A Bitter Taste Receptor as a Novel Molecular Target on Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma.

Cancer-associated fibroblasts (CAFs) execute diverse and complex functions in cancer progression. While reprogramming the crosstalk between CAFs and cancer epithelial cells is a promising avenue to evade the adverse effects of stromal depletion, drugs are limited by their suboptimal pharmacokinetics and off-target effects. Thus, there is a need to elucidate CAF-selective cell surface markers that can improve drug delivery and efficacy. Here, functional proteomic pulldown with mass spectrometry was used to identify taste receptor type 2 member 9 (TAS2R9) as a CAF target. TAS2R9 target characterization included binding assays, immunofluorescence, flow cytometry, and database mining. Liposomes conjugated to a TAS2R9-specific peptide were generated, characterized, and compared to naked liposomes in a murine pancreatic xenograft model. Proof-of-concept drug delivery experiments demonstrate that TAS2R9-targeted liposomes bind with high specificity to TAS2R9 recombinant protein and exhibit stromal colocalization in a pancreatic cancer xenograft model. Furthermore, the delivery of a CXCR2 inhibitor by TAS2R9-targeted liposomes significantly reduced cancer cell proliferation and constrained tumor growth through the inhibition of the CXCL-CXCR2 axis. Taken together, TAS2R9 is a novel cell-surface CAF-selective target that can be leveraged to facilitate small-molecule drug delivery to CAFs, paving the way for new stromal therapies.

Correlation of 68Ga-FAPi-46 PET Biodistribution with FAP Expression by Immunohistochemistry in Patients with Solid Cancers: Interim Analysis of a Prospective Translational Exploratory Study.

Fibroblast activation protein (FAP)-expressing cancer-associated fibroblasts confer treatment resistance and promote metastasis and immunosuppression. Because FAP is overexpressed in many cancers, radiolabeled molecules targeting FAP are studied for their use as pancancer theranostic agents. This study aimed to establish the spectrum of FAP expression across various cancers by immunohistochemistry and to explore whether 68Ga FAP inhibitor (FAPi)-46 PET biodistribution faithfully reflects FAP expression from resected cancer and non-cancer specimens. Methods: We conducted a FAP expression screening using immunohistochemistry on a pancancer human tissue microarray (141 patients, 14 different types of cancer) and an interim analysis of a prospective exploratory imaging trial in cancer patients. Volunteer patients underwent 1 whole-body 68Ga-FAPi-46 PET/CT scan and, subsequently, surgical resection of their primary tumor or metastasis. 68Ga-FAPi-46 PET SUVmax and SUVmean was correlated with FAP immunohistochemistry score in cancer and tumor-adjacent non-cancer tissues for each patient. Results: FAP was expressed across all 14 cancer types on tissue microarray with variable intensity and frequency, ranging from 25% to 100% (mean, 76.6% ± 25.3%). Strong FAP expression was observed in 50%-100% of cancers of the bile duct, bladder, colon, esophagus, stomach, lung, oropharynx, ovary, and pancreas. Fifteen patients with various cancer types (colorectal [n = 4], head and neck [n = 3], pancreas [n = 2], breast [n = 2], stomach [n = 1], esophagus [n = 2], and uterus [n = 1]) underwent surgery after their 68Ga-FAPi-46 PET/CT scan within a mean interval of 16.1 ± 14.4 d. 68Ga-FAPi-46 SUVs and immunohistochemistry scores were higher in cancer than in tumor-adjacent non-cancer tissue: mean SUVmax 7.7 versus 1.6 (P < 0.001), mean SUVmean 6.2 versus 1.0 (P < 0.001), and mean FAP immunohistochemistry score 2.8 versus 0.9 (P < 0.001). FAP immunohistochemistry scores strongly correlated with 68Ga-FAPi 46 SUVmax and SUVmean: r = 0.781 (95% CI, 0.376-0.936; P < 0.001) and r = 0.783 (95% CI, 0.379-0.936; P < 0.001), respectively. Conclusion: In this interim analysis of a prospective exploratory imaging trial, 68Ga-FAPi-46 PET biodistribution across multiple cancers strongly correlated with FAP tissue expression. These findings support further exploration of FAPi PET as a pancancer imaging biomarker for FAP expression and as a stratification tool for FAP-targeted therapies.

