The clinical efficacy of chemotherapy in solid tumours is limited by systemic toxicity and the inability to deliver a cytotoxic concentration of anticancer drugs to all tumour cells. Temperature sensitive drug carriers provide a mechanism for triggering the rapid release of chemotherapeutic agents in a targeted region. Thermally mediated drug release also leverages the ability of hyperthermia to increase tumour blood flow, vessel permeability, and drug cytotoxicity. Drug release from thermosensitive liposome drug carriers in the tumour vasculature serves as a continuous intravascular infusion of free drug originating at the tumour site. However, localized drug release requires precise heating to improve drug delivery and efficacy in tumours while minimizing drug exposure in normal tissue. Focused ultrasound can noninvasively heat millimeter-sized regions deep within the body, and can be combined with MR thermometry for precise temperature control. This thesis describes the development of strategies to achieve localized hyperthermia using MRI-controlled focused ultrasound, for the purpose of image-guided heat-triggered drug release from thermosensitive drug carriers. First, a preclinical MRI-controlled focused ultrasound system was developed as a platform for studies of controlled hyperthermia and drug delivery in rabbits. The feasibility of using ultrasound hyperthermia to achieve localized doxorubicin release from thermosensitive liposomes was demonstrated in normal rabbit muscle. Second, strategies were described for using MR thermometry to control ultrasound heating at a muscle-bone interface based on MR temperature measurements in adjacent soft tissue, demonstrating localized drug delivery in adjacent muscle and bone marrow. Third, fluorescence microscopy was employed to demonstrate that increased overall drug accumulation in rabbit VX2 tumours corresponds to high levels of bioavailable drug reaching their active site in the nuclei of tumour cells. The results of this thesis demonstrate that image-guided drug delivery using thermosensitive liposomes and MRI-controlled focused ultrasound hyperthermia can be used to noninvasively achieve precisely localized drug deposition in soft tissue, at bone interfaces, and in solid tumours. Clinical application of this work could provide a noninvasive means of enhancing chemotherapy in a variety of solid tumours.

PKCe plays a very important role in mediating the protection against myocardial ischemia and reperfusion injury induced by ischemic preconditioning (IPC) and pharmacologic preconditioning (PPC). The redistribution of PKCe was assessed by subcellular fractionation and western blotting in the Langendorff-perfused rabbit heart. Either 5min ischemia or 5min administration of adenosine A1 and/or A3 agonists, bradykinin, angiotensin II, and d1-opioid agonists resulted in PKCe redistribution from the cytosol to the mitochondria. This effect of IPC on PKCe redistribution was visible up to at least 30min of reperfusion, while that of PPC was lost by 10min of drug washout, indicative of the transient nature of PKCe redistribution. PKCe redistribution to mitochondria by IPC was also visualized using immunogold electron microscopy. Thus, IPC and PPC caused PKCe redistribution from the cytosol to the mitochondria, which was longer-lasting in IPC than in PPC.

PKCe plays a very important role in mediating the protection against myocardial ischemia and reperfusion injury induced by ischemic preconditioning (IPC) and pharmacologic preconditioning (PPC). The redistribution of PKCe was assessed by subcellular fractionation and western blotting in the Langendorff-perfused rabbit heart. Either 5min ischemia or 5min administration of adenosine A1 and/or A3 agonists, bradykinin, angiotensin II, and d1-opioid agonists resulted in PKCe redistribution from the cytosol to the mitochondria. This effect of IPC on PKCe redistribution was visible up to at least 30min of reperfusion, while that of PPC was lost by 10min of drug washout, indicative of the transient nature of PKCe redistribution. PKCe redistribution to mitochondria by IPC was also visualized using immunogold electron microscopy. Thus, IPC and PPC caused PKCe redistribution from the cytosol to the mitochondria, which was longer-lasting in IPC than in PPC.

