Colony-stimulating factor 1 receptor (CSF1R, Fms) is an integral transmembrane glycoprotein with tyrosine specific protein kinase activity that it is found on the mononuclear phagocytes to promote their survival, proliferation and differentiation. Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R. There is a variable association of Fms with the stem cell marker CD34 on acute myeloid leukemia (AML) cells and this suggests different structures of the AML hierarchy in different patients. Mouse stromal cells (MS-5) were transduced with a plasmid containing human CSF-1 because mouse CSF-1 is inactive on human CSF1R. Results show that AML cells cultured with CSF-1-expressing stroma had a much better growth and survival than the control stroma, suggesting that CSF-1 might be a stimulating factor for the growth of leukemic stem cells.

This thesis is composed of three studies pertaining to the quality of care for radical cystectomy in Ontario between 1992 and 2004. In the first paper, the associations between provider volume and both operative and overall mortality were assessed. In the second paper, potential factors that could explain the association between volume and outcome were explored. In the final paper, the impact of waiting for cystectomy on survival outcomes was evaluated. Methods: A total of 3296 patients undergoing cystectomy for bladder cancer in Ontario between 1992 and 2004 were identified using the Canadian Institute for Health Information Discharge Abstract Database and the Ontario Cancer Registry. The effects of hospital and surgeon volume on operative mortality and overall survival were assessed using random effects logistic regression and marginal Cox Proportional Hazards modeling, respectively. To elucidate the factors underlying the volume-outcome association, the ability of a number of structure and process of care variables to attenuate the impact of volume was assessed. The effect of waiting for care, from transurethral resection to cystectomy, on overall survival was also assessed using marginal Cox models. Results: Neither hospital nor surgeon volume was significantly associated with operative mortality; however, both were associated with overall mortality. Of the measured structure/process measures, hospital factors caused the greatest attenuation of the volume hazard ratios, albeit to a limited degree. The wait time between the decision for surgery and cystectomy was also significantly associated with overall survival. The impact of delayed care was greatest for patients with lower stage disease. The data suggested a maximum wait time of 40 days for cystectomy. Conclusions: In this thesis, gaps in the quality of care for radical cystectomy in Ontario were identified. Patients treated by low volume hospitals and surgeons or those with long wait times all experienced worse outcomes. Since the underlying measures responsible for provider volume remain elusive, additional work is required to understand what these factors are. Initiatives to decrease wait times, however, are under way in Ontario. Whether these interventions decrease wait times and benefit patients remains to be seen.

Applied cancer research is heavily focused on the development of diagnostic tools with high sensitivity and specificity that are able to accurately detect the presence and anatomical location of neoplastic cells, as well as therapeutic strategies that are effective at curing or controlling the disease while being minimally invasive and having negligible side effects. Recently, much effort has been placed on the development of nanoparticles as diagnostic imaging and therapeutic agents, and several of these nanoplatforms have been successfully adopted in both the research and clinical arenas. This thesis describes the development of a nanoparticulate liposome system for use in a number of applications including multimodality imaging with computed tomography (CT) and magnetic resonance (MR), longitudinal vascular imaging, image-based biodistribution assessment, and CT detection of neoplastic and inflammatory lesions. Extensive in vitro and in vivo characterization was performed to determine the physico-chemical properties of the liposome agent, including its size, morphology, stability and agent loading, as well as its pharmacokinetics, biodistribution, tumor targeting and imaging performance. Emphasis was placed on the in vivo CT-based quantification of liposome accumulation and clearance from healthy and tumor tissues in a VX2 carcinoma rabbit model, gaining insight not only on the spatial but also the temporal biodistribution of the agent. The thesis concludes with a report that describes the performance of liposomes and CT imaging to detect and localize tumor and inflammatory lesions as compared to that of 18F-fluorodeoxyglucose (FDG) – positron emission tomography (PET). The outcome of the study suggests that liposome-CT could be employed as a competitive method for whole body image-based disease detection and localization. Overall, this work demonstrated that this liposome agent along with quantitative imaging systems and analysis tools, has the potential to positively impact cancer treatment outcome through improved diagnosis and staging, as well as enable personalization of treatment delivery via target delineation. However, in order to prove clinical benefit, steps must be taken to advance this agent through the regulatory stages and obtain approval for its use in humans. Ultimately, the clinical adoption of this multifunctional agent may offer improvements for disease detection, spatial delineation and therapy guidance.

