Evaluation of integrin αvß6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvß6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvß6.

Cellular-Based Selections Aid Yeast-Display Discovery of Genuine Cell-Binding Ligands: Targeting Oncology Vascular Biomarker CD276.

Yeast surface display is a proven tool for the selection and evolution of ligands with novel binding activity. Selections from yeast surface display libraries against transmembrane targets are generally carried out using recombinant soluble extracellular domains. Unfortunately, these molecules may not be good models of their true, membrane-bound form for a variety of reasons. Such selection campaigns often yield ligands that bind a recombinant target but not target-expressing cells or tissues. Advances in cell-based selections with yeast surface display may aid the frequency of evolving ligands that do bind true, membrane-bound antigens. This study aims to evaluate ligand selection strategies using both soluble target-driven and cellular selection techniques to determine which methods yield translatable ligands most efficiently and generate novel binders against CD276 (B7-H3) and Thy1, two promising tumor vasculature targets. Out of four ligand selection campaigns carried out using only soluble extracellular domains, only an affibody library sorted against CD276 yielded translatable binders. In contrast, fibronectin domains against CD276 and affibodies against CD276 were discovered in campaigns that either combined soluble target and cellular selection methods or used cellular selection methods alone. A high frequency of non target-specific ligands discovered from the use of cellular selection methods alone motivated the development of a depletion scheme using disadhered, antigen-negative mammalian cells as a blocking agent. Affinity maturation of CD276-binding affibodies by error-prone PCR and helix walking resulted in strong, specific cellular CD276 affinity ( Kd = 0.9 ± 0.6 nM). Collectively, these results motivate the use of cellular selections in tandem with recombinant selections and introduce promising affibody molecules specific to CD276 for further applications.

Ultrasound molecular imaging as a non-invasive companion diagnostic for netrin-1 interference therapy in breast cancer.

In ultrasound molecular imaging (USMI), ligand-functionalized microbubbles (MBs) are used to visualize vascular endothelial targets. Netrin-1 is upregulated in 60% of metastatic breast cancers and promotes tumor progression. A novel netrin-1 interference therapy requires the assessment of netrin-1 expression prior to treatment. In this study, we studied netrin-1 as a target for USMI and its potential as a companion diagnostic in breast cancer models. Methods: To verify netrin-1 expression and localization, an in vivo immuno-localization approach was applied, in which anti-netrin-1 antibody was injected into living mice 24 h before tumor collection, and revealed with secondary fluorescent antibody for immunofluorescence analysis. Netrin-1 interactions with the cell surface were studied by flow cytometry. Netrin-1-targeted MBs were prepared using MicroMarker Target-Ready (VisualSonics), and validated in in vitro binding assays in static conditions or in a flow chamber using purified netrin-1 protein or netrin-1-expressing cancer cells. In vivo USMI of netrin-1 was validated in nude mice bearing human netrin-1-positive SKBR7 tumors or weakly netrin-1-expressing MDA-MB-231 tumors using the Vevo 2100 small animal imaging device (VisualSonics). USMI feasibility was further tested in transgenic murine FVB/N Tg(MMTV/PyMT634Mul) (MMTV-PyMT) mammary tumors. Results: Netrin-1 co-localized with endothelial CD31 in netrin-1-positive breast tumors. Netrin-1 binding to the surface of endothelial HUVEC and cancer cells was partially mediated by heparan sulfate proteoglycans. MBs targeted with humanized monoclonal anti-netrin-1 antibody bound to netrin-1-expressing cancer cells in static and dynamic conditions. USMI signal was significantly increased with anti-netrin-1 MBs in human SKBR7 breast tumors and transgenic murine MMTV-PyMT mammary tumors compared to signals recorded with either isotype control MBs or after blocking of netrin-1 with humanized monoclonal anti-netrin-1 antibody. In weakly netrin-1-expressing human tumors and normal mammary glands, no difference in imaging signal was observed with anti-netrin-1- and isotype control MBs. Ex vivo analysis confirmed netrin-1 expression in MMTV-PyMT tumors. Conclusions: These results show that USMI allowed reliable detection of netrin-1 on the endothelium of netrin-1-positive human and murine tumors. Significant differences in USMI signal for netrin-1 reflected the significant differences in netrin-1 mRNA & protein expression observed between different breast tumor models. The imaging approach was non-invasive and safe, and provided the netrin-1 expression status in near real-time. Thus, USMI of netrin-1 has the potential to become a companion diagnostic for the stratification of patients for netrin-1 interference therapy in future clinical trials.

Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents.

MicroRNAs are critical regulators of cancer initiation, progression, and dissemination. Extensive evidence suggests that the inhibition of over-expressed oncogenic miRNA function can be a robust strategy for anticancer therapy. However, in vivo targeted delivery of miRNA therapeutics to various types of cancers remains a major challenge. Inspired by their natural synthesis and cargo delivery capabilities, researchers have exploited tumor cell-derived extracellular vesicles (TEVs) for the cancer-targeted delivery of therapeutics and theranostics. Here, we investigate a TEV-based nanoplatform for multimodal miRNA delivery and phototherapy treatments as well as the magnetic resonance imaging of cancer. We demonstrated loading of anti-miR-21 that blocks the function of endogenous oncogenic miR-21 over-expressed in cancer cells into and subsequent delivery by TEVs derived from 4T1 cells. We also produced Cy5-anti-miR-21-loaded TEVs from two other cancer cell lines (HepG2 and SKBR3) and confirmed their robust homologous and heterologous transfection efficiency and intracellular Cy5-anti-miR-21 delivery. Additionally, TEV-mediated anti-miR-21 delivery attenuated doxorubicin (DOX) resistance in breast cancer cells with a 3-fold higher cell kill efficiency than in cells treated with DOX alone. We then investigated TEVs as a biomimetic source for the functionalization of gold-iron oxide nanoparticles (GIONs) and demonstrated nanotheranostic properties of TEV-GIONs in vitro. TEV-GIONs demonstrated excellent T2 contrast in in vitro magnetic resonance (MR) imaging and resulted in efficient photothermal effect in 4T1 cells. We also evaluated the biodistribution and theranostic property of anti-miR-21 loaded TEV-GIONs in vivo by labeling with indocyanine green near-infrared dye. We further validated the tumor specific accumulation of TEV-GIONs using MR imaging. Our findings demonstrate that the distribution pattern of the TEV-anti-miR-21-GIONs correlated well with the tumor-targeting capability as well as the activity and efficacy obtained in response to doxorubicin combination treatments. TEVs and TEV-GIONs are promising nanotheranostics for future applications in cancer molecular imaging and therapy.

Advances in Diagnostic and Intraoperative Molecular Imaging of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. To improve outcomes, there is a critical need for improved tools for detection, accurate staging, and resectability assessment. This could improve patient stratification for the most optimal primary treatment modality. Molecular imaging, used in combination with tumor-specific imaging agents, can improve established imaging methods for PDAC. These novel, tumor-specific imaging agents developed to target specific biomarkers have the potential to specifically differentiate between malignant and benign diseases, such as pancreatitis. When these agents are coupled to various types of labels, this type of molecular imaging can provide integrated diagnostic, noninvasive imaging of PDAC as well as image-guided pancreatic surgery. This review provides a detailed overview of the current clinical imaging applications, upcoming molecular imaging strategies for PDAC, and potential targets for imaging, with an emphasis on intraoperative imaging applications.

Longitudinal assessment of ultrasound-guided complementary microRNA therapy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second-leading cause of cancer related deaths worldwide and new strategies to efficiently treat HCC are critically needed. The aim of this study was to assess the longitudinal treatment effects of two complementary miRNAs (miRNA-122 and antimiR-21) encapsulated in biodegradable poly lactic-co-glycolic acid (PLGA) - poly ethylene glycol (PEG) nanoparticles (PLGA-PEG-NPs), administered by an ultrasound-guided and microbubble-mediated delivery approach in doxorubicin-resistant and non-resistant human HCC xenografts. Using in vitro assays, we show that repeated miRNA treatments resulted in gradual reduction of HCC cell proliferation and reversal of doxorubicin resistance. Optimized US parameters resulted in a 9-16 fold increase (p = 0.03) in miRNA delivery in vivo in HCC tumors after two US treatments compared to tumors without US treatment. Furthermore, when combined with doxorubicin (10 mg/kg), longitudinal miRNA delivery showed a significant inhibition of tumor growth in both resistant and non-resistant tumors compared to non-treated, and doxorubicin treated controls. We also found that ultrasound-guided miRNA therapy was not only effective in inhibiting HCC tumor growth but also allowed lowering the dose of doxorubicin needed to induce apoptosis. In conclusion, the results of this study suggest that ultrasound-guided and MB-mediated delivery of miRNA-122 and antimiR-21, when combined with doxorubicin, is a highly effective approach to treat resistant HCC while reducing doxorubicin doses needed for treating non-resistant HCC in longitudinal treatment experiments. Further refinement of this strategy could potentially lead to better treatment outcomes for patients with HCC.

