nanoparticles cytotoxicity was evaluated in vivo studies with pancreatic tumors. The results show that the drug is more effective when is loaded into polymeric nanoparticles compared to drug alone, by reducing orthotopic pancreatic tumor growth. In addition, a selection of hydrophobic to hydrophilic drugs were encapsulated into polymeric nanoparticles to find optimal drug loadings by using single or double emulsification techniques. The release of these drugs from PLGA nanoparticles was evaluated to determine the overall release profile characteristics. The encapsulation of the drug pemetrexed was improved by using polyethileneimine. The high positive charge density of polyethileneimine causes a strong electrostatic interaction with the carboxylic acids of pemetrexed; this complex decreases the solubility of pemetrexed and boosts the encapsulation efficiency. Additionally, a drug release mathematical analysis that considers the effects of the temperature of release was effectively established. The analysis was performed by using two different models: the first one simultaneously incorporates the mechanisms of initial burst and nanoparticle degradation - relaxation, and the second model, besides of the mechanisms of the first model, includes the diffusion of the drug. Both models were successfully employed to describe the experimental release of rhodamine 6G from PEGylated nanoparticles at different temperatures. From the parameters obtained by the fit using each model, it was possible to define a set of new relations of the form of Arrhenius to estimate the parameters of release at other temperatures.

Metformin (MET) is a widely used drug indicated for type-2 diabetes management. Interestingly, numerous epidemiological studies show that MET may confer protective benefits from overall cancer risk and cancer-related mortality. Various pre-clinical studies show that MET also exerts chemotherapeutic properties using doses that far exceed those for glycemic control. Currently, there are numerous ongoing clinical trials testing the chemotherapeutic advantages of MET treatment in various cancer types using doses that are within the therapeutic range for diabetes management. We sought out to determine whether therapeutic doses of MET for diabetes management is chemopreventive or chemotherapeutic. We have shown that such doses are chemopreventive at best since they were unable to decrease cell viability in BxPC-3 (wild type K-RAS) and MIA PaCa-2 (mutant K-RAS) pancreatic cancer (PDAC) cells. Through a targeted metabolomics approach using 1,2-¹³C₂-D-glucose as the sole tracer, we have shown that a therapeutic dose (100 μM) of MET that is prescribed for diabetes management inhibits glucose-derived new palmitate synthesis when acetyl-CoA is dedicated towards de novo fatty acid synthesis. This occurs when a) K-RAS mutation is present (in MIA PaCa-2 cells) and b) cholesterol in the form of the more water-soluble derivative cholesteryl hemisuccinate (CHS) is supplemented in the media to prevent acetyl-CoA from being directed towards cholesterol synthesis. Immnunoblot analyses showed that this phenomenon is regulated by decreased protein expression of the fatty acid synthase (FAS) enzyme. We also showed that chronic (every two days in 30 days) CHS and MET treatment decreased triglyceride intracellular concentrations in BxPC-3 and MIA PaCa-2 cells. These treatments exerted no effect on FASN gene expression, indicating that the lipid-inhibitory effects of MET in PDAC is regulated at the metabolic flux and protein expression levels. Finally, we interrogated the metabolic effects of MET treatment using uniformly-labeled glucose tracer (¹³C₆-D-glucose) and gas chromatography/mass spectrometry (GC/MS) using a LSL-K-rasᴳ¹²ᴰ/⁺;LSL-Trp53ᴿ¹⁷²ᴴ/⁺;Pdx-1-Cre (KPC) mouse model of PDAC. We showed that acute (5 days), high-dose treatment (250 mg/kg body weight administered intraperitoneally) of MET reversed the glycolytic metabolism of pancreatic tumor into an oxidative one in the presence of K-ras and Tp53 mutation. MET, regardless of K-ras status, inhibited glucose-derived acetate enrichment towards palmitate synthesis. Immunohistochemistry analysis of KPC pancreases revealed that a decrease in the tumor cell proliferation marker Ki67 and in the FAS protein was observed in KPC mice treated with MET. In summary, we have identified a mechanism of action of the popular anti-hyperglycemic drug MET against PDAC. It is an inhibitor of de novo fatty acid synthesis that is dependent on the K-RAS and metabolic status of the tumor. Future pre-clinical and clinical studies should take this into consideration when performing mechanistic studies on MET and when investigating the potential chemotherapeutic effects of this drug. It is also important to determine what dose of MET is chemotherapeutic in the clinic. As shown by other pre-clinical studies and ours as well, this dose exceeds the range for diabetes management.

The domestic dog, Canis lupus familiaris, has emerged as a model system for the study of human hereditary diseases. Of the approximately 450 hereditary diseases described in the dog, half have clinical presentations that are quite similar to specific human diseases. Understanding the genetic bases of canine hereditary diseases will not only complement comparative genetics studies but also facilitate selective breeding practices to reduce incidences in the dog. Whole genome screens have great potential to identify the marker(s) that segregate with canine hereditary diseases for which no reasonable candidate genes exist. The Minimal Screening Set-1 (MSS-1) was the first set of microsatellite markers described for linkage analysis in the dog and was, until recently, the best tool for genome screens. The MSS-2 is the most recently described screening set and offers increased density and more polymorphic markers. The first objective of this work was to develop tools to streamline genomic analyses in the study of canine hereditary diseases. This was achieved through the development of 1) multiplexing strategies for the MSS-1, 2) a multiplex of microsatellite markers for use in canine forensics and parentage assays and 3) chromosome-specific multiplex panels for the MSS-2. Multiplexing is the simultaneous amplification and analysis of markers and significantly reduces the expense and time required to collect genotype information. Pancreatic acinar atrophy (PAA) is a disease characterized by the degeneration of acinar cells of the exocrine pancreas and is the most important cause of exocrine pancreatic insufficiency (EPI) in the German Shepherd Dog (GSD). Although the prognosis for dogs having EPI is typically good with treatment, many dogs are euthanized because the owners are unable to afford the expensive enzyme supplements. The second objective of this work was to determine the mode of transmission of EPI in the GSD and conduct a whole genome screen for linkage. Two extended families of GSDs having PAA were assembled and used to determine the pattern of transmission. The results of this indicate that PAA is an autosomal recessive disease. The multiplexed MSS-1 was used to conduct an initial whole genome screen, although no markers were suggestive of linkage.