Migration speed of captured breast cancer subpopulations correlates with metastatic fitness.

The genetic alterations contributing to migration proficiency, a phenotypic hallmark of metastatic cells required for colonizing distant organs, remain poorly defined. Here, we used single-cell magneto-optical capture (scMOCa) to isolate fast cells from heterogeneous human breast cancer cell populations, based on their migratory ability alone. We show that captured fast cell subpopulations retain higher migration speed and focal adhesion dynamics over many generations as a result of a motility-related transcriptomic profile. Upregulated genes in isolated fast cells encoded integrin subunits, proto-cadherins and numerous other genes associated with cell migration. Dysregulation of several of these genes correlates with poor survival outcomes in people with breast cancer, and primary tumors established from fast cells generated a higher number of circulating tumor cells and soft tissue metastases in pre-clinical mouse models. Subpopulations of cells selected for a highly migratory phenotype demonstrated an increased fitness for metastasis.

Adaptive settings for the nearest-neighbor particle tracking algorithm.

BACKGROUND: The performance of the single particle tracking (SPT) nearest-neighbor algorithm is determined by parameters that need to be set according to the characteristics of the time series under study. Inhomogeneous systems, where these characteristics fluctuate spatially, are poorly tracked when parameters are set globally. RESULTS: We present a novel SPT approach that adapts the well-known nearest-neighbor tracking algorithm to the local density of particles to overcome the problems of inhomogeneity. CONCLUSIONS: We demonstrate the performance improvement provided by the proposed method using numerical simulations and experimental data and compare its performance with state of the art SPT algorithms. AVAILABILITY AND IMPLEMENTATION: The algorithms proposed here, are released under the GNU General Public License and are freely available on the web at http://sourceforge.net/p/adaptivespt. CONTACT: javier.mazzaferri@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Analysis of a novel field dilution method for testing samples that exceed the analytic range of point-of-care blood lead analyzers.

Investigators developed and evaluated a dilution method for the LeadCare II analyzer (LCII) for blood lead levels >65 µg/dL, the analyzer's maximum reporting value. Venous blood samples from lead-poisoned children were initially analyzed in the field using the dilution method. Split samples were analyzed at the US Centers for Disease Control and Prevention (CDC) laboratory using both the dilution method and inductively coupled plasma-mass spectrometry (ICP-MS). The concordance correlation coefficient of CDC LCII vs. ICP-MS values (N = 211) was 0.976 (95 % confidence interval (CI) 0.970-0.981); of Field LCII vs. ICP-MS (N = 68) was 0.910 (95% CI 0.861-0.942), and CDC LCII vs. Field LCII (N = 53) was 0.721 (95% CI 0.565-0.827). Sixty percent of CDC and 54% of Field LCII values were within ±10% of the ICP-MS value. Results from the dilution method approximated ICP-MS values and were useful for field-based decision-making. Specific recommendations for additional evaluation are provided.

C3a elicits unique migratory responses in immature low-density neutrophils.

Neutrophils represent the immune system's first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

Opto-magnetic Selection and Isolation of Single Cells.

Capturing single cells from large heterogenous populations based solely on observable traits is necessary for many cell biology applications and remains a major technical challenge. The protocol we present allows the isolation of viable and metabolically active cells selected for their shape, migration speed, contact to other cells, or intracellular protein localization. We previously introduced a method termed Cell Labeling via Photobleaching (CLaP) for the efficient tagging of cells chosen for visual criteria. Here we describe a new protocol for capturing such cells using ferromagnetic beads termed single-cell magneto-optical capture (scMOCa). This technology is especially useful when the number of target cells represents an extremely low fraction of the total population (potentially one single cell), a situation in which conventional sorting techniques like fluorescent or magnetic activated cell sorting (F/MACS) cannot provide satisfactory results in terms of capture efficiency and specificity. scMOCa uses the lasers of a confocal microscope to photobleach and crosslink biotin-4-fluorecein molecules to cell membranes. Streptavidin coated magnetic beads then adhere to biotin moieties and a magnet allows the capture of illuminated cells. By precisely controlling liquid volumes and spacing between the different parts of a simple setup, high cell selectivity and capture efficacy can be achieved. scMOCA allows visual selection and isolation of any number of cells in a microscopy field and captured cells remain viable to generate new colonies of chosen phenotypes for downstream analyses.