A Novel Monoclonal Antibody Targeting Cancer-Specific Plectin Has Potent Antitumor Activity in Ovarian Cancer.

Cancer-specific plectin (CSP) is a pro-tumorigenic protein selectively expressed on the cell surface of major cancers, including ovarian cancer (OC). Despite its assessable localization, abundance, and functional significance, the therapeutic efficacy of targeting CSP remains unexplored. Here, we generated and investigated the anticancer effects of a novel CSP-targeting monoclonal antibody, 1H11, in OC models. Its therapeutic efficacy as a monotherapy and in combination with chemotherapy was evaluated in vitro using two OC cell lines and in vivo by a subcutaneous ovarian cancer model. 1H11 demonstrated rapid internalization and high affinity and specificity for both human and murine CSP. Moreover, 1H11 induced significant and selective cytotoxicity (EC50 = 260 nM), G0/G1 arrest, and decreased OC cell migration. Mechanistically, these results are associated with increased ROS levels and reduced activation of the JAK2-STAT3 pathway. In vivo, 1H11 decreased Ki67 expression, induced 65% tumor growth inhibition, and resulted in 30% tumor necrosis. Moreover, 1H11 increased chemosensitivity to cisplatin resulting in 60% greater tumor growth inhibition compared to cisplatin alone. Taken together, CSP-targeting with 1H11 exhibits potent anticancer activity against ovarian cancer and is deserving of future clinical development.

Plectin in Cancer: From Biomarker to Therapeutic Target.

The cytolinker and scaffolding protein, plectin, has emerged as a potent driver of malignant hallmarks in many human cancers due to its involvement in various cellular activities contributing to tumorigenesis, including cancer cell proliferation, adhesion, migration, invasion, and signal transduction. Evidence shows that beyond plectin's diverse protein interactome, its cancer-specific mislocalization to the cell surface enables its function as a potent oncoprotein. As such, therapeutic targeting of plectin, its protein interactors, and, in particular, cancer-specific plectin (CSP) presents an attractive opportunity to impede carcinogenesis directly. Here, we report on plectin's differential gene and protein expression in cancer, explore its mutational profile, and discuss the current understanding of plectin's and CSP's biological function in cancer. Moreover, we review the landscape of plectin as a prognostic marker, diagnostic biomarker, and target for imaging and therapeutic modalities. We highlight how, beyond their respective biological importance, plectin's common overexpression in cancer and CSP's cancer-specific bioavailability underscore their potential as high-value druggable targets. We discuss how recent evidence of the potent anti-cancer effects of CSP therapeutic targeting opens the door for cell-surface mislocalized proteins as novel therapeutic targets.

X-aptamers targeting Thy-1 membrane glycoprotein in pancreatic ductal adenocarcinoma.

Modified DNA aptamers incorporated with amino-acid like side chains or drug-like ligands can offer unique advantages and enhance specificity as affinity ligands. Thy-1 membrane glycoprotein (THY1 or CD90) was previously identified as a biomarker candidate of neovasculature in pancreatic ductal adenocarcinoma (PDAC). The current study developed and evaluated modified DNA X-aptamers targeting THY1 in PDAC. The expression and glycosylation of THY1 in PDAC tumor tissues were assessed using immunohistochemistry and quantitative proteomics. Bead-based X-aptamer library that contains 108 different sequences was used to screen for high affinity THY1 X-aptamers. The sequences of the X-aptamers were analyzed with the next-generation sequencing. The affinities of the selected X-aptamers to THY1 were quantitatively evaluated with flow cytometry. Three high affinity THY1 X-aptamers, including XA-B217, XA-B216 and XA-A9, were selected after library screening and affinity binding evaluation. These three X-aptamers demonstrated a high binding affinity and specificity to THY1 protein and the THY1 expressing cell lines, using THY1 antibody as a comparison. The development of these X-aptamers provides highly specific and non-immunogenic affinity ligands for THY1 binding in the context of biomarker development and clinical applications. They could be further exploited to assist molecular imaging of PDAC targeting THY1.