The clinical efficacy of chemotherapy in solid tumours is limited by systemic toxicity and the inability to deliver a cytotoxic concentration of anticancer drugs to all tumour cells. Temperature sensitive drug carriers provide a mechanism for triggering the rapid release of chemotherapeutic agents in a targeted region. Thermally mediated drug release also leverages the ability of hyperthermia to increase tumour blood flow, vessel permeability, and drug cytotoxicity. Drug release from thermosensitive liposome drug carriers in the tumour vasculature serves as a continuous intravascular infusion of free drug originating at the tumour site. However, localized drug release requires precise heating to improve drug delivery and efficacy in tumours while minimizing drug exposure in normal tissue. Focused ultrasound can noninvasively heat millimeter-sized regions deep within the body, and can be combined with MR thermometry for precise temperature control. This thesis describes the development of strategies to achieve localized hyperthermia using MRI-controlled focused ultrasound, for the purpose of image-guided heat-triggered drug release from thermosensitive drug carriers. First, a preclinical MRI-controlled focused ultrasound system was developed as a platform for studies of controlled hyperthermia and drug delivery in rabbits. The feasibility of using ultrasound hyperthermia to achieve localized doxorubicin release from thermosensitive liposomes was demonstrated in normal rabbit muscle. Second, strategies were described for using MR thermometry to control ultrasound heating at a muscle-bone interface based on MR temperature measurements in adjacent soft tissue, demonstrating localized drug delivery in adjacent muscle and bone marrow. Third, fluorescence microscopy was employed to demonstrate that increased overall drug accumulation in rabbit VX2 tumours corresponds to high levels of bioavailable drug reaching their active site in the nuclei of tumour cells. The results of this thesis demonstrate that image-guided drug delivery using thermosensitive liposomes and MRI-controlled focused ultrasound hyperthermia can be used to noninvasively achieve precisely localized drug deposition in soft tissue, at bone interfaces, and in solid tumours. Clinical application of this work could provide a noninvasive means of enhancing chemotherapy in a variety of solid tumours.

Background: Functional magnetic resonance imaging (fMRI) can require extensive preprocessing to minimize noise and maximize signal. There is evidence suggesting that fixed-subject preprocessing pipelines, the current standard in fMRI preprocessing, are suboptimal compared to individual-subject pipelines. Aim: We sought to test if individual-subject preprocessing pipeline optimization, compared to fixed, resulted in stronger and more reliable brain-patterns in episodic recognition. Methodology: 27 young healthy controls were scanned via fMRI while performing forced-choice episodic recognition. Several sets of fMRI preprocessing pipelines were tested and optimized in a fixed and individual-subject manner, using methods outlined by Churchill et al. (2011). Results: Individual-subject pipeline optimization, compared to fixed, significantly increased reproducibility, significantly increased the detection of positively and negatively activated voxels, and resulted in a brain-pattern with significant correlation to a task behavioral measure. Conclusions: Individual-subject pipeline optimization, compared to fixed, led to stronger and more reliable brain-patterns that are significantly correlated with behavior.

Background: Functional magnetic resonance imaging (fMRI) can require extensive preprocessing to minimize noise and maximize signal. There is evidence suggesting that fixed-subject preprocessing pipelines, the current standard in fMRI preprocessing, are suboptimal compared to individual-subject pipelines. Aim: We sought to test if individual-subject preprocessing pipeline optimization, compared to fixed, resulted in stronger and more reliable brain-patterns in episodic recognition. Methodology: 27 young healthy controls were scanned via fMRI while performing forced-choice episodic recognition. Several sets of fMRI preprocessing pipelines were tested and optimized in a fixed and individual-subject manner, using methods outlined by Churchill et al. (2011). Results: Individual-subject pipeline optimization, compared to fixed, significantly increased reproducibility, significantly increased the detection of positively and negatively activated voxels, and resulted in a brain-pattern with significant correlation to a task behavioral measure. Conclusions: Individual-subject pipeline optimization, compared to fixed, led to stronger and more reliable brain-patterns that are significantly correlated with behavior.

The effective diagnosis of leukemia subtypes requires the detection of multiple cell surface markers. Current methods of detection use mostly fluorophores, which are limited by their large spectral bandwidths, photobleaching, and incompatibility with histological stains used for morphological assessments. Antibody-conjugated Surface enhanced Raman scattering (SERS) nanoparticles is an alternative tool that overcomes these limitations. A current drawback of SERS is the lack of available tools to analyze the bioconjugation of antibodies to nanoparticles following EDC/sulfo-NHS cross-linking, which produces inconsistent results and determines the efficacy of SERS probe targeting. This study uses the flow cytometry approach to evaluate SERS particles by incorporating FITC and DyLight650 secondary antibodies. Flow cytometry was also used to assess targeting of particles to markers on LY10 cells and CLL cells and to detect SERS signals by inserting a 710 BP 10nm FWHM filter specific for MGITC.

