Three modes of cancer cell movement. Similar to that of other cells, the migration of cancer cells is categorized into three modes: (a)–(c). Binding of extracellular cues to receptors and integrins transduces changes of the intercellular organization and morphology of the cell. A mesenchymal single cancer cell (a) forms a protrusion at the LE toward high chemoattractant levels and traction at the rear edge. The LE contains focal complexes that firmly adhere to stimuli through membrane receptors and integrins. An amoeboid single cancer cell (b) has dynamic focal complexes and high deformability. It migrates through blebbing movement induced by ECM and other stimuli. Collective cancer cells (c) migrate in a manner similar to mesenchymal cell, except that the intercellular connection is maintained during migration. Collective cell migration can change to single cell migration via epithelial-mesenchymal transition (C→D→A+B). Single cells can also switch from one mode to another.

Schematic of cell interaction with mechanical environment. (a) Sequential frames over 4 h show the displacement of cells, one with amoeboid (faster) and one with mesenchymal migration mode, inside the same channel. Cell migration speed can be quantified. Reproduced and adapted with permission from D. Irimia and M. Toner, Integrative Biology 1, 506 (2009). Copyright © 2009 by Royal Society of Chemistry Publishing group. (b) Cell spontaneously “squeezes” through microchannel. (b1) The dual-layered microfluidic device is fabricated with PDMS such that microchannels bridge between two parallel cell culture channels. (b2) Images of a time-lapse with a SPC-treated Panc-1 cell, migrating from left to right through a micron channel (7×11×150 μm), exhibit cell deformation in five stages. (b3) The corresponding localizations of cytokeratin (in green), filamentous actin (magenta), and the nuclei (blue) are visualized by staining. Scale bar: 50 μm. Reproduced and adapted with permission from C. G. Rolli et al., PLoS ONE 5, e8726 (2010). Copyright © 2010 by Public Library of Science. (c) Arrays of microposts can be used for measurement of cell adhesion during migration process. (c1) Under proper geometric constraints of postheight and width, cell exerting traction forces would deflect the elastomeric posts. The corresponding force can be quantified by measuring the displacement of micropoles (c2). (c3) Differential interference contrast micrographs of a smooth muscle cell cultured for 2 h on a postarray. These demonstrate loss of traction forces in response to the treatment of 2,3-butanedione monoxime or cytochalasin D, inhibitors to myosin contractility. [(c4)–(c6)] Phase contrast and immunofluorescence images of a smooth muscle cells on the microposts. Localization of fibronectin (in red) and focal adhesion protein vinculin (in green) indicates a strong correlation between direction and magnitude of traction force and focal adhesion area. Reproduced and adapted with permission from J. L. Tan et al., Proc. Natl. Acad. Sci. U.S.A. 100, 1484 (2003). Copyright © 2003 by National Academy of Sciences; I. Schoen et al., Nano Lett. 10, 1823 (2010). Copyright © 2010 by American Chemistry Society Publications.

Diffusion-based gradient generator using microchannels. (a) Gradient generator is composed of two parallel perfusion chamber connected with an array of microchannels. Two streams are mixed in bridging microchannels through diffusion predominantly. As illustrated in simulation (a1), the linear gradient of content B is created in microchannels. (a2) Cytoskeleton polarity is observed with cells that migrating along this attractant gradient. Actin (in red) is mainly located at the leading edge and on the side in contract with the microchannel wall. Microtubules (in green) are localized behind the nucleus but extend from the leading edge to the uropod. No thin lamellipodium is seen at the leading edge. Scale bar: 5 μm. Reproduced and adapted with permission from D. Irimia et al., Lab Chip 7, 1783 (2007). Copyright © 2007 by Royal Society of Chemistry. (b) Microjets gradient generator. (b1) A 3D schematic of the device shows the parallel source and sink chambers that are connected with microchannel and an open structure of the cell culture/gradient chamber. (b2) Top view of the device with a fluorescence image overlay of an Alexa 488 gradient that is generated through diffusion (see Ref. 75). Reproduced and adapted with permission from T. M. Keenan et al., Lab Chip 10, 116 (2010). Copyright © 2010 by Royal Society of Chemistry. (c) Additional manipulation of perfusion chamber can introduce further control over convection in cell culture/gradient chamber. (c1) Simulation of gradient. (c2) Fluid flow streamlines showing the cross-current flow pattern produced due to the pressure drop between inlets/outlets. (c3) The resulting fluid velocity along the dashed line is shown in (C2) (see Ref. 76). Reproduced and adapted with permission from A. Shamloo et al., Lab Chip 8, 1292 (2008). Copyright © 2008 by Royal Society of Chemistry.