In vivo liposomal delivery of PPARα/γ dual agonist tesaglitazar in a model of obesity enriches macrophage targeting and limits liver and kidney drug effects.

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism. Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.

Precision Medicine in Pancreatic Disease-Knowledge Gaps and Research Opportunities: Summary of a National Institute of Diabetes and Digestive and Kidney Diseases Workshop.

A workshop on research gaps and opportunities for Precision Medicine in Pancreatic Disease was sponsored by the National Institute of Diabetes and Digestive Kidney Diseases on July 24, 2019, in Pittsburgh. The workshop included an overview lecture on precision medicine in cancer and 4 sessions: (1) general considerations for the application of bioinformatics and artificial intelligence; (2) omics, the combination of risk factors and biomarkers; (3) precision imaging; and (4) gaps, barriers, and needs to move from precision to personalized medicine for pancreatic disease. Current precision medicine approaches and tools were reviewed, and participants identified knowledge gaps and research needs that hinder bringing precision medicine to pancreatic diseases. Most critical were (a) multicenter efforts to collect large-scale patient data sets from multiple data streams in the context of environmental and social factors; (b) new information systems that can collect, annotate, and quantify data to inform disease mechanisms; (c) novel prospective clinical trial designs to test and improve therapies; and (d) a framework for measuring and assessing the value of proposed approaches to the health care system. With these advances, precision medicine can identify patients early in the course of their pancreatic disease and prevent progression to chronic or fatal illness.

Importance of thorough tissue and cellular level characterization of targeted drugs in the evaluation of pharmacodynamic effects.

Targeted nanoparticle delivery is a promising strategy for increasing efficacy and limiting side effects of therapeutics. When designing a targeted liposomal formulation, the in vivo biodistribution of the particles must be characterized to determine the value of the targeting approach. Peroxisome proliferator-activated receptor (PPAR) agonists effectively treat metabolic syndrome by decreasing dyslipidemia and insulin resistance but side effects have limited their use, making them a class of compounds that could benefit from targeted liposomal delivery. The adipose targeting sequence peptide (ATS) could fit this role, as it has been shown to bind to adipose tissue endothelium and induce weight loss when delivered conjugated to a pro-apoptotic peptide. To date, however, a full assessment of ATS in vivo biodistribution has not been reported, leaving important unanswered questions regarding the exact mechanisms whereby ATS targeting enhances therapeutic efficacy. We designed this study to evaluate the biodistribution of ATS-conjugated liposomes loaded with the PPARα/γ dual agonist tesaglitazar in leptin-deficient ob/ob mice. The ATS-liposome biodistribution in adipose tissue and other organs was examined at the cellular and tissue level using microscopy, flow cytometry, and fluorescent molecular tomography. Changes in metabolic parameters and gene expression were measured by target and off-target tissue responses to the treatment. Unexpectedly, ATS targeting did not increase liposomal uptake in adipose relative to other tissues, but did increase uptake in the kidneys. Targeting also did not significantly alter metabolic parameters. Analysis of the liposome cellular distribution in the stromal vascular fraction with flow cytometry revealed high uptake by multiple cell types. Our findings highlight the need for thorough study of in vivo biodistribution when evaluating a targeted therapy.

Plectin-targeted liposomes enhance the therapeutic efficacy of a PARP inhibitor in the treatment of ovarian cancer.

