The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity.

1National Research Council Halifax, 1411 Oxford Street, Halifax, NS B3H 3Z1, Canada 2Department of Pathology, Queen Elizabeth II Health Science Center and Dalhousie University, 5788 University Ave., Halifax, NS B3H 1V8, Canada *Corresponding author Rama K. Singh, PhD Human Health Therapeutics Portfolio National Research Council Canada 1411 Oxford Street Halifax, Nova Scotia B3H 3Z1, Canada Tel. (902) 426-3802 Fax: (902) 426-9413 E-mail: rama.singh@nrc-cnrc.gc.ca

Lung cancer is the leading cause of cancer-related death worldwide, and the majority of cases (77%) are not diagnosed until the disease has spread to regional lymph nodes or distant sites.Even among Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma patients who have been diagnosed early and where there has been no spread to lymph nodes, recurrence after surgical intervention is high.Improved understanding of the molecular alterations involved in aggressivity and recurrence of these tumors may provide better biomarkers for the identification of patients who would benefit from adjuvant chemotherapy.By comparing the expression of microRNAs in advanced Stage II/III tumors with those expressed in earlier Stage I tumors, we aimed to identify differentially expressed molecular biomarkers that could underly progression and recurrence of Stage I tumors.This pilot study utilized TaqMan qPCR assays to assess the expression of microRNAs in tumor tissue, matched normal tissue and plasma samples from Stage I and Stage II/III lung adenocarcinoma patients.Seven microRNAs were identified from plasma that could distinguish between patients with Stage I and Stage II/III adenocarcinoma.The up-regulation of miR-29a in plasma of patients with later-stage adenocarcinoma would result in enhanced expression of several molecules involved in integrin signaling, migration and proliferation.Analysis of differential expression of microRNAs in later-stage compared to early-stage lung adenocarcinoma implicates focal adhesion and ECM-receptor pathways in progression and recurrence.Plasma miR-29a is a promising biomarker that can be assessed non-invasively and whose clinical utility should be pursued.

Personalized treatment of lung cancer using therapies that target activating oncogenic mutations such as EGFR and ALK has become the standard of care.Current molecular testing is routinely performed for single genes and increasingly in a multiplex format.However, the scarcity of sufficient biopsy material has necessitated a more high-throughput and comprehensive testing approach.Next Generation Sequencing (NGS) offers great promise as a highly sensitive method of detection for a variety of biopsy sources (tissue, blood, pleural effusions).However, there are multiple NGS platforms and panels with varying advantages and disadvantages.This pilot study compared four different library construction methods (Ion AmpliSeq, Illumina TruSeq, and Raindance Thunderbolts amplicon-based methods and Roche EZSeq sequence capture method) and two different sequencing instruments (Ion Torrent PGM and Illumina MiSeq).A common set of ten tumor/normal pairs from lung adenocarcinoma patients were analysed by all platforms.Additional samples were analysed in subsets of the platforms.To assess the feasibility of sequencing circulating free DNA (cfDNA) from plasma and pleural effusions, two additional samples were analysed on two amplicon-based platforms.A bioinformatic pipeline for automated sequence data analysis was developed using the Galaxy environment.To determine the most cost-effective, technically streamlined library construction and sequencing method, we compared coverage statistics, sensitivity, variant detection, and workflow for all platforms.

Objectives Papillary thyroid cancer (PTC) is increasing in incidence. Fine needle aspiration is the gold standard for diagnosis, but results can be indeterminate. Identifying tissue and serum biomarkers, like microRNA, is therefore desirable. We sought to identify miRNA that is differentially expressed in the serum of patients with PTC. Methods Serum miRNA was quantified in 31 female thyroidectomy patients: 13 with benign disease and 18 with PTC. qPCR results were compared for significant fold-changes in 175 miRNAs, against a pooled control. Results 128 miRNA qualified for analysis. There were identifiable fold-changes in miRNA levels between benign and control, and between PTC and control. There were statistically significant fold changes in the level of four miRNAs between benign and PTC: hsa-miR-146a-5p and hsa-miR-199b-3p were down-regulated, while hsa-let7b-5p and hsa-miR-10a-5p were up-regulated. Conclusions MicroRNA is differentially expressed in the serum of patients with PTC. Serum miRNA has the potential to aid in thyroid cancer diagnosis.

