Metabolic reprogramming by mutant GNAS creates an actionable dependency in intraductal papillary mucinous neoplasms of the pancreas.

BACKGROUND: Oncogenic 'hotspot' mutations of KRAS and GNAS are two major driver alterations in intraductal papillary mucinous neoplasms (IPMNs), which are bona fide precursors to pancreatic ductal adenocarcinoma. We previously reported that pancreas-specific Kras G12D and Gnas R201C co-expression in p48Cre; KrasLSL-G12D; Rosa26LSL-rtTA; Tg (TetO-GnasR201C) mice ('Kras;Gnas' mice) caused development of cystic lesions recapitulating IPMNs. OBJECTIVE: We aim to unveil the consequences of mutant Gnas R201C expression on phenotype, transcriptomic profile and genomic dependencies. DESIGN: We performed multimodal transcriptional profiling (bulk RNA sequencing, single-cell RNA sequencing and spatial transcriptomics) in the 'Kras;Gnas' autochthonous model and tumour-derived cell lines (Kras;Gnas cells), where Gnas R201C expression is inducible. A genome-wide CRISPR/Cas9 screen was conducted to identify potential vulnerabilities in KrasG12D;GnasR201C co-expressing cells. RESULTS: Induction of Gnas R201C-and resulting G(s)alpha signalling-leads to the emergence of a gene signature of gastric (pyloric type) metaplasia in pancreatic neoplastic epithelial cells. CRISPR screening identified the synthetic essentiality of glycolysis-related genes Gpi1 and Slc2a1 in Kras G12D;Gnas R201C co-expressing cells. Real-time metabolic analyses in Kras;Gnas cells and autochthonous Kras;Gnas model confirmed enhanced glycolysis on Gnas R201C induction. Induction of Gnas R201C made Kras G12D expressing cells more dependent on glycolysis for their survival. Protein kinase A-dependent phosphorylation of the glycolytic intermediate enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) was a driver of increased glycolysis on Gnas R201C induction. CONCLUSION: Multiple orthogonal approaches demonstrate that Kras G12D and Gnas R201C co-expression results in a gene signature of gastric pyloric metaplasia and glycolytic dependency during IPMN pathogenesis. The observed metabolic reprogramming may provide a potential target for therapeutics and interception of IPMNs.

Loss of Rnf43 Accelerates Kras-Mediated Neoplasia and Remodels the Tumor Immune Microenvironment in Pancreatic Adenocarcinoma.

BACKGROUND & AIMS: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been characterized sufficiently. In this study, we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. METHODS: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43flox/flox (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months, and at moribund status followed by analysis of pancreata by single-cell RNA sequencing. Comparative analyses between moribund KRC and a moribund Kras/Tp53-driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC sequencing, and in vitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. RESULTS: We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared with KC. Importantly, KRC mice have a significantly decreased survival compared with KC mice. Using single-cell RNA sequencing, we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes, with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine CXCL5 was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. CONCLUSIONS: The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared with previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

Case Report: Long-term complete response to PSMA-targeted radioligand therapy and abiraterone in a metastatic prostate cancer patient.

Despite decades of research and clinical trials, metastatic castration-resistant prostate cancer (mCRPC) remains incurable and typically fatal. Current treatments may provide modest increases in progression-free survival but can come with significant adverse effects and are disaggregated from the diagnostic imaging needed to fully assess the spread of metastatic disease. A theranostic approach, using radiolabeled ligands that target the cell surface protein PSMA, simplifies the visualization and disease treatment process by enabling both to use similar agents. Here, we describe an exemplary case wherein a gentleman in his 70s with mCRPC on diagnosis was treated with 177Lu-PSMA-617 and abiraterone, and remains disease-free to date, over five years later.

High accuracy of mesoscopic epi-fluorescence tomography for non-invasive quantitative volume determination of fluorescent protein-expressing tumours in mice.