[Contrast-enhanced ultrasound: Liver Imaging Reporting and Data System (CEUS LI-RADS)]. / Standardisierte Befundung und Dokumentation der Kontrastmittelsonografie der Leber (CEUS LI-RADS).

The American College of Radiology (ACR) endorsed the Liver Imaging Reporting and Data System (LI-RADS) for standardized reporting and data collection of computed tomography (CT) and magnetic resonance (MR) imaging for hepatocellular carcinoma (HCC) in high-risk patients (liver cirrhosis). The LI-RADS imaging criteria are used to classify 'observations' from 'definitely benign' (LR-1) to 'definitely HCC' (LR-5) based on imaging criteria.Coincidently, the recent approval in the United States of a microbubble contrast agent for liver imaging (Lumason®, known as SonoVue® in Europe and elsewhere), LI-RADS. is being expanded to include contrast-enhanced ultrasound (CEUS). An international working group was initiated in 2014. Herewith, the most current version of CEUS-LI-RADS is presented.

Structured Reporting of Multiphasic CT for Hepatocellular Carcinoma: Effect on Staging and Suitability for Transplant.

OBJECTIVE: The purpose of this study is to evaluate whether use of a standardized radiology report template would improve the ability of liver transplant surgeons to diagnose stage T2 hepatocellular carcinoma (HCC) and determine patient suitability to undergo orthotopic liver transplant (OLT). MATERIALS AND METHODS: In this retrospective study, a standardized template was devised, and its use was mandated for reporting of liver CT findings for patients with cirrhosis and HCC. Two surgeons analyzed 200 reports (100 before and 100 after template implementation) for descriptions of cirrhosis, portal hypertension, lesion enhancement characteristics, tumor thrombus, portal and superior mesenteric vein patency, and Organ Procurement Transplantation Network (OPTN) class. Ability to determine Milan criteria and surgeon satisfaction were also assessed. Data obtained before and after template implementation were statistically analyzed using the Cochran-Mantel-Haenszel test. RESULTS: Template implementation increased the percentage of reports documenting the presence or absence of portal hypertension (74% to 88% for surgeon 1 and 86% to 87% for surgeon 2; p = 0.042); lesion number (76% to 88% for surgeon 2 [no change for surgeon 1]; p = 0.038), size (95% to 96% for surgeon 1 and 82% to 93% for surgeon 2; p = 0.03), and enhancement (93% to 94% for surgeon 1 and 80% to 91% for surgeon 2; p = 0.049); presence of tumor thrombus (10% to 57% for surgeon 1 and 31% to 63% for surgeon 2; p < 0.001); and OPTN class (8% to 82% for surgeon 1 and 2% to 81% for surgeon 2; p < 0.001). The surgeons were significantly more able to determine the presence of T2 disease and qualification for exception points after implementation of the template (increasing from 80% to 94%; p = 0.025). Satisfaction with reports also improved (p < 0.0001). CONCLUSION: The reporting template improved determination of patient suitability to undergo transplant according to the Milan criteria.

How to perform Contrast-Enhanced Ultrasound (CEUS).

"How to perform contrast-enhanced ultrasound (CEUS)" provides general advice on the use of ultrasound contrast agents (UCAs) for clinical decision-making and reviews technical parameters for optimal CEUS performance. CEUS techniques vary between centers, therefore, experts from EFSUMB, WFUMB and from the CEUS LI-RADS working group created a discussion forum to standardize the CEUS examination technique according to published evidence and best personal experience. The goal is to standardise the use and administration of UCAs to facilitate correct diagnoses and ultimately to improve the management and outcomes of patients.

Development and Preclinical Validation of a Cysteine Knottin Peptide Targeting Integrin αvß6 for Near-infrared Fluorescent-guided Surgery in Pancreatic Cancer.

