Circulating tumor cell technologies.

Circulating tumor cells, a component of the "liquid biopsy", hold great potential to transform the current landscape of cancer therapy. A key challenge to unlocking the clinical utility of CTCs lies in the ability to detect and isolate these rare cells using methods amenable to downstream characterization and other applications. In this review, we will provide an overview of current technologies used to detect and capture CTCs with brief insights into the workings of individual technologies. We focus on the strategies employed by different platforms and discuss the advantages of each. As our understanding of CTC biology matures, CTC technologies will need to evolve, and we discuss some of the present challenges facing the field in light of recent data encompassing epithelial-to-mesenchymal transition, tumor-initiating cells, and CTC clusters.

Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis.

Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (10(0)-10(3) minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 10(2) minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 µm h(-1)) than either fibronectin (10.6 µm h(-1)) or collagen (7.6 µm h(-1)) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 µm h(-1) across all ECMs tested. We observed relatively mild chemoattraction (∼67% of myoblast population) in response to bFGF gradients of 3.2 ng mL(-1) mm(-1) regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and proliferation.

Microfluidic gradients reveal enhanced neurite outgrowth but impaired guidance within 3D matrices with high integrin ligand densities.

The density of integrin-binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell-adhesive ligands on neurite chemoattraction is not well understood. A platform is presented here that combines gradient-generating microfluidic devices with 3D protein-engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia-derived spheroids. Spheroids are encapsulated in elastin-like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade-off between neurite extension and neurite guidance is reminiscent of the well-known trade-off between adhesive forces at the leading and trailing edges of a migrating cell, implying that a similar matrix-mediated balance of forces regulates neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels.

Small-molecule axon-polarization studies enabled by a shear-free microfluidic gradient generator.

A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron-friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 h exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.