OBJECTIVES: To compare mesoscopic epi-fluorescence tomography (MEFT) and EPRI-illumination reflectance imaging (EPRI) for quantitative tumour size assessment in mice. METHODS: Tumour xenografts of green/red fluorescent protein (GFP/RFP)-expressing colon cancer cells were measured using MEFT, EPRI, ultrasound (US) and micro computed tomography (µCT) at day 14 post-injection (n = 6). Results from MEFT and EPRI were correlated with each other and with US and µCT (reference methods). Tumour volumes were measured ex vivo by GFP and RFP fluorescence imaging on cryoslices and compared with the in vivo measurements. RESULTS: High correlation and congruency were observed between MEFT, US and µCT (MEFT/US: GFP: r (2) = 0.96; RFP: r (2) = 0.97, both P < 0.05; MEFT/µCT: GFP: r (2) = 0.93; RFP: r (2) = 0.90; both P < 0.05). Additionally, in vivo MEFT data were highly correlated and congruent with ex vivo cryoslice imaging results (GFP: r (2) = 0.96; RFP: r (2) = 0.99; both P < 0.05). In comparison, EPRI significantly overestimated tumour volumes (P < 0.05), although there was a significant correlation with US and µCT (EPRI/US: GFP: r (2) = 0.95; RFP: r (2) = 0.94; both P < 0.05; EPRI/µCT GFP: r (2) = 0.86; RFP: r (2) = 0.86; both P < 0.05). CONCLUSIONS: Fluorescence distribution reconstruction using MEFT affords highly accurate three-dimensional (3D) tumour volume data showing superior accuracy compared to EPRI. Thus, MEFT is a very suitable technique for quantitatively assessing fluorescence distribution in superficial tumours at high spatial resolution.

Blood Flow Imaging in the Neonatal Brain Using Angular Coherence Power Doppler.

Using ultrasound to image small vessels in the neonatal brain can be difficult in the presence of strong clutter from the surrounding tissue and with a neonate motion during the scan. We propose a coherence-based beamforming method, namely the short-lag angular coherence (SLAC) beamforming that suppresses incoherent noise and motion artifacts in Ultrafast data, and we demonstrate its applicability to improve detection of blood flow in the neonatal brain. Instead of estimating spatial coherence across the receive elements, SLAC utilizes the principle of acoustic reciprocity to estimate angular coherence from the beamsummed signals from different plane-wave transmits, which makes it computationally efficient and amenable to advanced beamforming techniques, such as f-k migration. The SLAC images of a simulated speckle phantom show similar edge resolution and texture size as the matching B-mode images, and reduced random noise in the background. We apply SLAC power Doppler (PD) to free-hand imaging of neonatal brain vasculature with long Doppler ensembles and show that: 1) it improves visualization of small vessels in the cortex compared to conventional PD and 2) it can be used for tracking of blood flow in the brain over time, meaning it could potentially improve the quality of free-hand functional ultrasound.

Seeing the Invisible-Ultrasound Molecular Imaging.

Ultrasound molecular imaging has been developed in the past two decades with the goal of non-invasively imaging disease phenotypes on a cellular level not depicted on anatomic imaging. Such techniques already play a role in pre-clinical research for the assessment of disease mechanisms and drug effects, and are thought to in the future contribute to earlier diagnosis of disease, assessment of therapeutic effects and patient-tailored therapy in the clinical field. In this review, we first describe the chemical composition and structure as well as the in vivo behavior of the ultrasound contrast agents that have been developed for molecular imaging. We then discuss the strategies that are used for targeting of contrast agents to specific cellular targets and protocols used for imaging. Next we describe pre-clinical data on imaging of thrombosis, atherosclerosis and microvascular inflammation and in oncology, including the pathophysiological principles underlying the selection of targets in each area. Where applicable, we also discuss efforts that are currently underway for translation of this technique into the clinical arena.

Ultrasound and microbubble mediated therapeutic delivery: Underlying mechanisms and future outlook.

