Investigating the Mechanisms of Breast Cancer Metastasis Using Multimodality Molecular Imaging

Introduction: Breast cancer recurrence continues to be a significant challenge in the clinic. Despite successful removal and/or treatment of the original tumour, many patients experience relapse in the breast or at distant sites. Furthermore, the diagnosis of metastatic disease often occurs too late for effective treatment. Methods: In this thesis, we combine iron-based cellular MRI and longitudinal BLI to noninvasively track the fate of cancer cells into overt tumours in the mouse brain. We then apply this imaging model to study the effect of a primary breast tumour on the growth of secondary metastases in an immune competent mouse model. Finally, we utilized dual-luciferase BLI to investigate the potential of self-homing circulating tumour cells (CTCs) as a novel cancer theranostic in both orthotopic and metastatic models of breast cancer. Results: BLI complemented our cellular MRI technologies well by providing longitudinal measures of cancer cell viability. Using in vivo BLI/MRI, we demonstrated the presence of a 4T1 primary tumour significantly enhances total brain tumour burden. Finally, using dual-luciferase BLI, we demonstrated the ability of experimental CTCs to home to and treat primary tumours and disseminated breast cancer lesions. Conclusion: MRI and BLI are complementary technologies to noninvasively study the fate of breast cancer cells, as well as the mechanisms contributing to metastasis including CTR/CTE and tumour self-homing. Furthermore, we provide evidence that CTCs are a novel theranostic platform for the visualization and treatment of pre-established tumour sites throughout the body

Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models

Confocal and two-photon microscopy has been widely used in bone research to not only produce high quality, three-dimensional images but also to provide valuable structural and quantitative information. In this article, we describe step-by-step protocols for confocal and two-photon microscopy to investigate earlier cellular events during colonisation of cancer cells in bone using xenograft mouse models. This includes confocal/two-photon microscopy imaging of paraformaldehyde fixed thick bone sections and frozen bone samples

Advancing fluorescent contrast agent recovery methods for surgical guidance applications

Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

Developing a novel spheroid-on-chip microfluidic device for investigations into metastasis

One in two people born after 1960 will develop cancer, with 90% of all cancer deaths arising from metastasis. Conventional 2D in vitro metastasis models do not fully replicate tumour complexity. In vivo models can address tumour complexity, but do not fully represent human tumour biology. Multicellular spheroids are widely used 3D models of cancer. Spheroids contain internal zonal differentiation of oxygen, metabolite, and nutrient gradients, associated with regions of proliferative, quiescent, and necrotic cells. Current in vitro static spheroid methodologies do not recapitulate factors of cellular spread including continuous flow or shear stress. Therefore, better 3D in vitro models to investigate metastasis represent an area of unmet need. This research aims to develop spheroid-on-chip models, providing a novel strategy to investigate cancer spread. The microfluidic devices used in this study feature weirs for spheroid inclusion, and a borosilicate base coverslip for optical clarity. The devices feature an access port allowing direct access to the microwell. Spheroids derived from established cancer cell lines, MCF7 and U-87 MG, were formed off-chip and incorporated into the device by pipetting, before being perfused with complete media at 3 μL min-1 for 72 h. Cell viability was assessed in effluent, using the CytoTox Glo assay, demonstrating spheroid viability is robustly maintained on-chip. In situ analysis of cell viability, through FDA/PI live/dead staining indicated an increased proportion of viable cells and decreased dead cells on-chip compared to off-chip. VEGF (Vascular Endothelial Growth Factor) ELISA showed that VEGF secretion, as evaluated by its presence in conditioned media, was comparable across all testing conditions. The chip model has been further developed to allow spheroids to be embedded in ECM (extracellular matrix)-like matrices. VEGF ELISA and IL-6 ELISA showed that both VEGF and IL-6 secretion, as evaluated by its presence in conditioned media, was comparable across all testing conditions. ELISA also showed that IL-6 and VEGF was increased in the on-chip models within hydrogel conditions. The device has also allowed the direct imaging of spheroids on chip over 72 h. The analysis of U-87 MG spheroids on chip showed that invasion through Matrigel was comparable to the off-chip static models. Whilst migration on chip, through collagen analysis, was increased over the off-chip counterparts. The work shown offers a novel insight into cancer metastasis; on a more replicative model than the current conventional in vitro techniques