This study builds on previous research showing that breast cancer survival is poorer for First Nations (FN) women compared to other Ontario women. Few studies have examined breast cancer survival in Indigenous populations compared to general populations; all of these report poorer survival among Indigenous people. Fewer still have examined potential factors related to the poorer survival, but these often suggest poorer prognosis even after adjustment for them. Study objectives were: to compare the distribution of demographic, prognostic and treatment factors between FN and non-FN women; to investigate factors associated with later diagnosis in FN women; to compare stage specific survival for FN and non-FN women controlling for important factors potentially associated with breast cancer survival; and to examine potential determinants of survival for FN women by stage at diagnosis. A case-case design was employed to compare FN women (n=287) diagnosed with invasive breast cancer to a frequency-matched random sample of women (n=671) from the general population diagnosed with breast cancer within the Ontario Cancer Registry. Women were matched (2:1) on period of diagnosis (1995-1999 and 2000-2004), age at diagnosis (<50 vs. 50≥), and Regional Cancer Centre (RCC). Stage at diagnosis and data relevant to the determinants of breast cancer survival were collected from medical charts at the RCCs. FN women were diagnosed with breast cancer at later stages compared to non-FN women. Having a non-screened method of detection and increasing BMI were associated with a later breast cancer diagnosis. FN women with comorbidity however were less likely to be diagnosed at a later stage. An unforeseen novel finding was that the survival disadvantage occurred after an early breast cancer diagnosis, whereas the survival experiences for those diagnosed at stages II+ were similar. In a multivariate analysis, elevated risk was observed for FN women in stage I, and significant risk was seen in women with comorbidity. These findings are actionable and can be used to improve the prognosis of FN women with breast cancer. It is likely that the same or similar factors are largely responsible for the survival disadvantage observed among Ontario FN people for most other major cancers.

Heat shock protein 90 (Hsp90) is an essential and conserved chaperone, required for the conformational maturation and stability of many signaling kinases. We hypothesized that the functional pleiotropism of Hsp90 can be exploited during pharmacological inhibition causing simultaneous restraint of tumor growth as well as suppression of distant spread. Recognizing the lack of therapeutic options in advanced and metastatic squamous cell carcinomas (SCC) of the uterine cervix as well as the oral cavity, this dual concept was tested in corresponding cell lines and xenografts, and correlated with clinical data on client protein expression. Examination of the cell cycle response to Hsp90 inhibition revealed a G2/M-arrest in a panel of four cervical cancer cell lines and a contribution of abnormal mitosis to apoptosis induction in vitro. Although limited to intraperitoneal application, in vivo evidence of biological activity including heat shock response and decreased client kinase phosphorylation was seen with the geldanamycin derivative 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). Importantly, focal adhesion kinase (FAK) signaling and associated functional parameters were inhibited by the drug treatment. Functional significance of FAK as a client was confirmed using a molecular model based on FAK-related non-kinase (FRNK) expression. Dependency on FAK appeared to be a requirement for full response to FRNK as well as 17-DMAG, and was observed in the mesenchymal-like cervical cell line SiHa. FAK expression and E-cadherin loss were features found in both cervical and oral malignancies, but absent from normal mucosa of either anatomic site. Particularly high FAK expression was noted in oral SCC with sarcomatoid features. Thus, we conclude that Hsp90 inhibition has potential in the treatment of advanced and metastatic SCC of cervical and oral origin. The further examination of novel Hsp90-targeting compounds as well as strategies focused on other components of the Hsp90 chaperone complex seems warranted.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