Beyond the emerging field of oncological ultrasound molecular imaging, the recent significant advancements in ultrasound and contrast agent technology have paved the way for therapeutic ultrasound mediated microbubble oscillation and has shown that this approach is capable of increasing the permeability of microvessel walls while also initiating enhanced extravasation and drug delivery into target tissues. In addition, a large number of preclinical studies have demonstrated that ultrasound alone or combined with microbubbles can efficiently increase cell membrane permeability resulting in enhanced tissue distribution and intracellular drug delivery of molecules, nanoparticles, and other therapeutic agents. The mechanism behind the enhanced permeability is the temporary creation of pores in cell membranes through a phenomenon called sonoporation by high-intensity ultrasound and microbubbles or cavitation agents. At low ultrasound intensities (0.3-3 W/cm2), sonoporation may be caused by microbubbles oscillating in a stable motion, also known as stable cavitation. In contrast, at higher ultrasound intensities (greater than 3 W/cm2), sonoporation usually occurs through inertial cavitation that accompanies explosive growth and collapse of the microbubbles. Sonoporation has been shown to be a highly effective method to improve drug uptake through microbubble potentiated enhancement of microvascular permeability. In this review, the therapeutic strategy of using ultrasound for improved drug delivery are summarized with the special focus on cancer therapy. Additionally, we discuss the progress, challenges, and future of ultrasound-mediated drug delivery towards clinical translation.

Nondestructive Detection of Targeted Microbubbles Using Dual-Mode Data and Deep Learning for Real-Time Ultrasound Molecular Imaging.

Ultrasound molecular imaging (UMI) is enabled by targeted microbubbles (MBs), which are highly reflective ultrasound contrast agents that bind to specific biomarkers. Distinguishing between adherent MBs and background signals can be challenging in vivo. The preferred preclinical technique is differential targeted enhancement (DTE), wherein a strong acoustic pulse is used to destroy MBs to verify their locations. However, DTE intrinsically cannot be used for real-time imaging and may cause undesirable bioeffects. In this work, we propose a simple 4-layer convolutional neural network to nondestructively detect adherent MB signatures. We investigated several types of input data to the network: "anatomy-mode" (fundamental frequency), "contrast-mode" (pulse-inversion harmonic frequency), or both, i.e., "dual-mode", using IQ channel signals, the channel sum, or the channel sum magnitude. Training and evaluation were performed on in vivo mouse tumor data and microvessel phantoms. The dual-mode channel signals yielded optimal performance, achieving a soft Dice coefficient of 0.45 and AUC of 0.91 in two test images. In a volumetric acquisition, the network best detected a breast cancer tumor, resulting in a generalized contrast-to-noise ratio (GCNR) of 0.93 and Kolmogorov-Smirnov statistic (KSS) of 0.86, outperforming both regular contrast mode imaging (GCNR = 0.76, KSS = 0.53) and DTE imaging (GCNR = 0.81, KSS = 0.62). Further development of the methodology is necessary to distinguish free from adherent MBs. These results demonstrate that neural networks can be trained to detect targeted MBs with DTE-like quality using nondestructive dual-mode data, and can be used to facilitate the safe and real-time translation of UMI to clinical applications.

Efficacy of Affibody-Based Ultrasound Molecular Imaging of Vascular B7-H3 for Breast Cancer Detection.

PURPOSE: Human B7-H3 (hB7-H3) is a promising molecular imaging target differentially expressed on the neovasculature of breast cancer and has been validated for preclinical ultrasound (US) imaging with anti-B7-H3-antibody-functionalized microbubbles (MB). However, smaller ligands such as affibodies (ABY) are more suitable for the design of clinical-grade targeted MB. EXPERIMENTAL DESIGN: Binding of ABYB7-H3 was confirmed with soluble and cell-surface B7-H3 by flow cytometry. MB were functionalized with ABYB7-H3 or anti-B7-H3-antibody (AbB7-H3). Control and targeted MB were tested for binding to hB7-H3-expressing cells (MS1hB7-H3) under shear stress conditions. US imaging was performed with MBABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 coimplanted with MS1hB7-H3 or control MS1WT cells and a transgenic mouse model of breast cancer development. RESULTS: ABYB7-H3 specifically binds to MS1hB7-H3 and murine-B7-H3-expressing monocytes. MBABY-B7-H3 (8.5 ± 1.4 MB/cell) and MBAb-B7-H3 (9.8 ± 1.3 MB/cell) showed significantly higher (P < 0.0001) binding to the MS1hB7-H3 cells compared with control MBNon-targeted (0.5 ± 0.1 MB/cell) under shear stress conditions. In vivo, MBABY-B7-H3 produced significantly higher (P < 0.04) imaging signal in orthotopic tumors coengrafted with MS1hB7-H3 (8.4 ± 3.3 a.u.) compared with tumors with MS1WT cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MBABY-B7-H3 (9.6 ± 2.0 a.u.) produced higher (P < 0.0002) imaging signal compared with MBNon-targeted (1.3 ± 0.3 a.u.), whereas MBABY-B7-H3 signal in normal mammary glands and tumors with B7-H3 blocking significantly reduced (P < 0.02) imaging signal. CONCLUSIONS: MBABY-B7-H3 enhances B7-H3 molecular signal in breast tumors, improving cancer detection, while offering the advantages of a small size ligand and easier production for clinical imaging.

Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma.

Early detection of pancreatic ductal adenocarcinoma (PDAC) represents the most significant step toward the treatment of this aggressive lethal disease. Previously, we engineered a preclinical Thy1-targeted microbubble (MBThy1) contrast agent that specifically recognizes Thy1 antigen overexpressed in the vasculature of murine PDAC tissues by ultrasound (US) imaging. In this study, we adopted a single-chain variable fragment (scFv) site-specific bioconjugation approach to construct clinically translatable MBThy1-scFv and test for its efficacy in vivo in murine PDAC imaging, and functionally evaluated the binding specificity of scFv ligand to human Thy1 in patient PDAC tissues ex vivo. MATERIALS AND METHODS: We recombinantly expressed the Thy1-scFv with a carboxy-terminus cysteine residue to facilitate its thioether conjugation to the PEGylated MBs presenting with maleimide functional groups. After the scFv-MB conjugations, we tested binding activity of the MBThy1-scFv to MS1 cells overexpressing human Thy1 (MS1Thy1) under liquid shear stress conditions in vitro using a flow chamber setup at 0.6 mL/min flow rate, corresponding to a wall shear stress rate of 100 seconds, similar to that in tumor capillaries. For in vivo Thy1 US molecular imaging, MBThy1-scFv was tested in the transgenic mouse model (C57BL/6J - Pdx1-Cre; KRas; Ink4a/Arf) of PDAC and in control mice (C57BL/6J) with L-arginine-induced pancreatitis or normal pancreas. To facilitate its clinical feasibility, we further produced Thy1-scFv without the bacterial fusion tags and confirmed its recognition of human Thy1 in cell lines by flow cytometry and in patient PDAC frozen tissue sections of different clinical grades by immunofluorescence staining. RESULTS: Under shear stress flow conditions in vitro, MBThy1-scFv bound to MS1Thy1 cells at significantly higher numbers (3.0 ± 0.8 MB/cell; P < 0.01) compared with MBNontargeted (0.5 ± 0.5 MB/cell). In vivo, MBThy1-scFv (5.3 ± 1.9 arbitrary units [a.u.]) but not the MBNontargeted (1.2 ± 1.0 a.u.) produced high US molecular imaging signal (4.4-fold vs MBNontargeted; n = 8; P < 0.01) in the transgenic mice with spontaneous PDAC tumors (2-6 mm). Imaging signal from mice with L-arginine-induced pancreatitis (n = 8) or normal pancreas (n = 3) were not significantly different between the two MB constructs and were significantly lower than PDAC Thy1 molecular signal. Clinical-grade scFv conjugated to Alexa Fluor 647 dye recognized MS1Thy1 cells but not the parental wild-type cells as evaluated by flow cytometry. More importantly, scFv showed highly specific binding to VEGFR2-positive vasculature and fibroblast-like stromal components surrounding the ducts of human PDAC tissues as evaluated by confocal microscopy. CONCLUSIONS: Our findings summarize the development and validation of a clinically relevant Thy1-targeted US contrast agent for the early detection of human PDAC by US molecular imaging.

FN3 Protein Conjugates for Cancer Diagnosis and Imaging Studies.