Development of targeted therapeutic strategies for metastatic lung cancer

El cáncer de pulmón es el cáncer que se diagnostica con más frecuencia y la principal causa de muerte por cáncer en todo el mundo. Es importante destacar que alrededor del 75% de los pacientes son diagnosticados en estadios metastásicos avanzados, cuando la cirugía ya no es posible, lo que supone una caída dramática de la tasa de supervivencia a 5 años al 6%. El principal objetivo de esta tesis es definir nuevas estrategias terapéuticas inspiradas en la biología para pacientes con cáncer de pulmón metastásico. Para ello, se exploraron diferentes estrategias terapéuticas inspiradas en la biología del tumor, una de ellas proviene de los exosomas tumorales y la otra de las células que diseminan desde el tumor primario para formar las metástasis

Design and Development of Polymeric Nanoparticles for the Delivery of Therapeutics

The development of nanoscale drug delivery systems is a rapidly growing field within the realm of nanomedicine, as it has the potential to improve therapeutic efficacy and minimize side effects of various drugs. This dissertation focuses on the rational design, development and application of well-defined polymeric nanoparticles, capable of high loading of both hydrophobic and hydrophilic therapeutic agents, toward the effective treatment of lung diseases. In the first study, cisplatin was loaded into non-degradable poly(acrylic acid)-b-polystyrene-based SCKs through the formation of coordination bonds between platinum and carboxylate groups in the nanoparticle shell domain. The effects of crosslinking were investigated by comparing drug loading & release, in vitro cytotoxicities, and immunotoxicities. In another study, degradable polyphosphoester-based polymeric micelles and SCKs, each derived from non-cytotoxic, amphiphilic block-graft terpolymers, were specifically designed and synthesized for anti-cancer drug paclitaxel (PTX) delivery toward the treatment of osteosarcoma lung metastases. PTX could be encapsulated into either micelles or SCKs, with overall PTX concentration as high as 4.8 mg/mL vs. the low solubility for free PTX in water of less than 2.0 μg/mL. In vivo biodistribution indicated that both micelles & SCKs underwent extravasation from the lung in a controlled manner, while crosslinking slowed the rate of extravasation significantly. Moreover, hydrophilic silver cations were also attached to the nanoparticles via the interaction between silver and alkyne as a potential treatment for bacterial pulmonary infections. The well-defined Ag-loaded nanoparticles released silver in a controlled and sustained manner over 5 days, and displayed enhanced in vitro antibacterial activities against cystic fibrosis-associated pathogens and decreased cytotoxicity to human bronchial epithelial cells, in comparison to silver acetate

Smart design and in vitro testing of nanoparticles for microenvironmentally-triggered extracellular drug release