Advances in genomics and proteomics drive precision medicine by providing actionable genetic alterations and molecularly targeted therapies, respectively. While genomic analysis and medicinal chemistry have advanced patient stratification with treatments tailored to the genetic profile of a patient's tumor, proteomic targeting has the potential to enhance the therapeutic index of drugs like poly(ADP-ribose) polymerase (PARP) inhibitors. PARP inhibitors in breast and ovarian cancer patients with BRCA1/2 mutations have shown promise. About 10% of the patients who received Olaparib (PARP inhibitor) showed adverse side effects including neutropenia, thrombocytopenia and in some cases resulted in myelodysplastic syndrome, indicating that off-target effects were substantial in these patients. Through proteomic analysis, our lab previously identified plectin, a cytolinker protein that mislocalized onto the cell surface during malignant transformation of healthy ovarian tissue. This cancer specific phenotype allowed us to image pancreatic cancer successfully using plectin targeted peptide (PTP) conjugated to nanoparticles or displayed on capsid protein of adeno-associated virus (AAV) particles. Objective: The goal of this study was to integrate the available pharmacogenomics and proteomic data to develop effective anti-tumor therapies using a targeted drug delivery approach. Methods: Plectin expression and localization in human ovarian tumor specimens were analyzed followed by in vitro confirmation of cell surface plectin localization in healthy and ovarian cancer cell lines. PTP-conjugated liposomes were prepared and their specificity for plectin+ cells was determined in vitro and in vivo. A remote loading method was employed to encapsulate a PARP inhibitor (AZ7379) into liposomes. An ideal buffer exchange method and remote loading conditions were determined based on the amount of lipid and drug recovered at the end of a remote loading process. Finally, in vivo tumor growth studies were performed to determine the efficacy of PTP liposomes in preventing PARP activity in mice bearing OVCAR8 (high grade epithelial ovarian cancer (EOC)) tumors. Results: PTP liposomal AZ7379 delivery not only enhanced PARP inhibition but also resulted in decelerated tumor growth in mice bearing subcutaneous and intraperitoneal OVCAR8 tumors. In mice bearing subcutaneous or intraperitoneal tumors, treatment with PTP liposomes resulted in a 3- and 1.7-fold decrease in tumor volume, respectively, compared to systemic drug treatment. Conclusion: Targeted drug delivery assisted by genomic and proteomic data provides an adaptable model system that can be extended to effectively treat other cancers and diseases.

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.

Evaluation of pharmacokinetic and pharmacodynamic profiles of liposomes for the cell type-specific delivery of small molecule drugs.

Liposome-based drug formulations represent an exciting avenue of research as they increase efficacy to toxicity ratios. Current formulations rely on passive accumulation to the disease site where drug is taken up by the cells. Ligand mediated targeting increases the net accumulation of liposomes, however, an unexplored benefit is to potentially refine pharmacodynamics (PD) of a drug specifically to different cell types within diseased tissue. As a model system, we engineered cardiomyocyte- (I-1) and endothelial-targeted (B-40) liposomes to carry a VEGFR2 inhibitor (PTK787), and examined the effect of cell type-specific delivery on both pharmacokinetics (PK) and PD. Neovascularization in post-myocardial infarction was significantly reduced by B-40 liposomes loaded with PTK787 as compared to animals injected with I-1 liposomes, and profoundly more as compared to free PTK787. This study thus shows that the intraorgan targeting of drugs through cell type-specific delivery holds substantial promise towards lowering the minimal efficacious dose administered systemically.

Corticosterone primes the neuroinflammatory response to Gulf War Illness-relevant organophosphates independently of acetylcholinesterase inhibition.