Activity of the hypoxia inducible HIF-alpha transcription factors drive the hypoxic response, resulting in enhancement of angiogenesis, tumour growth, invasion and metastasis. Seeking to uncover a role for Tid1 in control of HIF2-alpha, we used lentiviral shRNA to knock-down Tid1 in 786-0 RCC cells with and without pVHL. In 786-0 cells stably expressing pVHL30, Tid1 knock-down resulted in a dramatic reduction in HIF2-alpha levels relative to controls. Adenoviral-mediated overexpression of Tid1S rescued this decline in HIF2-alpha levels, while overexpression of Tid1L enhanced this decline. A protective role of Tid1S for HIF2-alpha was reproduced in a HEK293 cell model. Immunoprecipitations in HEK293 cells revealed a lack of direct binding between HIF2-alpha and Tid1 in vivo, while adenoviral-mediated overexpression of Tid1 in this model failed to alter in vitro binding between HIF2-alpha and pVHL30. We present a model in which Tid1 regulates HIF2-alpha stability through regulation of pVHL30 nuclear import.

Non-Small-Cell-Lung Cancer (NSCLC) patients with mutations in EGFR have greater response rates and survival when treated with the tyrosine kinase inhibitor erlotinib. To elucidate how erlotinib inhibits EGFR, this study included: 1) inhibiting an EGFR mutant cell line to reveal EGFR regulated phosphotyrosine (pY) sites; 2) comparing erlotinib sensitive and insensitive cell lines to reveal functionally important pY sites; 3) revealing novel pY sites. Observations were collected using the LTQ-Orbitrap mass spectrometer. This study identified five new EGFR regulated pY sites and five pY sites that correlated with erlotinib sensitivity; the majority of them are related to cell-cell interactions. By comparing all observed pY sites to the Phosphosite and PhosphoELM database, our results included 67 unregistered sites. This study has identified novel biomarkers and potential therapeutic targets, many of which were associated with cell migration and adhesion function. Further functional validation is necessary.

In an attempt to identify agents that specifically target neuroblastoma (NB) tumour-initiating cells (TIC) we performed drug screens using libraries of bioactive compounds. Cardiac glycosides (CGs) were the largest class of drugs identified with antitumour activity. At high CG doses inhibitory effects on the Na+/K+-ATPase induce cardiotoxicity; therefore, CG analogues were designed in an attempt to separate the effects on NB cells from cardiotoxicity. We identified RIDK34 as our lead compound from a structure-activity-relationship analysis (IC50 8 nM). RIDK34 contains a unique oxime group and shows increasing potency against NB TICs. The Na+/K+-ATPase is a target for the apoptotic activity of digoxin and RIDK34, whereby a signaling cascade involving Src and ERK may induce apoptosis. Furthermore, we predict that signaling activation does not require inactivation of the Na+/K+-ATPase and subsequent deregulation of [Na+]i and [K+]I gradients. Thus CGs and particularly RIDK34 may be expected to display diminished cardiotoxicity and greater therapeutic potential.

Dynamic nuclear polarization and dissolution of 13C-labeled metabolite allows dynamic imaging of metabolism in-vivo. However, the spatial and temporal resolutions of magnetic resonance spectroscopic imaging are limited by the duration of free-induction decay acquisitions and the T1-based, non-recoverable polarization decay. This thesis describes the implementation of a spectral-spatial radiofrequency excitation pulse with a `flyback' echo-planar readout trajectory to dynamically image [1-13C]-pyruvate and [1-13C]-lactate in an interleaved manner. This technique excites a single resonance of either [1-13C]-pyruvate or [1-13C]-lactate and generates dynamic images with 5mm in-plane resolution. Metabolite dynamics extracted from the images and the corresponding non-localized spectroscopic data reveal similar kinetic rates upon fitting to a kinetic model. This demonstrates the feasibility of probing metabolism in heterogeneous tissues in-vivo with dynamic interleaved 13C MR imaging.