Purpose: Intraoperative near-infrared fluorescence (NIRF) imaging could help stratification for the proper primary treatment for patients with pancreatic ductal adenocarcinoma (PDAC), and achieve complete resection, as it allows visualization of cancer in real time. Integrin αvß6, a target specific for PDAC, is present in >90% of patients, and is able to differentiate between pancreatitis and PDAC. A clinically translatable αvß6-targeting NIRF agent was developed, based on a previously developed cysteine knottin peptide for PET imaging, R01-MG, and validated in preclinical mouse models.Experimental Design: The applicability of the agent was tested for cell and tissue binding characteristics using cell-based plate assays, subcutaneous, and orthotopic pancreatic models, and a transgenic mouse model of PDAC development (Pdx1-Cretg/+;KRasLSL G12D/+;Ink4a/Arf-/-). IRDye800CW was conjugated to R01-MG in a 1:1 ratio. R01-MG-IRDye800, was compared with a control peptide and IRDye800 alone.Results: In subcutaneous tumor models, a significantly higher tumor-to-background ratio (TBR) was seen in BxPC-3 tumors (2.5 ± 0.1) compared with MiaPaCa-2 (1.2 ± 0.1; P < 0.001), and to the control peptide (1.6 ± 0.4; P < 0.005). In an orthotopic tumor model, tumor-specific uptake of R01-MG-IRDye800 was shown compared with IRDye800 alone (TBR 2.7 vs. 0.86). The fluorescent signal in tumors of transgenic mice was significantly higher, TBR of 3.6 ± 0.94, compared with the normal pancreas of wild-type controls, TBR of 1.0 ± 0.17 (P < 0.001).Conclusions: R01-MG-IRDye800 shows specific targeting to αvß6, and holds promise as a diagnostic and therapeutic tool to recognize PDAC for fluorescence-guided surgery. This agent can help improve the stratification of patients for a potentially curative, margin-negative resection. Clin Cancer Res; 24(7); 1667-76. ©2018 AACR.

Improved Sensitivity in Ultrasound Molecular Imaging With Coherence-Based Beamforming.

Ultrasound molecular imaging (USMI) is accomplished by detecting microbubble (MB) contrast agents that have bound to specific biomarkers, and can be used for a variety of imaging applications, such as the early detection of cancer. USMI has been widely utilized in preclinical imaging in mice; however, USMI in humans can be challenging because of the low concentration of bound MBs and the signal degradation caused by the presence of heterogenous soft tissue between the transducer and the lesion. Short-lag spatial coherence (SLSC) beamforming has been proposed as a robust technique that is less affected by poor signal quality than standard delay-and-sum (DAS) beamforming. In this paper, USMI performance was assessed using contrast-enhanced ultrasound imaging combined with DAS (conventional CEUS) and with SLSC (SLSC-CEUS). Each method was characterized by flow channel phantom experiments. In a USMI-mimicking phantom, SLSC-CEUS was found to be more robust to high levels of additive thermal noise than DAS, with a 6dB SNR improvement when the thermal noise level was +6dB or higher. However, SLSC-CEUS was also found to be insensitive to increases in MB concentration, making it a poor choice for perfusion imaging. USMI performance was also measured in vivo using VEGFR2-targeted MBs in mice with subcutaneous human hepatocellular carcinoma tumors, with clinical imaging conditions mimicked using a porcine tissue layer between the tumor and the transducer. SLSC-CEUS improved the SNR in each of ten tumors by an average of 41%, corresponding to 3.0dB SNR. These results indicate that the SLSC beamformer is well-suited for USMI applications because of its high sensitivity and robust properties under challenging imaging conditions.

CEUS LI-RADS: algorithm, implementation, and key differences from CT/MRI.

Contrast-enhanced ultrasound (CEUS) is a specialized form of ultrasound (US) performed with an intravenous injection of microbubble contrast agents. It has been successfully used for a variety of applications including characterization of liver tumors. In April 2014, the American College of Radiology (ACR) convened a working group of international experts to develop ACR CEUS Liver Imaging Reporting and Data System (CEUS LI-RADS). An initial version of CEUS LI-RADS was published in August 2016. Although the CEUS LI-RADS concept and principles for liver lesion characterization, using dynamic contrast enhancement features, are similar to those for CT or MRI, there are significant differences between CT/MRI and CEUS LI-RADS. Therefore, CEUS LI-RADS has different diagnostic features and a unique characterization algorithm. The size of a lesion, the type and degree of arterial phase enhancement, the presence of washout, and the timing and degree of washout are the major features used for categorization. This paper describes key differences between CT/MRI and CEUS, and provides a diagnostic algorithm of CEUS LI-RADS with detailed, step-by-step instructions and imaging examples of CEUS LI-RADS categories.

Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents.

