Comparative performance of different methods for circulating tumor cell enrichment in metastatic breast cancer patients.

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.

Transcriptional Feedback Links Lipid Synthesis to Synaptic Vesicle Pools in Drosophila Photoreceptors.

Neurons can maintain stable synaptic connections across adult life. However, the signals that regulate expression of synaptic proteins in the mature brain are incompletely understood. Here, we describe a transcriptional feedback loop between the biosynthesis and repertoire of specific phospholipids and the synaptic vesicle pool in adult Drosophila photoreceptors. Mutations that disrupt biosynthesis of a subset of phospholipids cause degeneration of the axon terminal and loss of synaptic vesicles. Although degeneration of the axon terminal is dependent on neural activity, activation of sterol regulatory element binding protein (SREBP) is both necessary and sufficient to cause synaptic vesicle loss. Our studies demonstrate that SREBP regulates synaptic vesicle levels by interacting with tetraspanins, critical organizers of membranous organelles. SREBP is an evolutionarily conserved regulator of lipid biosynthesis in non-neuronal cells; our studies reveal a surprising role for this feedback loop in maintaining synaptic vesicle pools in the adult brain.

A mass spectrometry-based plasma protein panel targeting the tumor microenvironment in patients with breast cancer.

Proteins secreted or shed by cancerous cells are seen as a rich source of biomarkers and novel therapeutic targets. Recently, the importance of the tumor microenvironment, which comprises the surrounding non-tumor cells, has received increased attention for its role in tumor progression. We developed a targeted proteomics assay to monitor a panel of plasma proteins postulated to be present in the tumor microenvironment. The plasma of 76 breast cancer patients was depleted of abundant circulating proteins, enzymatically digested and labeled by reductive methylation. The labeled digests were analyzed by tandem mass spectrometry using a multiple reaction monitoring acquisition method. The protein targets were correlated with the tumor characteristics, the extent of the disease and the clinical staging of the patients. Linear discriminant analysis revealed that infiltrating ductal and invasive mammary breast carcinomas could be grouped based on distinctive peptide levels of fibronectin, clusterin, gelsolin and α-1-microglobulin/Inter-α-trypsin inhibitor light chain precursor (AMBP). These proteins have been previously associated with breast cancer at the tissue level, however, this is the first study to measure plasma levels of these proteins and correlate these levels with clinical features. Significant variability was seen between unique peptides belonging to the same protein. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Iodination on tyrosine residues during oxidation with sodium periodate in solid phase extraction of N-linked glycopeptides.

Solid-phase extraction of N-linked glycopeptides (SPEG) using hydrazide-modified supports has become a common sample preparation procedure in glycoproteomic experiments. We demonstrate that iodination of tyrosine residues occur in SPEG as a side reaction during an oxidation step with sodium periodate. MS/MS analysis of oxidized bovine serum albumin and carbonic anhydrase digests revealed a characteristic shift of m/z 125.9 on all y and b fragment ions containing the modified tyrosine residues. Selected reaction monitoring (SRM) measurements showed that the peak intensity from of the iodinated peptides increased during the course of oxidation. After an hour of oxidation, SRM analysis revealed that the strongest signal from an iodinated peptide was approximately one-tenth of the intensity of the corresponding unmodified peptide. Iodinated tyrosine residues were also identified in serum samples subjected to SPEG and analyzed by LC-ESI-MS/MS. We recommend assessing this side reaction by including iodotyrosine as a variable modification when performing database searches on SPEG experiments. For SRM-based acquisitions, we encourage the avoidance of tyrosine-containing glycopeptides or, if this is not practical, monitoring transitions that contain the potential modified iodinated tyrosine residue to monitor the presence of the iodinated form of the glycopeptide.

Overexpressing temperature-sensitive dynamin decelerates phototransduction and bundles microtubules in Drosophila photoreceptors.

shibire(ts1), a temperature-sensitive mutation of the Drosophila gene encoding a Dynamin orthologue, blocks vesicle endocytosis and thus synaptic transmission, at elevated, or restrictive temperatures. By targeted Gal4 expression, UAS-shibire(ts1) has been used to dissect neuronal circuits. We investigated the effects of UAS-shibire(ts1) overexpression in Drosophila photoreceptors at permissive (19 degrees C) and restrictive (31 degrees C) temperatures. At 19 degrees C, overexpression of UAS-shi(ts1) causes decelerated phototransduction and reduced neurotransmitter release. This phenotype is exacerbated with dark adaptation, age and in white mutants. Photoreceptors overexpressing UAS-shibire(ts1) contain terminals with widespread vacuolated mitochondria, reduced numbers of vesicles and bundled microtubules. Immuno-electron microscopy reveals that the latter are dynamin coated. Further, the microtubule phenotype is not restricted to photoreceptors, as UAS-shibire(ts1) overexpression in lamina cells also bundles microtubules. We conclude that dynamin has multiple functions that are interrupted by UAS-shibire(ts1) overexpression in Drosophila photoreceptors, destabilizing their neural communication irreversibly at previously reported permissive temperatures.