Cationic antimicrobial (host defense) peptides (CAPs) are able to kill microorganisms and cancer cells, leading to their consideration as novel candidate therapeutic agents in human medicine. CAPs can physically associate with anionic membrane structures, such as those found on cancer cells, causing pore formation, intracellular disturbances, and leakage of cell contents. In contrast, normal cells are less negatively-charged and are typically not susceptible to CAP-mediated cell death. Because the interaction of CAPs with cells is based on charge properties rather than cell proliferation, both rapidly dividing and quiescent cancer cells, as well as multidrug-resistant cancer cells, are targeted by CAPs, making CAPS potentially valuable as anti-cancer agents. CAPs often exist as families of peptides with slightly different amino acid sequences. In addition, libraries of synthetic peptide variants based on naturally occurring CAP templates can be generated in order to improve upon their action. High-throughput screens are needed to quickly and efficiently assess the suitability of each CAP variant. Here we present the methods for assessing CAP-mediated cytotoxicity against cancer cells (suspension and adherent) and untransformed cells (measured using the tritiated thymidine-release or MTT assay), and for discriminating between cell death caused by necrosis (measured using lactate dehydrogenase- or 51Cr-release assays), or apoptosis and necrosis (single-stranded DNA content measured by flow cytometry). In addition the clonogenic assay, which assesses the ability of single transformed cells to multiply and produce colonies, is described.

Recurrence after lung cancer surgery is high, even among Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma patients diagnosed early as Stage I, where there has been no spread to lymph nodes.Understanding the biological underpinnings of aggressivity and recurrence in this subset of tumours may enable the identification of patients who would benefit from adjuvant therapy.The purpose of this study was to identify differentially expressed molecular biomarkers that might underlie recurrence of Stage I tumours by comparing gene expression in later-stage tumours with those expressed in early-stage tumours.Gene expression in tissue biopsy samples from five Stage I and five Stage II/III NSCLC adenocarcinoma patients was analysed using an oligonucleotide microarray containing 17,000 probes printed in duplicate.Analyses were performed on total RNA isolated from tumour tissue of each patient using universal human RNA as a reference.Compared to normal tissues, the transcriptome of Stage I NSCLC adenocarcinomas showed enrichment in general pathways in cancer, whereas in Stage II/III more specific cancer pathways such as focal adhesion and ECM-receptor interaction pathways were enriched and components of the PPAR signalling pathway were depleted.Relative to early-stage NSCLC, Stage II/III adenocarcinomas showed up-regulation of genes of the basic transcriptional and translational machinery, particularly the "cancer testis antigen" PASD1 transcription factor.The actin cytoskeleton re-organisation and interleukin-6 pathways were also up-regulated whereas there was a generalized down-regulation of immune effectors and genes involved in immune system development.This small-scale transcriptome study provides important information about the pathways and molecules likely to be involved in the more metastatic propensity of those Stage I NSCLC adenocarcinomas that recur.

Pleurocidins are a novel family of α-helical cationic antimicrobial peptides (CAPs) that are structurally and functionally similar to cathelicidins, one of the major CAP families. As cathelicidins stimulate mast cell chemotaxis and mediator release, we postulated that pleurocidins similarly activate mast cells. A screen of 20 pleurocidin peptides revealed that some were capable of degranulating the human mast cell line LAD2 (Laboratory of Allergic Diseases 2). Pleurocidin NRC-04 caused LAD2 to adhere, migrate, degranulate, and release cysteinyl leukotrienes and prostaglandin D2. Moreover, pleurocidin increased intracellular Ca2+ mobilization in mast cells and induced the production of proinflammatory chemokines such as monocyte chemotactic protein-1/C-C motif chemokine ligand 2 (CCL2) and macrophage inflammatory protein-1β/CCL4. Our evaluation of possible cellular mechanisms suggested that G proteins, phosphoinositol-3 kinase (PI3K), phospholipase C (PLC), and phosphokinase C (PKC) were involved in pleurocidin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G protein inhibitor), wortmanin (PI3K inhibitor), U-73122 (PLC inhibitor), and Ro-31-8220 (PKC inhibitor), respectively. We also found that human mast cells expressed the N-formyl-peptide receptor 1 (FPRL1) receptor and FPRL1-specific inhibitor affected pleurocidin-mediated activation of mast cell. Our finding that the novel CAP pleurocidin activated human mast cell through G protein–coupled receptor signaling suggests that this peptide might have immunomodulatory functions.