Gulf War Illness (GWI) is a chronic multi-symptom disorder affecting veterans of the 1991 Gulf War. Among the symptoms of GWI are those associated with sickness behavior, observations suggestive of underlying neuroinflammation. We have shown that exposure of mice to the stress hormone, corticosterone (CORT), and to diisopropyl fluorophosphate (DFP), as a nerve agent mimic, results in marked neuroinflammation, findings consistent with a stress/neuroimmune basis of GWI. Here, we examined the contribution of irreversible and reversible acetylcholinesterase (AChE) inhibitors to neuroinflammation in our mouse model of GWI. Male C57BL/6J mice received 4 days of CORT (400 mg/L) in the drinking water followed by a single dose of chlorpyrifos oxon (CPO; 8 mg/kg, i.p.), DFP (4 mg/kg, i.p.), pyridostigmine bromide (PB; 3 mg/kg, i.p.), or physostigmine (PHY; 0.5 mg/kg, i.p.). CPO and DFP alone caused cortical and hippocampal neuroinflammation assessed by qPCR of tumor necrosis factor-alpha, IL-6, C-C chemokine ligand 2, IL-1ß, leukemia inhibitory factor and oncostatin M; CORT pretreatment markedly augmented these effects. Additionally, CORT exposure prior to DFP or CPO enhanced activation of the neuroinflammation signal transducer, signal transducer and activator of transcription 3 (STAT3). In contrast, PHY or PB alone or with CORT pretreatment did not produce neuroinflammation or STAT3 activation. While all of the CNS-acting AChE inhibitors (DFP, CPO, and PHY) decreased brain AChE activity, CORT pretreatment abrogated these effects for the irreversible inhibitors. Taken together, these findings suggest that irreversible AChE inhibitor-induced neuroinflammation and particularly its exacerbation by CORT, result from non-cholinergic effects of these compounds, pointing potentially to organophosphorylation of other neuroimmune targets.

High-Throughput Approaches to the Development of Molecular Imaging Agents.

Molecular imaging allows for the visualization of changes at the cellular level in diseases such as cancer. A successful molecular imaging agent must rely on disease-selective targets and ligands that specifically interact with those targets. Unfortunately, the translation of novel target-specific ligands into the clinic has been frustratingly slow with limitations including the complex design and screening approaches for ligand identification, as well as their subsequent optimization into useful imaging agents. This review focuses on combinatorial library approaches towards addressing these two challenges, with particular focus on phage display and one-bead one-compound (OBOC) libraries. Both of these peptide-based techniques have proven successful in identifying new ligands for cancer-specific targets and some of the success stories will be highlighted. New developments in screening methodology and sequencing technology have pushed the bounds of phage display and OBOC even further, allowing for even faster and more robust discovery of novel ligands. The combination of multiple high-throughput technologies will not only allow for more accurate identification, but also faster affinity maturation, while overall streamlining the process of translating novel ligands into clinical imaging agents.

Imaging in pancreatic disease.

Pancreatic diseases, chronic pancreatitis, pancreatic cancer and diabetes mellitus, taken together, occur in >10% of the world population. Pancreatic diseases, as with other diseases, benefit from early intervention and appropriate diagnosis. Although imaging technologies have given clinicians an unprecedented toolbox to aid in clinical decision-making, advances in these technologies and development of molecular-based diagnostic tools could enable physicians to identify diseases at an even earlier stage and, thereby, improve patient outcomes. In this Review, we discuss and identify gaps in the use of imaging techniques for the early detection and appropriate treatment stratification of various pancreatic diseases, including diabetes mellitus, acute and chronic pancreatitis and pancreatic cancer. Imaging techniques discussed are MRI, CT, PET and ultrasonography. Additionally, the identification of new molecular targets for imaging and the development of contrast agents that are able to give molecular information in noninvasive radionuclear imaging and ultrasonography are emerging areas of innovation that could lead to increased diagnostic accuracy and improved patient outcomes.

Hybridization-Induced Aggregation Technology for Practical Clinical Testing: KRAS Mutation Detection in Lung and Colorectal Tumors.

KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA-a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways.

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing.

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective-targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use.

Plectin-1 as a Biomarker of Malignant Progression in Intraductal Papillary Mucinous Neoplasms: A Multicenter Study.

