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

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.

Our objective was to examine the impact of palliative care (PC) on aggressive care near death for patients with advanced pancreatic cancer. Measures of aggressive care included (i) chemotherapy within 14 days of death; (ii) more than one emergency department (ED) visit; (iii) more than one hospitalization; and (iv) at least one intensive care unit (ICU) admission, all within 30 days of death. A retrospective population-based cohort study was conducted with patients diagnosed with advanced pancreatic cancer in Ontario. Multivariable logistic analyses were performed. Our final cohort consisted of 5,381 patients (median survival of 75 days). 52% received a PC consultation. PC consultation was associated with decreased use of chemotherapy near death (OR=0.34); and lower risk of ICU admission (OR=0.12), multiple ED visits (OR=0.19), and multiple hospitalizations near death (OR=0.24). A per unit increase in the monthly rate of PC visits was associated with lower odds of aggressive care.

Our objective was to examine the impact of palliative care (PC) on aggressive care near death for patients with advanced pancreatic cancer. Measures of aggressive care included (i) chemotherapy within 14 days of death; (ii) more than one emergency department (ED) visit; (iii) more than one hospitalization; and (iv) at least one intensive care unit (ICU) admission, all within 30 days of death. A retrospective population-based cohort study was conducted with patients diagnosed with advanced pancreatic cancer in Ontario. Multivariable logistic analyses were performed. Our final cohort consisted of 5,381 patients (median survival of 75 days). 52% received a PC consultation. PC consultation was associated with decreased use of chemotherapy near death (OR=0.34); and lower risk of ICU admission (OR=0.12), multiple ED visits (OR=0.19), and multiple hospitalizations near death (OR=0.24). A per unit increase in the monthly rate of PC visits was associated with lower odds of aggressive care.

Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.

No description available.

This thesis describes a study undertaken between 1992 and 2001 to explore the possible health impacts of human exposure to pesticides. The study followed the health outcomes of approximately 4000 outdoor workers over a period of up to sixty-one years. These workers comprised two subcohorts of approximately even size, one composed of agricultural workers with high insecticide exposures, and the other made up of outdoor staff from local councils in the same area with little or no occupational exposure to insecticides. Mortality and morbidity were compared between the two groups, and with the general Australian community. The study identifies significantly increased mortality among both exposed and control subjects when compared to the Australian population. The major cause of this increase was mortality from smoking related diseases. The study also identifies significant increases in mortality among exposed subjects for a number of conditions that do not appear to be the result of smoking patterns, both when compared to the control group and the Australian population. These include pancreatic cancer in some DDT exposed subjects and asthma, diabetes, and leukaemia in subjects working with more modern chemicals. There was also an increase in self reported chronic illness and asthma, and lower neuropsychological functioning scores among surviving exposed subjects when compared to controls. Diabetes was also reported more commonly by subjects reporting occupational use of herbicides.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet need with a 5-year survival following diagnosis of 3% with limited surgical, radiotherapy and chemotherapy treatment options. Central to PDAC tumorigenesis is acquisition of an oncogenic Kras mutation to drive acinar-to-ductal metaplasia and progression to PDAC that is potentiated by NF-kB deregulation. However, PDAC requires the additional loss of tumour suppressors such as p53, SMAD4 or p16. ASPP family members ASPP2 and iASPP regulate both p53 and NF-kB, and are classified as a tumour suppressor and oncogene respectively. However, the precise roles of ASPP2 and iASPP in pancreatic cancer are unknown. In this thesis I demonstrate that ASPP2 suppresses metastasis and iASPP suppresses the pro-inflammatory tumour microenvironment. In a mouse model of PDAC development, ASPP2-deficiency does not alter metaplasia, PanIN progression or primary PDAC onset. However, median survival due to metastasis is significantly reduced in an ASPP2-deficient PDAC model. I demonstrate ASPP2-deficient PDAC can result in increased squamous differentiation defined histologically or via increased p63 expression. I propose ASPP2 is a key suppressor ΔNp63 and the squamous PDAC subtype in vivo. Conversely, iASPP is a putative oncogene and high expression in cancer associates with poor prognosis. However, in a mouse model of PDAC, loss of iASPP accelerates PDAC onset and metastasis. I demonstrate that iASPP is a functional tumour suppressor of a pro-inflammatory phenotype in response to oncogenic Kras and pancreatitis. I propose ASPP2- and iASPP-deficient mouse models of PDAC represent in vivo the squamous and immunogenic subtypes of PDAC respectively; and are relevant tools to study mechanisms of metastasis and inflammation-driven carcinogenesis.