The effective diagnosis of leukemia subtypes requires the detection of multiple cell surface markers. Current methods of detection use mostly fluorophores, which are limited by their large spectral bandwidths, photobleaching, and incompatibility with histological stains used for morphological assessments. Antibody-conjugated Surface enhanced Raman scattering (SERS) nanoparticles is an alternative tool that overcomes these limitations. A current drawback of SERS is the lack of available tools to analyze the bioconjugation of antibodies to nanoparticles following EDC/sulfo-NHS cross-linking, which produces inconsistent results and determines the efficacy of SERS probe targeting. This study uses the flow cytometry approach to evaluate SERS particles by incorporating FITC and DyLight650 secondary antibodies. Flow cytometry was also used to assess targeting of particles to markers on LY10 cells and CLL cells and to detect SERS signals by inserting a 710 BP 10nm FWHM filter specific for MGITC.

Radiation therapy is administered to more than 50% of patients diagnosed with cancer. Mechanisms of interaction between radiation and tumour cells are relatively well understood on a molecular level, but much remains uncertain regarding how radiation interacts with the tumour as a whole. Recent studies have suggested that tumour response to radiation may in fact be regulated by endothelial cell response, consequently stressing the role of tumour blood vessels in radiation treatment response. As a result, various treatment regimens have been proposed to strategically combine radiation with vascular targeting agents. A great deal of effort has been aimed towards developing efficient vascular targeting agents. Nonetheless, no optimal method has yet been devised to strategically deliver such agents. Recent evidence suggesting that these drugs may “normalize” tumour blood vessels and enhance radiosensitivity, is supporting experiments where anti-angiogenic drugs are combined with cytotoxic therapies such as radiotherapy. In contrast, ultrasound-stimulated microbubbles have recently been demonstrated to enhance radiation therapy by biophysically interacting with endothelial cells. When combined with single radiation doses, these microbubbles are believed to cause localized vascular destruction followed by tumour cell death. Finally, a new form of ‘pro-angiogenics’ has also been demonstrated to induce a therapeutic tumour response. The overall aim of this thesis is to study the role of tumour blood vessels in treatment responses to single-dose radiation therapy and to investigate radiation-based vascular targeting strategies. Using pharmacological and biophysical agents, blood vessels were altered to determine how they influence tumour cell death, clonogenicity, and tumour growth, and to study how these may be optimally combined with radiation. Three-dimensional high-frequency power Doppler ultrasound was used throughout these studies to investigate vascular response to therapy.

Statins, common cholesterol control drugs, are appreciated to have promising anticancer activity through inhibition of the mevalonate pathway. Several lines of evidence suggest that certain tumours are susceptible to statins, but the underlying molecular features arbitrating this sensitivity remain unknown. We hypothesize that (i) not all statins will behave equivalently in the context of anticancer therapy, and (ii) a molecularly-defined subset of tumours are intrinsically sensitive to statins. My objectives have therefore been to further our understanding of functional differences between statins influencing their anticancer effects, and to investigate molecular features associated with statin sensitivity in breast cancer. Specifically, this thesis addresses two aims: (i) to characterize differential interactions between four statins and the xenobiotic transporter P-glycoprotein (P-gp; also known as ABCB1), and (ii) to identify molecular features associated with fluvastatin and lovastatin sensitivity in breast tumour cell lines. We first characterized the interactions of statins with P-gp in vitro and in multidrug-resistant (MDR) tumour cells. While lovastatin could directly bind to P-gp and modulate MDR, no significant interactions were observed with fluvastatin. Fluvastatin may therefore be appropriate for use in unselected patients, to avoid adverse drug interactions with coadministered P-gp substrate chemotherapeutics. Fluvastatin has also shown promise in breast cancer treatment, where molecular features predictive of statin sensitivity would be particularly valuable. A panel of 19 immortalized breast cell lines was therefore characterized for sensitivity to fluvastatin and lovastatin. Relatively statin-sensitive cells underwent apoptosis upon statin treatment, and were more likely to have an estrogen receptor alpha (ERα)-negative, basal-like phenotype. By mining available baseline gene expression data, a candidate 10-gene signature predictive of fluvastatin sensitivity was also generated. Taken together, this research provides insight into molecular markers of statin sensitivity that may facilitate fast-tracking of these drugs to clinical trials in subsets of cancer patients most likely to respond.