OBJECTIVE:To identify serum biomarkers of papillary thyroid cancer.METHODS:Prospective analysis was performed of banked tumor and serum specimens from 99 patients with thyroid masses. Enzyme-linked immunosorbent assay (ELISA) was employed to measure levels of five serum proteins previously demonstrated to be up-regulated in papillary thyroid cancer (PTC): angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase 3 like-1 (YKL-40), and galectin-3 (GAL-3). Serum levels were compared between patients with PTC and those with benign tumors.RESULTS:A total of 99 patients were enrolled in the study (27 men, 72 women), with a median age of 54 years. Forty-three patients had PTC and 58 cases were benign tumors. There were no statistically significant differences when comparing all five different biomarkers between PTC and other benign thyroid tumors. The p-values were 0.94, 0.48, 0.72, 0.48, and 0.90 for YKL-40, Gal-3, CK19, TIMP-1, and Ang-1, respectively.CONCLUSION:Serum levels of four of the five proteins were elevated in patients with thyroid masses relative to normal values. However, the difference between benign and PTC was not significant. Two of the markers (Gal-3 & TIMP-1) displayed a greater potential difference, which may warrant further investigation. This study suggests that other serum markers should be sought. This is the first study to investigate potential serum biomarkers based on over-expressed proteins in thyroid cancer versus benign pathology.

Infectious diseases and cancer are leading causes of mortality and our ability to combat them is compromised due to the emergence of antibiotic-resistant strains of bacteria and chemotherapy-resistant cancer cells. Combined with the scarcity of new classes of antibiotics due to the abandonment of antibacterial research by pharmaceutical companies (Williams and Bax, 2009) and the lengthy development time lines to market (Projan and Bradford, 2007), there is an urgent need for alternative therapeutics. Cationic antimicrobial peptides (CAPs) have emerged as a promising source of novel therapeutics. Not only do they rapidly kill microbes and cancer cells, they also can modulate host innate immunity to augment clearance of microbes and promote tissue healing resulting from inflammation. Further‐ more, they are less prone to resistance than traditional antibiotics (McPhee and Hancock, 2005). Synthetic variants of naturally occurring CAPs, have been designed that exhibit great‐ er selectivity and stability. Here I summarize some of the key features of CAPs, directing the reader to recent pertinent reviews, and focus on characteristics of pleurocidin CAPs that make them attractive for clinical applications.

Growth of Streptomyces venezuelae ISP5230 with O-propargyl-L-serine led to the efficient production of an alkyne-containing jadomycin. The installed alkyne functionality provided a uniquely reactive handle within the natural product and was subsequently reacted with a series of azides to afford an eight-member library of jadomycin triazoles. The compounds were evaluated for their DNA cleavage, antibacterial and cytotoxic properties.

INTRODUCTION:Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells.METHODS:MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) and acid phosphatase cell-viability assays were used to assess NRC-03- and NRC-07-mediated killing of breast carcinoma cells. Erythrocyte lysis was determined with hemolysis assay. NRC-03 and NRC-07 binding to breast cancer cells and normal fibroblasts was assessed with fluorescence microscopy by using biotinylated-NRC-03 and -NRC-07. Lactate dehydrogenase-release assays and scanning electron microscopy were used to evaluate the effect of NRC-03 and NRC-07 on the cell membrane. Flow-cytometric analysis of 3,3'-dihexyloxacarbocyanine iodide- and dihydroethidium-stained breast cancer cells was used to evaluate the effects of NRC-03 and NRC-07 on mitochondrial membrane integrity and reactive oxygen species (ROS) production, respectively. Tumoricidal activity of NRC-03 and NRC-07 was evaluated in NOD SCID mice bearing breast cancer xenografts.RESULTS:NRC-03 and NRC-07 killed breast cancer cells, including drug-resistant variants, and human mammary epithelial cells but showed little or no lysis of human dermal fibroblasts, umbilical vein endothelial cells, or erythrocytes. Sublethal doses of NRC-03 and, to a lesser extent, NRC-07 significantly reduced the median effective concentration (EC50) of cisplatin for breast cancer cells. NRC-03 and NRC-07 bound to breast cancer cells but not fibroblasts, suggesting that killing required peptide binding to target cells. NRC-03- and NRC-07-mediated killing of breast cancer cells correlated with expression of several different anionic cell-surface molecules, suggesting that NRC-03 and NRC-07 bind to a variety of negatively-charged cell-surface molecules. NRC-03 and NRC-07 also caused significant and irreversible cell-membrane damage in breast cancer cells but not in fibroblasts. NRC-03- and NRC-07-mediated cell death involved, but did not require, mitochondrial membrane damage and ROS production. Importantly, intratumoral administration of NRC-03 and NRC-07 killed breast cancer cells grown as xenografts in NOD SCID mice.CONCLUSIONS:These findings warrant the development of stable and targeted forms of NRC-03 and/or NRC-07 that might be used alone or in combination with conventional chemotherapeutic drugs for the treatment of breast cancer.