Opto-magnetic capture of individual cells based on visual phenotypes.

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.

Unusual mitochondrial genome structures throughout the Euglenozoa.

Mitochondrial DNA of Kinetoplastea is composed of different chromosomes, the maxicircle (bearing 'regular' genes) and numerous minicircles (specifying guide RNAs involved in RNA editing). In trypanosomes [Kinetoplastea], DNA circles are compacted into a single dense body, the kinetoplast. This report addresses the question whether multi-chromosome mitochondrial genomes and compacted chromosome organization are restricted to Kinetoplastea or rather occur throughout Euglenozoa, i.e., Kinetoplastea, Euglenida and Diplonemea. To this end, we investigated the diplonemid Rhynchopus euleeides and the euglenids Petalomonas cantuscygni, Peranema trichophorum and Entosiphon sulcatum, using light and electron microscopy and molecular techniques. Our findings together with previously published data show that multi-chromosome mitochondrial genomes prevail across Euglenozoa, while kinetoplast-like mtDNA packaging is confined to trypanosomes.

A Haptotaxis Assay for Neutrophils using Optical Patterning and a High-content Approach.

Neutrophil recruitment guided by chemotactic cues is a central event in host defense against infection and tissue injury. While the mechanisms underlying neutrophil chemotaxis have been extensively studied, these are just recently being addressed by using high-content approaches or surface-bound chemotactic gradients (haptotaxis) in vitro. Here, we report a haptotaxis assay, based on the classic under-agarose assay, which combines an optical patterning technique to generate surface-bound formyl peptide gradients as well as an automated imaging and analysis of a large number of migration trajectories. We show that human neutrophils migrate on covalently-bound formyl-peptide gradients, which influence the speed and frequency of neutrophil penetration under the agarose. Analysis revealed that neutrophils migrating on surface-bound patterns accumulate in the region of the highest peptide concentration, thereby mimicking in vivo events. We propose the use of a chemotactic precision index, gyration tensors and neutrophil penetration rate for characterizing haptotaxis. This high-content assay provides a simple approach that can be applied for studying molecular mechanisms underlying haptotaxis on user-defined gradient shape.

Engineered cell culture substrates for axon guidance studies: moving beyond proof of concept.

Promoting axon regeneration following injury is one of the ultimate challenges of neuroscience, and understanding the mechanisms that regulate axon growth and guidance is essential to achieve this goal. During development axons are directed over relatively long distances by a precise extracellular distribution of chemical signals in the embryonic nervous system. Multiple guidance proteins, including netrins, slits, semaphorins, ephrins and neurotrophins have been identified as key players in this process. During the last decade, engineered cell culture substrates have been developed to investigate the cellular and molecular mechanisms underlying axon guidance. This review is focused on the biological insights that have been achieved using new techniques that attempt to mimic in vitro the spatial patterns of proteins that growth cones encounter in vivo.

Description of Rhynchopus euleeides n. sp. (Diplonemea), a free-living marine euglenozoan.

We describe Rhynchopus euleeides n. sp., using light and electron microscopy. This free-living flagellate, which was isolated earlier from a marine habitat, can be grown axenically in a rich medium based on modified seawater. In the trophic stage, cells are predominantly elliptical and laterally flattened, but frequently change their shape (metaboly). Gliding is the predominant manner of locomotion. The two flagella, which are typically concealed in their pocket, are short stubs of unequal length, have conventional axonemes, but apparently lack a paraxonemal rod. Swarmer cells, which form only occasionally, are smaller in size and carry two conspicuous flagella of more than 2 times the body length. Cells are decorated with a prominent apical papillum. Both the flagellar pocket and the adjacent feeding apparatus seem to merge together into a single sub-apical opening. The mitochondrion, which is most likely single, is located peripherally. It is reticulated in shape and contains only a few lamellar cristae. Mitochondrial DNA is abundant and evenly distributed throughout the organelle. Morphological synapomorphies confirm the affiliation of the species with the genus Rhynchopus (Diplonemea, Euglenozoa). We discuss the characters that distinguish Rhynchopus from Diplonema corroborating the validity of the two genera.