Bioconjugation of biologically useful proteins is in great demand (e.g., conjugation to biotins, metal chelators, and drug carriers to target specific tissues for both in vitro and in vivo use). These conjugates provide widespread opportunities for various biological and biomedical applications. Evolving state-of-the-art protein conjugation strategies have led to the development of many affinity ligands, including for cancer imaging and diagnosis. However, to achieve the desirable protein conjugates, there are many challenges that remain to be addressed in order to obtain a reproducible procedure for all proteins and ligands. These include a control over the protein modification and the efficiency of the conjugation while retaining the original biological protein affinity postmodification. Here we present detailed conjugation methods for the human fibronectin tenth type III domain (FN3) protein scaffold for use in preclinical PET imaging. More specifically, this chapter provides detailed methods to produce a FN3 and a FN3-chelator-conjugate, its labeling with the radionuclide 64-Cu, and its use for noninvasive PET imaging in mice.

Bioinspired Preservation of Natural Killer Cells for Cancer Immunotherapy.

The ability to cryopreserve natural killer (NK) cells has a significant potential in modern cancer immunotherapy. Current cryopreservation protocols cause deterioration in NK cell viability and functionality. This work reports the preservation of human cytokine-activated NK cell viability and function following cryopreservation using a cocktail of biocompatible bioinspired cryoprotectants (i.e., dextran and carboxylated ε-poly-L-lysine). Results demonstrate that the recovered NK cells after cryopreservation and rewarming maintain their viability immediately after thawing at a comparable level to control (dimethyl sulfoxide-based cryopreservation). Although, their viability drops in the first day in culture compared to controls, the cells grow back to a comparable level to controls after 1 week in culture. In addition, the anti-tumor functional activity of recovered NK cells demonstrates higher cytotoxic potency against leukemia cells compared to control. This approach presents a new direction for NK cell preservation, focusing on function and potentially enabling storage and distribution for cancer immunotherapy.

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.

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.

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.

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.

Thy1-Targeted Microbubbles for Ultrasound Molecular Imaging of Pancreatic Ductal Adenocarcinoma.

Purpose: To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbubble-enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC).Experimental Design: Thy1-scFv were engineered using yeast-surface-display techniques. Binding to soluble human and murine Thy1 and to Thy1-expressing cells was assessed by flow cytometry. Thy1-scFv was then attached to gas-filled microbubbles to create MBThy1-scFv Thy1 binding of MBThy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MBscFv-scrambled and MBNon-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice in vivoResults: Thy1-scFv had a KD of 3.4 ± 0.36 nmol/L for human and 9.2 ± 1.7 nmol/L for murine Thy1 and showed binding to both soluble and cellularly expressed Thy1. MBThy1-scFv was attached to Thy1 with high affinity compared with negative control microbubbles (P < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (P < 0.01) higher binding of MBThy1-scFv (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MBscFv-scrambled (0.57 ± 0.53) and MBNon-targeted (0.43 ± 0.53). In vivo ultrasound molecular imaging using MBThy1-scFv demonstrated significantly higher signal (P < 0.01) in both orthotopic (5.32 ± 1.59 a.u.) and transgenic PDAC (5.68 ± 2.5 a.u.) mice compared with chronic pancreatitis (0.84 ± 0.6 a.u.) and normal pancreas (0.67 ± 0.71 a.u.). Ex vivo immunofluorescence confirmed significantly (P < 0.01) increased Thy1 expression in PDAC compared with chronic pancreatitis and normal pancreas tissue.Conclusions: A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound. Clin Cancer Res; 24(7); 1574-85. ©2018 AACR.

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.

Spectroscopic Photoacoustic Molecular Imaging of Breast Cancer using a B7-H3-targeted ICG Contrast Agent.