OBJECTIVE: This study aimed to evaluate Plectin-1 expression as a biomarker of malignant risk for intraductal papillary mucinous neoplasms (IPMNs). METHODS: Plectin-1 immunohistochemistry (IHC) was performed retrospectively on surgical (n = 71) and cytological (n = 33) specimens from Mayo Clinic Jacksonville and UCLA Medical Center, including IPMNs with low-grade dysplasia, high-grade dysplasia (HGD), or an associated invasive adenocarcinoma. RESULTS: Plectin-1 expression was increased in invasive adenocarcinoma compared with adjacent in situ IPMN (P = 0.005), as well as the in situ HGD component of IPMNs with invasive cancer compared with HGD of IPMNs without invasive cancer (P = 0.02). Plectin IHC discriminated IPMNs with invasive adenocarcinoma from noninvasive IPMN (area under the curve [AUC] of 0.79, 75% sensitivity, and 85% specificity) but was insufficient for discriminating HGD IPMN from low-grade dysplasia IPMNs in surgical resections (AUC of 0.67, 56% sensitivity, and 64% specificity) or fine-needle aspiration specimens (AUC of 0.45). CONCLUSIONS: Although Plectin-1 IHC has insufficient accuracy to be used as a definitive biomarker for malignant risk in the evaluation of IPMN biopsy or cytological specimens, increased Plectin-1 expression observed in both invasive cancer and in situ HGD of malignant IPMNs suggests that it might be successfully leveraged as a cyst fluid biomarker or molecular imaging target.

Advances in Biomedical Imaging, Bioengineering, and Related Technologies for the Development of Biomarkers of Pancreatic Disease: Summary of a National Institute of Diabetes and Digestive and Kidney Diseases and National Institute of Biomedical Imaging and Bioengineering Workshop.

A workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Biomedical Imaging and Bioengineering focused on research gaps and opportunities in the development of new biomarkers of pancreatic disease. The session was held on July 22, 2015, and structured into 6 sessions: 1) Introduction and Overview; 2) Keynote Address; 3) New Approaches to the Diagnosis of Chronic Pancreatitis; 4) Biomarkers of Pain and Inflammation; 5) New Approaches to the Detection of Pancreatic Cancer; and 6) Shed Exosomes, Shed Cells, and Shed Proteins. Recent advances in the fields of pancreatic imaging, functional markers of pancreatic disease, proteomics, molecular and cellular imaging, and detection of circulating cancer cells and exosomes were reviewed. Knowledge gaps and research needs were highlighted. The development of new methods for the noninvasive determination of pancreatic pathology; the use of cellular markers of pancreatic function, inflammation, pain, and malignancy; and the refinement of methods to identify cells and cellular constituents of pancreatic cancer were discussed. The further refinement of sophisticated technical methods and the need for clinical studies to validate these new approaches in large-scale studies of patients at risk for the development of pancreatic disease were repeatedly emphasized.

Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads.

Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating KRAS mutations. Following the development and optimization of the assay, we apply it for KRAS mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common KRAS mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis.

Development of target-specific liposomes for delivering small molecule drugs after reperfused myocardial infarction.

Although reperfusion is essential in restoring circulation to ischemic myocardium, it also leads to irreversible events including reperfusion injury, decreased cardiac function and ultimately scar formation. Various cell types are involved in the multi-phase repair process including inflammatory cells, vascular cells and cardiac fibroblasts. Therapies targeting these cell types in the infarct border zone can improve cardiac function but are limited by systemic side effects. The aim of this work was to develop liposomes with surface modifications to include peptides with affinity for cell types present in the post-infarct myocardium. To identify peptides specific for the infarct/border zone, we used in vivo phage display methods and an optical imaging approach: fluorescence molecular tomography (FMT). We identified peptides specific for cardiomyocytes, endothelial cells, myofibroblasts, and c-Kit + cells present in the border zone of the remodeling infarct. These peptides were then conjugated to liposomes and in vivo specificity and pharmacokinetics were determined. As a proof of concept, cardiomyocyte specific (I-1) liposomes were used to deliver a PARP-1 (poly [ADP-ribose] polymerase 1) inhibitor: AZ7379. Using a targeted liposomal approach, we were able to increase AZ7379 availability in the infarct/border zone at 24h post-injection as compared with free AZ7379. We observed ~3-fold higher efficiency of PARP-1 inhibition when all cell types were assessed using I-1 liposomes as compared with negative control peptide liposomes (NCP). When analyzed further, I-1 liposomes had 9-fold and 1.5-fold higher efficiencies in cardiomyocytes and macrophages, respectively, as compared with NCP liposomes. In conclusion, we have developed a modular drug delivery system that can be targeted to cell types of therapeutic interest in the infarct border zone.