PURPOSE: Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo. PROCEDURES: By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking. This work presents a biomedical imaging characterization of MEs including: MRI transverse relaxivity (r 2) for MEs and ME-labeled cells (compared to a commercially available iron oxide nanoparticle); microscopic validation of labeling efficiency and subcellular locations; and in vivo imaging of a MDA-MB-231BR (231BR) human breast cancer cells in a mouse brain. RESULTS: At 7T, r 2 relaxivity of bare MEs was higher (250 s-1 mM-1) than that of conventional SPIO (178 s-1 mM-1). Optimized in vitro loading of MEs into 231BR cells yielded 1-4 pg iron/cell (compared to 5-10 pg iron/cell for conventional SPIO). r 2 relaxivity dropped by a factor of ~3 upon loading into cells, and was on the same order of magnitude for ME-loaded cells compared to SPIO-loaded cells. In vivo, ME-labeled cells exhibited strong MR contrast, allowing as few as 100 cells to be detected in mice using an optimized 3D SPGR gradient-echo sequence. CONCLUSIONS: Our results demonstrate the potential of magneto-endosymbionts as living MR contrast agents. They have r 2 relaxivity values comparable to traditional iron oxide nanoparticle contrast agents, and provide strong MR contrast when loaded into cells and implanted in tissue.

Contrast-enhanced ultrasound of the liver: technical and lexicon recommendations from the ACR CEUS LI-RADS working group.

Contrast-enhanced ultrasound (CEUS) is a specific form of ultrasound imaging performed with intravenous administration of microbubble contrast agents. It has been extensively used for liver tumor characterization and was recently added to the American College of Radiology Liver Imaging Reporting and Data System (CEUS LI-RADS). This paper describes technical recommendations for successful liver CEUS lesion characterization, and provides imaging protocol and Lexicon of imaging findings.

Contrast-enhanced ultrasound (CEUS) liver imaging reporting and data system (LI-RADS) 2017 - a review of important differences compared to the CT/MRI system.

Medical imaging plays an important role in the diagnosis and management of hepatocellular carcinoma (HCC). The Liver Imaging Reporting and Data System (LI-RADS) was initially created to standardize the reporting and data collection of CT and MR imaging for patients at risk for HCC. As contrast-enhanced ultrasound (CEUS) has been widely used in clinical practice, it has recently been added to the LI-RADS. While CEUS LI-RADS shares fundamental concepts with CT/MRI LI-RADS, there are key differences between the modalities reflecting dissimilarities in the underlying methods of image acquisition and types of contrast material. This review introduces a recent update of CEUS LI-RADS and explains the key differences from CT/MRI LI-RADS.

Ultrasound Elastography: Review of Techniques and Clinical Applications.

Elastography-based imaging techniques have received substantial attention in recent years for non-invasive assessment of tissue mechanical properties. These techniques take advantage of changed soft tissue elasticity in various pathologies to yield qualitative and quantitative information that can be used for diagnostic purposes. Measurements are acquired in specialized imaging modes that can detect tissue stiffness in response to an applied mechanical force (compression or shear wave). Ultrasound-based methods are of particular interest due to its many inherent advantages, such as wide availability including at the bedside and relatively low cost. Several ultrasound elastography techniques using different excitation methods have been developed. In general, these can be classified into strain imaging methods that use internal or external compression stimuli, and shear wave imaging that use ultrasound-generated traveling shear wave stimuli. While ultrasound elastography has shown promising results for non-invasive assessment of liver fibrosis, new applications in breast, thyroid, prostate, kidney and lymph node imaging are emerging. Here, we review the basic principles, foundation physics, and limitations of ultrasound elastography and summarize its current clinical use and ongoing developments in various clinical applications.

In vitro characterization and in vivo ultrasound molecular imaging of nucleolin-targeted microbubbles.

Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p < 0.001. We then set out to create a method to extract both the tumor wash-in and wash-out kinetics and tumor accumulation following a single injection of targeted MBs. In order to accomplish this, a series of ultrasound frames (a clip) was recorded at the time of injection and subsequent time points. Each pixel within this clip was analyzed for the minimum intensity projection (MinIP) and average intensity projection (AvgIP). We found that the MinIP robustly demonstrates enhanced accumulation of F3-conjugated MBs over the range of tumor diameters evaluated here (2-8 mm), and the difference between the AvgIP and the MinIP quantifies inflow and kinetics. The inflow and clearance were similar for unbound F3-conjugated MBs, control (non-targeted) and scrambled control agents. Targeted agent accumulation was confirmed by a high amplitude pulse and by a two-dimensional Fourier Transform technique. In summary, F3-conjugated MBs provide a new imaging agent for ultrasound molecular imaging of cancer vasculature, and we have validated metrics to assess performance using low mechanical index strategies that have potential for use in human molecular imaging studies.