In the field of nanotechnology, one of the most operative research areas is nanomedicine, which applies nanotechnology to highly specific medical interventions for the prevention, diagnosis and treatment of diseases. Currently, the major issue that nanomedicine needs to face is the smart design and production of nanoparticles (NPs) based drug delivery systems for cancer therapy. Highly efficient drug delivery based on nanoparticles could potentially reduce the drug dose needed to achieve therapeutic benefit, thus reducing the side effects associated with the systemic delivery of drugs, whit great benefit to the patient. Indeed, a site-specific delivery of the active compound can be obtained manipulating NP surface by attaching ligands, such as peptides, antibodies or aptamers. Moreover, both passive and active targeting of the drug can be easily obtained by manipulating NP size and surface characteristics. NPs can also control and sustain the release of a drug during transport to, or at, the site of localization, altering drug distribution and subsequent clearance. At present, a new family of nanovectors, defined as stimuli-responsive nanocarriers (SRNs), is emerging. The key point in their mechanism of action lay in the fact that a specific cellular or extracellular endogenous stimulus of chemical, biochemical, or physical origin can modify NP conformation thus promoting the release of the active agent in a specific biological environment [1] [2]. In particular, a large variety of enzymes, such as proteases, glucuronidase, or carboxylesterases can be used as biochemical triggers. Generally the proteases, that are extracellularly expressed, such as the matrix metalloproteases (MMPs), are up-regulated in tumour microenvironment and are responsible for the proteolysis of the extracellular matrix (ECM) and of the basement membranes along with tissue remodelling and metastasis invasion. Since that, they are commonly identified as biomarkers of malignant tissues [3]. In the light of these considerations, Chapter.1 points out a smart approach in NPs design that takes benefits from the MMPs over-expression at tumour site, in order to produce a stimuli-responsive nanocarrier that allows a site specific drug release. To this aim, we proposed the use of a novel nanoparticle able to carry safely doxorubicin (Dox) at tumour tissues, and to respond to MMP-2 enzyme. The produced NPs are made up of a biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) – block – PEG copolymer (namely PELGA), blended with a TAP (Tumour Activated Pro-drug) composed by a MMP-2-sensitive peptide bound to Dox at the C-terminus and to PLGA molecule at the N-terminus. These NPs are named PELGA-TAP NPs. The presence of the MMP-2 enzyme in situ, leads to the destruction of the bond between the peptide and the Dox, with the consequent diffusion and accumulation of the drug in the extracellular environment. This mechanism allows the drug delivery only in presence of an endogenous stimulus that comes from the very nature of the tumour tissue itself. Furthermore, the same NPs were prepared without the presence of the peptide sequence, as negative control, and were named PELGA-Dox. Spheroids of U87 (Human Glioma cells) and HDF (Human Dermal Fibroblast) cells were used as in vitro models of tumour and healthy tissue, respectively, to demonstrate NPs ability to “sense” the differences in the expression levels of endogenous MMP-2 enzymes [4]. Since the production process and effectiveness of PELGA-TAP and PELGA-Dox NPs was well established and consolidate, in Chapter.2 we tested them in a new three-dimensional microtissue (3D µTP) model, which is an in vitro tissue equivalent proposed by Brancato et al. [5]. They fabricate µTPs with the aim to replicate in vitro the composition and the functionalities of the tumour microenvironment. In this work they clearly show that µTPs better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the tumour microenvironment. In particular, the 3D model was developed using normal fibroblasts (NF) and human epithelial cell lines (MCF10), or cancer-activated fibroblasts (CAF) and human breast adenocarcinoma cells (MCF7), to produce healthy and cancer microtissues, respectively. In this scenario, PELGA-TAP and PELGA-Dox NPs were tested in terms of Dox release on these µTPs in order to further validate their efficacy and selective drug release in a more realistic in vitro model, which better resemble tumour microenvironment, closer to the in vivo conditions [6]. Moreover, Chapter.3 shows an upgrade of the PELGA-TAP NP presented above. The approach used for the production of the nanocarrier takes advantages from the layer by layer polymer deposition technique developed and optimized by Vecchione et al. [7]. This technique allows the production of a very stable nanocarrier able to load large amounts of hydrophobic drugs and prevents their systemic leakage. The delivery system we proposed is a crosslinked polyelectrolytes nanocapsule (NC) based on an oil-core and a matrix metalloproteases-2-sensitive shell. MMP-2 enzymes catalyse the disassembly of the NC, which is stabilized by a MMP-2-cleavable peptide sequence as cross-linker. Also in this case, the drug release occurs in a spatially-controlled fashion upon an endogenous stimulus coming from the very nature of the tumour itself. The same NC was also produced with a scrambled peptide sequence as negative control. These NCs were tested on a spheroidal in vitro model, in order to proof their selective shell destabilization and consequent stimuli-responsive drug release in tumour microenvironment. Spheroids of U87 and HDF were used as models of tumour and healthy tissue, respectively. Cell viability was evaluated by means of Alamar Blue Assay. Moreover, the selective disassembly of the NC shell was followed using confocal microscopy and colocalization analyses were also performed. Finally, in Chapter.4 preliminary studies aimed to point out the advantages of an extracellular drug delivery are presented