Purpose: Breast cancer imaging methods lack diagnostic accuracy, in particular for patients with dense breast tissue, and improved techniques are critically needed. The purpose of this study was to evaluate antibody-indocyanine green (ICG) conjugates, which undergo dynamic absorption spectrum shifts after cellular endocytosis and degradation, and spectroscopic photoacoustic (sPA) imaging to differentiate normal breast tissue from breast cancer by imaging B7-H3, a novel breast cancer associated molecular target. Methods: Quantitative immunohistochemical staining of endothelial and epithelial B7-H3 expression was assessed in 279 human breast tissue samples, including normal (n=53), benign lesions (11 subtypes, n=129), and breast cancers (4 subtypes, n=97). After absorption spectra of intracellular and degraded B7-H3-ICG and Isotype control-ICG (Iso-ICG) were characterized, sPA imaging in a transgenic murine breast cancer model (FVB/N-Tg(MMTVPyMT)634Mul) was performed and compared to imaging of control conditions [B7-H3-ICG in tumor negative animals (n=60), Iso-ICG (n=30), blocking B7-H3+B7-H3-ICG (n=20), and free ICG (n=20)] and validated with ex vivo histological analysis. Results: Immunostaining showed differential B7-H3 expression on both the endothelium and tumor epithelium in human breast cancer with an area under the ROC curve of 0.93 to differentiate breast cancer vs non-cancer. Combined in vitro/in vivo imaging showed that sPA allowed specific B7-H3-ICG detection down to the 13 nM concentration and differentiation from Iso-ICG. sPA molecular imaging of B7-H3-ICG showed a 3.01-fold (P<0.01) increase in molecular B7-H3-ICG signal in tumors compared to control conditions. Conclusions: B7-H3 is a promising target for both vascular and epithelial sPA imaging of breast cancer. Leveraging antibody-ICG contrast agents and their dynamic optical absorption spectra allows for highly specific sPA imaging of breast cancer.

A Cystine Knot Peptide Targeting Integrin αvß6 for Photoacoustic and Fluorescence Imaging of Tumors in Living Subjects.

Photoacoustic imaging is a nonionizing biomedical imaging modality with higher resolution and imaging depth than fluorescence imaging, which has greater sensitivity. The combination of the 2 imaging modalities could improve the detection of cancer. Integrin αvß6 is a cell surface marker overexpressed in many different cancers. Here, we report the development and evaluation of a dye-labeled cystine knot peptide, which selectively recognizes integrin αvß6 with high affinity, for photoacoustic and fluorescence imaging. The new dual-modality probe may find clinical application in cancer diagnosis and intraoperative imaging of integrin αvß6-positive tumors. METHODS: An engineered cystine knot peptide, R01, that recognizes integrin αvß6 was labeled with Atto 740 (A740) and evaluated for its specific cell uptake and its sensitivity threshold. A740-R01 was injected via the tail vein into nude mice xenografted with A431 (integrin αvß6-positive) or 293T (integrin αvß6-negative) tumors. Photoacoustic and fluorescence scans of tumors were acquired before and at 0.5, 1, 2, and 4 h after injection of A740-R01. Dynamic photoacoustic scans of various normal organs were also acquired. Ex vivo fluorescence imaging of tissues was performed 1 h after injection. RESULTS: The A740-R01 demonstrated integrin αvß6-dependent binding to A431 cells in culture. Sensitivity studies indicated that the probe may potentially detect lesions as small as 1 or 6 mm3 by fluorescence or photoacoustic imaging, respectively. The photoacoustic and fluorescence signals of A431 xenografts at 1 h after injection were 1.87 ± 0.25 arbitrary units (AU) and 8.27 ± 0.87 AU, respectively. Target specificity was confirmed by low tumor uptake in 293T tumors at 1 h after injection (1.07 ± 0.15 AU and 1.10 ± 0.14 AU for photoacoustic and fluorescence signals, respectively). A740-R01 exhibited hepatobiliary clearance marked by high uptake in the liver, spleen, and intestine but low uptake in the kidneys. CONCLUSION: A740-R01 specifically targeted integrin αvß6 with low nanomolar affinity. A740-R01 was able to detect integrin αvß6 both in vitro and in vivo by photoacoustic and fluorescence imaging. A740-R01 is able to detect αvß6-positive tumors in living subjects and may have clinical application in cancer diagnosis and real-time image-guided surgery.