Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging.

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

Dual-Isotope Cryoimaging Quantitative Autoradiography: Investigating Antibody-Drug Conjugate Distribution and Payload Delivery Through Imaging.

In vitro properties of antibody-drug conjugates (ADCs) such as binding, internalization, and cytotoxicity are often well characterized before in vivo studies. Interpretation of in vivo studies might be significantly enhanced by molecular imaging tools. We present here a dual-isotope cryoimaging quantitative autoradiography (CIQA) methodology combined with advanced 3-dimensional imaging and analysis allowing for the simultaneous study of both antibody and payload distribution in tissues of interest in a preclinical setting. Methods: TAK-264, an investigational ADC targeting anti-guanylyl cyclase C (GCC), was synthesized using tritiated monomethyl auristatin E. The tritiated ADC was then conjugated to diethylenetriaminepentaacetic acid, labeled with 111In, and evaluated in vivo in animals bearing GCC-positive and GCC-negative tumors. Results: CIQA revealed the time course of drug release from ADC and its distribution into various tumor regions that are less accessible to the antibody. For GCC-positive tumors, a representative section obtained 96 h after tracer injection showed only 0.8% of the voxels to have colocalized signal, versus over 15% of the voxels for a GCC-negative tumor section, suggesting successful and specific cleaving of the toxin in the GCC-positive lesions. Conclusion: The combination of a veteran established autoradiography technology with advanced image analysis methodologies affords an experimental tool that can support detailed characterization of ADC tumor penetration and pharmacokinetics.

Identification of the S100 fused-type protein hornerin as a regulator of tumor vascularity.

Sustained angiogenesis is essential for the development of solid tumors and metastatic disease. Disruption of signaling pathways that govern tumor vascularity provide a potential avenue to thwart cancer progression. Through phage display-based functional proteomics, immunohistochemical analysis of human pancreatic ductal carcinoma (PDAC) specimens, and in vitro validation, we reveal that hornerin, an S100 fused-type protein, is highly expressed on pancreatic tumor endothelium in a vascular endothelial growth factor (VEGF)-independent manner. Murine-specific hornerin knockdown in PDAC xenografts results in tumor vessels with decreased radii and tortuosity. Hornerin knockdown tumors have significantly reduced leakiness, increased oxygenation, and greater apoptosis. Additionally, these tumors show a significant reduction in growth, a response that is further heightened when therapeutic inhibition of VEGF receptor 2 (VEGFR2) is utilized in combination with hornerin knockdown. These results indicate that hornerin is highly expressed in pancreatic tumor endothelium and alters tumor vessel parameters through a VEGF-independent mechanism.Angiogenesis is essential for solid tumor progression. Here, the authors interrogate the proteome of pancreatic cancer endothelium via phage display and identify hornerin as a critical protein whose expression is essential to maintain the pancreatic cancer vasculature through a VEGF-independent mechanism.

Cardiac-selective expression of extracellular superoxide dismutase after systemic injection of adeno-associated virus 9 protects the heart against post-myocardial infarction left ventricular remodeling.

BACKGROUND: Cardiac magnetic resonance imaging has not been used previously to document the attenuation of left ventricular (LV) remodeling after systemic gene delivery. We hypothesized that targeted expression of extracellular superoxide dismutase (EcSOD) via the cardiac troponin-T promoter would protect the mouse heart against both myocardial infarction (MI) and subsequent LV remodeling. METHODS AND RESULTS: Using reporter genes, we first compared the specificity, time course, magnitude, and distribution of gene expression from adeno-associated virus (AAV) 1, 2, 6, 8, and 9 after intravenous injection. The troponin-T promoter restricted gene expression largely to the heart for all AAV serotypes tested. AAV1, 6, 8, and 9 provided early-onset gene expression that approached steady-state levels within 2 weeks. Gene expression was highest with AAV9, which required only 3.15×10(11) viral genomes per mouse to achieve an 84% transduction rate. AAV9-mediated, cardiac-selective gene expression elevated EcSOD enzyme activity in heart by 5.6-fold (P=0.015), which helped protect the heart against both acute MI and subsequent LV remodeling. In acute MI, infarct size in EcSOD-treated mice was reduced by 40% compared with controls (P=0.035). In addition, we found that cardiac-selective expression of EcSOD increased myocardial capillary fractional area and decreased neutrophil infiltration after MI. In a separate study of LV remodeling, after a 60-minute coronary occlusion, cardiac magnetic resonance imaging revealed that LV volumes at days 7 and 28 post-MI were significantly lower in the EcSOD group compared with controls. CONCLUSIONS: Cardiac-selective expression of EcSOD from the cardiac troponin-T promoter after systemic administration of AAV9 provides significant protection against both acute MI and LV remodeling.

Rapid analysis of vessel elements (RAVE): a tool for studying physiologic, pathologic and tumor angiogenesis.

Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI) that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity), and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.

Development of Secreted Protein and Acidic and Rich in Cysteine (SPARC) Targeted Nanoparticles for the Prognostic Molecular Imaging of Metastatic Prostate Cancer.

Prostate cancer is the most commonly diagnosed non-skin malignancy in the United States and presents with a wide range of aggressiveness from extremely slow-growing to highly aggressive. There is a clinical need to determine the metastatic potential of the primary tumor to design the most appropriate treatment plan ranging from watchful waiting to more aggressive, invasive surgical treatments. In this study we have developed a nanoparticle based imaging agent that targets SPARC (Secreted Protein Acidic Rich in Cysteine), a molecular marker of prostate cancer metastatic potential. Previous studies by this group used phage display to identify a peptide with high binding affinity and specificity for SPARC. In this study, the SPARC-targeted peptide sequence was used to design a biomaterial with improved pharmacokinetic properties by attaching it to a biocompatible nanoparticle that is also coupled to a fluorophore for in vivo imaging. Prostate cancer cell lines with varying degrees of SPARC expression were used to show the ability of the targeted nanoparticle to bind specifically to SPARC in vitro and in vivo including the clinically relevant bone and lung metastases. We show that in vivo imaging information correlates with the metastatic potential of the prostate tumor. This prognostic information could enable doctors to stratify patients and design personalized treatment strategies.

Molecular imaging agents: impact on diagnosis and therapeutics in oncology.

Imaging has become a crucial tool in oncology throughout the course of disease detection and management, and is an integral part of clinical trials. Anatomical and functional imaging led the way, providing valuable information used in the diagnosis of disease, including data regarding the size and location of the tumour and on physiological processes such as blood flow and perfusion. As understanding of cancer pathogenesis has advanced through the identification of genetic, biochemical and cellular alterations in evolving tumours, emphasis has been put on developing methods to detect and serially monitor such alterations. This class of approaches is referred to as molecular imaging. Molecular imaging offers the potential for increasingly sensitive and specific visualisation and quantification of biological processes at the cellular and molecular level. These approaches have become established as essential tools for cancer research, early cancer detection and staging, and monitoring and predicting response to targeted therapies. Here, we discuss recent advances in the development of molecular imaging agents and their implementation in basic cancer research as well as in more rationalised approaches to cancer care.