Optimization of Affibody molecules would allow for high contrast imaging of cancer associated surface receptors using molecular imaging. The primary aim of the thesis was to develop Affibody-based PET imaging agents to provide the highest possible sensitivity of RTK detection in vivo. The thesis evaluates the effect of radiolabelling chemistry on biodistribution and targeting properties of Affibody molecules directed against HER2 and PDGFRβ. The thesis is based on five published papers (I-V). Paper I. The targeting properties of maleimido derivatives of DOTA and NODAGA for site-specific labelling of a recombinant HER2-binding Affibody molecule radiolabelled with 68Ga were compared in vivo. Favourable in vivo properties were seen for the Affibody molecule with the combination of 68Ga with NODAGA. Paper II. The aim was to compare the biodistribution of 68Ga- and 111In-labelled HER2-targeting Affibody molecules containing DOTA, NOTA and NODAGA at the N-terminus. This paper also demonstrated favourable in vivo properties for Affibody molecules in combination with 68Ga and NODAGA placed on the N-terminus. Paper III.  The influence of chelator positioning on the synthetic anti-HER2 affibody molecule labelled with 68Ga was investigated. The chelator DOTA was conjugated either at the N-terminus, the middle of helix-3 or at the C-terminus of the Affibody molecules. The N-terminus placement provided the highest tumour uptake and tumour-to-organ ratios. Paper IV. The aim of this study was to evaluate if the 68Ga labelled PDGFRβ-targeting Affibody would provide an imaging agent suitable for PDGFRβ visualization using PET. The 68Ga labelled conjugate provided high-contrast imaging of PDGFRβ-expressing tumours in vivo using microPET as early as 2h after injection. Paper V. This paper investigated if the replacement of IHPEM with IPEM as a linker molecule for radioiodination of Affibody molecules would reduce renal retention of radioactivity. Results showed that the use of the more lipophilic linker IPEM reduced the renal radioactivity retention for radioiodinated Affibody molecules. In conclusion, this thesis clearly demonstrates that the labelling strategy is of great importance with a substantial influence on the targeting properties of Affibody molecules and should be taken under serious considerations when developing new imaging agents.

Molecular photoacoustic imaging is an exciting new field that promises to visualize molecular indicators of disease. The objective of this dissertation is to progress molecular imaging by providing a photoacoustic microscopy platform to better validate in vivo molecular photoacoustic imaging, diagnose disease, and study fundamental photoacoustic processes. Initially, a custom photoacoustic microscope was developed to provide high-sensitivity and high-resolution of both endogenous and exogenous contrast agents in thin cell or tissue samples. After characterization, the photoacoustc microscope was first used to image the hemoglobin distribution in the spleen and liver. The photoacoustic microscope was then used to image nanoparticles in injured and diseased cell and tissues samples. These images can be used for in vivo photoacoustic image validation or, independently, as a diagnostic tool for disease. To enhance the utility of photoacoustic microscopy, a quantitation technique was developed for nanoparticles in cells and tissues. Quantitative photoacoustic imaging has the potential to replace mass spectrometry and histology for a wide array of molecular imaging and targeting studies. Finally, photoacoustic microscopy was used to study the nonlinear dependence of the photoacoustic pressure with laser fluence of nanoparticle-loaded cells. New discoveries about the nonlinear dependence with nanoparticle concentration and cell type are presented. These new discoveries may provide the framework for a new type of photoacoustic imaging with contrast that is cell-type specific. Overall, the work described in this dissertation can be used to improve diagnosis and accelerate clinical translation of new and emerging molecular imaging techniques. / text

The criteria for diagnosing Alzheimer’s disease (AD) have recently been revised to include the use of biomarkers for the in vivo presence of β-amyloid, one of the neuropathological hallmarks of AD. Examples of such biomarkers are positron emission tomography (PET) β-amyloid specific ligands, including [18F]flutemetamol. The aim of this thesis was to characterize the binding properties of [18F]flutemetamol from a tracer kinetic perspective as well as by validating binding measures through comparison with tissue pathology assessments. The applicability of previously developed kinetic models of tracer binding for voxel-based analysis was examined and compared to arterial input compartment modelling, the “gold standard” for PET quantification. Several voxel-based methods were found to exhibit high correlations with compartment modelling, including the semi-quantitative standardized uptake value ratio (SUVR). The kinetic components of [18F]flutemetamol uptake were also investigated without model assumptions using the data driven method spectral analysis, with binding to β-amyloid shown to relate to a slow kinetic component. The same component was also found to predominate in the uptake of white matter, known to be free of β-amyloid accumulation. White matter uptake was however possible to separate from β-amyloid binding based on the relative contribution of the slow component to the total volume of distribution. Uptake of [18F]flutemetamol as quantified using SUVR or assessed visually was found to correlate well with tissue pathology assessments. Classifying the brains of 68 deceased subjects who had undergone [18F]flutemetamol PET scanning ante mortem, based on the spatial distribution of β-amyloid according to pre-defined phases, revealed that abnormal uptake patterns of [18F]flutemetamol were only certain to be found in the last phase of β-amyloid accumulation. In the same cohort however, [18F]flutemetamol was also shown to accurately distinguish between subjects with AD and non-AD dementia. While this supports the use of [18F]flutemetamol in clinical settings for ruling out AD, the association of abnormal [18F]flutemetamol uptake to late phases of β-amyloid accumulation may limit the detection of early accumulation and pre-clinical stages of AD. It remains to be investigated whether application of voxel-based methods and slow component filtering may increase sensitivity, particularly in the context of clinical trials.