e21156 Background: The most common methods for CTC isolation from peripheral blood depend on positive selection based on tumor cells' surface EpCAM expression. However, EpCAM expression may vary between CTCs and may be downregulated in cells undergoing epithelial-to-mesenchymal transition, potentially resulting in the failure to capture CTC subsets having a critical role as intermediates in cancer progression. METHODS We previously reported on a feasibility study using an unbiased approach with three different enrichment methods, involving both positive and negative selection, and obtained consistently high recovery of small numbers of MDA-MB-231 cancer cells spiked into donor human peripheral blood using the RosetteSep Human Circulating Epithelial Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC Canada). Cytospins prepared from recovered cells were used for identification and enumeration by immunocytochemistry using fluorescently laballed anti-CD45 and anti-CK19 antibodies. We optimized RNA amplification from the equivalent of one MDA-MB-231 cell (~10 pg) and used the resulting amplified RNA in microarray experiments. Peripheral blood from MBC patients was enriched for CTC, RNA was isolated and amplified, and microarray and qPCR analysis was used to investigate global and gene-specific transcription changes, respectively. RESULTS Numbers of CTC enriched from eight MBC patients ranged from 1 to 125 per 5 mL blood. EpCAM, CK19, vimentin, and CD45 expression by CTC was determined by qPCR. Preliminary results from microarray analysis of enriched CTC from four of six initial patients showed ~ 130 common genes upregulated more than twofold, including genes involved in tumorigenesis. Two of six patients had insufficiently amplified RNA. Gene enrichment analysis showed an abundance of genes involved in translation. CONCLUSIONS Although preliminary, these methods may lead to a more thorough characterization of micrometastases in advanced breast cancer and potentially early stage disease. Further microarray analysis and optimization methods are ongoing in MBC patients.

SUMMARY The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 μg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 μg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 μM; ∼64 μg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 μM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 μM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 μM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.

ABSTRACT In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans , a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 μM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii . The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.

Streptomyces venezuelae ISP5230 is recognized for the production of chloramphenicol and the jadomycin family of natural products. The jadomycins are angucycline natural products containing a unique oxazolone ring incorporating an amino acid present in the minimal culture media. Substitution of different amino acids results in products of varying biological activity. Analysis of cultures of S. venezuelae ISP5230 incubated with l- and d-norvaline and l- and d-norleucine indicated that only the d-configured amino acids were incorporated into the natural products. Subsequently, jadomycin DNV and jadomycin DNL were isolated and characterized (titers 4 and 9 mg L(-1), respectively). The compounds were evaluated in the National Cancer Institute cell line cancer growth inhibition and cytotoxicity screens, for antimicrobial activity against selected Gram-positive and Gram-negative bacteria, and as DNA-cleavage agents in vitro.

This tutorial presents a basic introduction to DNA microarrays as employed for gene expression analysis, approaching the subject from a chemometrics perspective. The emphasis is on describing the nature of the measurement process, from the platforms used to a few of the standard higher-level data analysis tools employed. Topics include experimental design, detection, image processing, measurement errors, ratio calculation, background correction, normalization, and higher-level data processing. The objective is to present the chemometrician with as clear a picture as possible of an evolving technology so that the strengths and limitations of DNA microarrays are appreciated. Although the focus is primarily on spotted, two-color microarrays, a significant discussion of single-channel, lithographic arrays is also included.