A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 μL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.

Arachidonate 5-lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP-9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP-9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)-hydroxyeicosatetraenoic acid also increased MMP-9 protein expression by BCPAP in a dose-dependent manner. Inhibitors of MMP-9 and ALOX5 reversed ALOX5-enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP-9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC.

These data provide new functional evidence of the uPA/uPAR system's role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets.

Pyrrole-imidazole (Py–Im) polyamides are synthetic non-genotoxic minor groove-binding small molecules. We hypothesized that Py–Im polyamides can modulate the cellular response to ionizing radiation. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. RNA-sequencing of cell lines exposed to the polyamide showed induction of genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes directly with DNA ligation in vitro. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies.

Of the 44,670 new cases of thyroid cancer predicted for this year in the US by the American Cancer Society, 80% will be papillary thyroid carcinoma (PTC). PTC typically arises from a gain-of-function mutation in the RET, RAS, or BRAF genes which make up a linear signaling cascade for ERK activation in over 90% of PTC cases. PTCs possessing the BRAFV600E mutation have been associated with greater local invasion and regional metastatic potential. Urokinase plasminogen activator (uPA) has been shown to be an important mediator of invasion and metastasis in several cancers. To determine the extent to which BRAFV600E mutation status predicts up-regulation of the uPA in PTC, a study using patient thyroid tissue samples obtained over a 6-year period at a single tertiary care center was undertaken. DNA and RNA were obtained from patient PTC and matched, normal thyroid tissue samples using the Trizol method. BRAFV600E mutational status of the DNA was determined using the TaqMan SNP genotyping assay, while RNA was analyzed for differences in uPA transcription levels (relative to matched, normal thyroid tissue) by qRT-PCR. Fifty-four percent of the PTC samples possessed the BRAFV600E mutation. PTC samples bearing the BRAFV600E mutation displayed significantly higher uPA RNA levels (relative to matched control tissue) than those samples with wild-type BRAF (5.883 vs. 1.27-fold, p<0.05, Wilcoxon signed-rank test). Additionally, uPA RNA levels were significantly higher in patients with nodal metastasis (p<0.05). These data provide new evidence of the roles of BRAFV600E and uPA in PTC invasive pathology and demonstrate for the first time that BRAFV600E status is able to predict higher uPA levels in PTC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2011-1063