Advances in optical imaging have provided methods for visualizing molecular expression in tumors in vivo, allowing the opportunity to study the complexity of the tumor microenvironment. The development of fluorescent contrast agents targeted to molecules expressed in cancer cells is critical for in vivo imaging of the tumors. Contrast agents emitting in the near infrared (NIR) allow for an increased depth of penetration in tissue due to decreased absorption and scattering. There is also significantly less autofluorescence from tissue in the NIR. Quantum dots are nanoscopic particles of semiconductors whose fluorescent emission wavelength is tunable by the size of the particle with desirable fluorescent qualities such as a wide range of excitation wavelengths, a narrow emission band, high quantum efficiency, high photostablility, and they can be produced to emit throughout the NIR imaging window. It has been shown that vascular endothelial growth factor receptor 2 (VEGFR2) is upregulated in many cancers, including colorectal, as it is important in tumor angiogenesis and is considered a predictor for clinical outcome and, in some instances, is used for targeted therapy with anti-angiogenic drugs. For these reasons, quantum dots bioconjugated to VEGFR2 antibodies have the potential to provide contrast between normal tissue and cancer, as well as a mechanism for evaluating the molecular changes associated with cancer in vivo. In this dissertation, we present on the design of two contrast agents using quantum dots targeted to VEGFR2 for use in the molecular imaging of colon cancer, both ex vivo and in vivo. First, as a preliminary ex vivo investigation into their efficacy, Qdot655® (655nm emission) were bioconjugated to anti-VEGFR2 antibodies through streptavidin/biotin linking. The resulting QD655-VEGFR2 contrast agent was used to label colon adenoma in vivo and imaged ex vivo with significant increase in contrast between diseased and undiseased tissue, allowing for fluorescence based visualization of the VEGFR2 expressing diseased areas of the colon with high sensitivity and specificity. Then, QD655-VEGFR2 was used in a longitudinal in vivo study to investigate ability to correlate fluorescence signal to tumor development over time using optical coherence tomography and laser induced fluorescence spectroscopy (OCT/LIF) dual-modality imaging. The contrast agent was able to target VGEFR2 expressing diseased areas of colon; however, challenges in fully flushing the unbound contrast agent from the colon before imaging arise when moving from ex vivo imaging to in vivo image. Lastly, lead sulfide (PbS) quantum dots were made by colloidal synthesis to emit at a 940 nm (QD940) and conjugated to anti-VEGFR2 primary antibodies through streptavidin/biotin linking. The resulting QD940-VEGFR2 contrast agent was then used to label cells in vitro. The QD940-VEGFR2 molecules were able to positively label VEGFR2 expressing cells and did not label VEGFR2 negative cells. Very low photoluminescence and large amounts of aggregation after conjugation of the quantum dot to streptavidin was detected. Improvements to the quantum dot stability through synthesis, capping and conjugation techniques must be made for this contrast agent to be effective as a contrast agent for cancer imaging.

No description available.

More and more researchers and clinicians are looking to molecular sensing to predict how cells will behave, seeking the answers to questions like "will these tumor cells become malignant?" or "how will these cells respond to chemotherapy?" Optical methods are attractive for answering these questions because optical radiation is safer and less expensive than alternative methods, such as CT which uses X-ray radiation, PET/SPECT which use gamma radiation, or MRI which is expensive and only available in a hospital setting. In this dissertation, three distinct optical methods are explored to detect at the molecular level: optical coherence tomography (OCT), laser-induced fluorescence (LIF), and optical polarimetry. OCT has the capability to simultaneously capture anatomical information as well as molecular information using targeted contrast agents such as gold nanoshells. LIF is less useful for capturing anatomical information, but it can achieve significantly better molecular sensitivity with the use of targeted fluorescent dyes. Optical polarimetry has potential to detect the concentration of helical molecules, such as glucose. All of these methods are noninvasive or minimally invasive.The work is organized into four specific aims. The first is the design and implementation of a fast, high resolution, endoscopic OCT system to facilitate minimally invasive mouse colon imaging. The second aim is to demonstrate the utility of this system for automatically identifying tumor lesions based on tissue microstructure. The third is to demonstrate the use of contrast agents to detect molecular expression using OCT and LIF. The last aim is to demonstrate a new method based on optical polarimetry for noninvasive glucose sensing.

The human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% of breast cancers (BCs) and confers an aggressive tumour phenotype with a poor prognosis. Trastuzumab (Herceptin®) is a humanized IgG1 monoclonal antibody (mAb) approved for treatment of HER2-positive breast cancer (BC), however many eligible patients do not respond. The hypothesis was that molecular imaging strategies that probe: i) the expression of HER2; ii) one of the mechanisms of action of trastuzumab or iii) evaluate the viability of tumour cells by their glucose utilization would be useful in predicting and monitoring the response of BC to treatment with trastuzumab. The relationship between tumour HER2 density, uptake of 111In-DTPA-trastuzumab and response to trastuzumab was evaluated by gamma camera imaging, biodistribution studies and monitoring tumour growth in mice implanted with BC xenografts. There was a non-linear relationship between HER2 expression and uptake of this radiopharmaceutical when tumour uptake was corrected for non-specific IgG accumulation and/or circulating blood radioactivity (r2=0.87-0.99). Tumour response corresponded better with the uncorrected tumour uptake of 111In-DTPA-trastuzumab. HER2 downregulation, a putative mechanism of action of trastuzumab, was noted as decreased tumour uptake on microSPECT/CT of mice bearing MDA-MB-361 xenografts administered 111In-DTPA-pertuzumab. Tumour uptake of 111In-DTPA-pertuzumab was reduced by 53% in mice treated for 3 days with trastuzumab (P<0.05) associated with an early molecular response to the drug. Furthermore, tumour uptake of 111In-DTPA-pertuzumab was reduced by 78% (P<0.001) in mice treated for 3 weeks, which corresponded with a reduction in HER2-positive tumour cells, indicating a therapeutic response. The relationship between changes in tumour uptake of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and response to trastuzumab was examined in mice bearing MDA-MB-361 and MDA-MB-231 BC xenografts, with high or very low HER2 expression, treated with trastuzumab. MicroPET imaging and biodistribution studies detected a 43-60% (P<0.03) reduction in tumour uptake of 18F-FDG in mice with MDA-MB-361 xenografts, treated with trastuzumab compared to PBS-treated controls. In contrast, there was no change in 18F-FDG uptake in MDA-MB-231 xenografts, that did not respond to trastuzumab. I conclude that molecular imaging is a promising tool for monitoring response of BC to treatment with trastuzumab.

Targeted imaging with microbubbles may resolve the molecular expression within the abnormal blood vessels of tumours. Optimal imaging requires understanding the interaction between targeted microbubbles and high-frequency ultrasound. Therefore, the subharmonic signal, and backscattering cross-section of individual bound microbubbles were examined with coaligned 30 MHz pulses and optical images. The peak subharmonic signal was generated for 1.6 µm diameter microbubbles for 20% and 11% bandwidth pulses and 1.8 µm for 45% bandwidth pulses at 200 kPa, consistent with estimations of the resonant size of microbubble's at 15 MHz. In order to measure the scattering cross-section, a new method was proposed to measure the receive transfer function of a transducer. Measurements of the backscattering cross-section scaled with the square of the radius, with signi cant size-independent variability. The results of this thesis will help optimise the parameters for targeted imaging, as well as further our understanding of the behaviour of microbubbles.

Statement of Problem Telomere dynamics in the three-dimensional (3D) space of the mammalian nucleus plays an important role in the maintenance of genomic stability. However, the telomere distribution in 3D nuclear space of normal and tumor cells was unknown when the study was initiated. Methods Telomere fluorescence in situ hybridization (FISH) and 3D molecular imaging, deconvolution, and analysis were used to investigate telomere organization in normal, immortalized and tumor cells from mouse and human cell lines, and primary tissues. Results Telomeres are organized in a non-overlapping manner and in a cell-cycle dependant fashion in normal cells. In the late G2 phase of cell cycle, telomeres are assembled into a flattened sphere that is termed the telomeric disk In contrast, the telomeric disk is disrupted in the tumor cells. Moreover, telomeric aggregates (TAs) are found in tumor cells. Conditional c-Myc over-expression induces telomeric aggregation leading to the onset of breakage-bridge-fusion cycles and subsequent chromosomal abnormality. Conclusions Telomeres are distributed in a nonrandom and dynamic fashion in the 3D space of a normal cell. Telomeric aggregates are present in cells with genomic instability such as tumor cells and cells with deregulation of c-Myc. Consequently, TA can be a useful biomarker for research in cancer and other disease processes.

Targeted imaging with microbubbles may resolve the molecular expression within the abnormal blood vessels of tumours. Optimal imaging requires understanding the interaction between targeted microbubbles and high-frequency ultrasound. Therefore, the subharmonic signal, and backscattering cross-section of individual bound microbubbles were examined with coaligned 30 MHz pulses and optical images. The peak subharmonic signal was generated for 1.6 µm diameter microbubbles for 20% and 11% bandwidth pulses and 1.8 µm for 45% bandwidth pulses at 200 kPa, consistent with estimations of the resonant size of microbubble's at 15 MHz. In order to measure the scattering cross-section, a new method was proposed to measure the receive transfer function of a transducer. Measurements of the backscattering cross-section scaled with the square of the radius, with signi cant size-independent variability. The results of this thesis will help optimise the parameters for targeted imaging, as well as further our understanding of the behaviour of microbubbles.