Microcontacting printing of surface cues. (a) A PDMS stamp that contains the desired microfeatures is made via soft lithography and is used for patterning through two techniques. Either by adsorbing on PDMS features (1→4→5→6) or filling in the PDMS channel (1→2→3), the biomolecule-contained ink makes contact with substrate and is geometrically patterned. The ink will then be coated on the substrate surface, according to these microsized features of the PDMS stamp, by self-assembly or surface adsorption. (b) The shape and distribution of vinculin-containing focal adhesions correspond to those of the micropatterned fibronectin islands. Immunofluorescence microscopic overlay images of cells cultured for 8 h on different micropatterned distributions of rhodamine-FN-coated islands (magenta) and stained with antivinculin antibodies (green). Reproduced and adapted with permission from N. Xia et al., FASEB J. 22, 1649 (2008). Copyright © 2008 by Federation of American Societies for Experimental Biology. (c) Time lapse of a fibroblast cell migrating along the surface gradient of fibronectin. Round-shaped fibronectin islands are patterned in gradient by varying interisland spacing. Cell is found to generate protrusion and polarity in response to the variation of interisland spacing and then migrate toward denser pattern. Personal communication/unpublished data with permission from Tatjana Autenrieth and Martin Bastmeyer [Universitaet Karlsruhe (TH), Germany].

BTSC migration through the microfluidic device. Real time microscopy demonstrating a BTSC (highlighted in red) migrating from seeding chamber to the receiving chamber through a bridging microchannel

Schematic of typical PDMS microfluidic device via soft lithography. (a) A mask with pattern designs printed on a transparency film is projected on a master coated with negative photoresist via optical lithography. [(b) and (c)] After the resulting master is made, a negative in bas-relief is made by casting a mixture of PDMS with a crosslinking reagent and allowing it to harden. [(d) and (e)] The resulting PDMS mold is released from the master and is sealed by another substrate, often a glass or other PDMS, to form channels. The sealing can be either irreversible through oxygen plasma bonding or reversible via the naturally adhesive surface of PDMS.

Schematic representation of the gradient generator and gradient characterization. (a) Top view of the device consisting of the gradient mixer and cell culture chamber. (b) 3D representation of the cell culture chamber where cells are exposed to gradients of chemoattractant. (c) Gradient profiles represented by fluorescent characterization of FITC-dextran (8 KDa): linear shape (top), hill shape (middle), and cliff shape (bottom). Reproduced and adapted with permission from N. L. Jeon et al., Nat. Biotechnol. 20, 826 (2002). Copyright © 2002 by Nature Publishing group.

Diffusion-based gradient generator using hydrogel. (a) Submillimeter-sized channel that connects between source and sink reservoirs can generate gradient profile in exponential shape (a1) and linear shape (a2), visualized with Alexa 488 solution. Reproduced and adapted with permission from V. V. Abhyanka et al., Lab Chip 8, 1507 (2008). Copyright © 2008 by Royal Society of Chemistry Publishing group. (b) Hydrogel-confined microchannels can generate gradients profile in a variety shapes. (b1) Hydrogel is confined in microchannels as indicated in red, while source and sink fluid flows toward the same outlet (in green), keeping constant molecule exchange with 3D hydrogel. Geometry of microchannel, therefore the hydrogel compartment determines the gradient profile in a concave down nonlinear shape (b2), linear shape (b3), and convex up nonlinear shape (b4). Reproduced and adapted with permission from B. Mosadegh et al., Langmuir 23, 10910 (2007). Copyright © 2007 by American Chemistry Society Publications. (c) A microfluidic gradient generator designed for the study of cancer cell directed invasion in a 3D environment. (c1) Two parallel perfusion channels were introduced with attractant-containing medium and control medium that served as source and sink reservoirs, respectively. Cell-BME mixture is loaded from cell inlets into cell culture chambers (shown in zoom view). (c2) The perfusion channels are introduced with FAM-DNA (MW 6000 D) and PBS, respectively. The dashed line indicates the channel location. In 20 min, a linear gradient is established and is maintained for over 24 h (see Ref. 77). Reproduced and adapted with permission from T. Liu et al., Electrophoresis 30, 4285 (2009). Copyright © 2009 by WILEY-VCH Verlag GmbH&Co.

Single cell migration in response to geometry confinement of surface pattern. (a) B16F1 cell on a pattern of two reservoirs (not shown) connected by ratchets. The cell migrates through the bridging ratchets (from right to left) by repeating a cycle of protrusion (at the cell’s leading edge, in the funneling direction) and retraction (at its rear). The polarity markers (Arp2/3 complex, seen as yellow on the overlay images with actin) and organization of actin cytoskeleton (fluorescent phalloidin, green) is stained to reveal the migration cycles. The scale bar represents 50 microns. Reproduced and adapted with permission from G. Mahmud et al., Nat. Phys. 5, 606 (2009). Copyright © 2009 by Nature Publishing group. (b) Polarized single cell in response to geometry constraint by surface pattern. (b) Top: cell membrane polarity propagates to cell internal polarity, as shown in the average of 75 cell fluorescent images. (b) Bottom, from left to right: carton illustration of this propagation process. In response to the anisotropic distribution of fibronectin in micropattern (gray), the distribution of adhesions (green) and actin network (red) becomes polarized with polymerizing meshwork on adhesive edges and stress fibers on nonadhesive edges. This in turn triggers the reorganization of actin-MT connectors and the location of Golgi apparatus (blue). Reproduced and adapted with permission from M. Théry et al., Proc. Natl. Acad. Sci. U.S.A. 103, 19771 (2006). Copyright © 2006 by National Academy of Sciences. (c) One-way microarrays direct the cell migration. Time-lapse phase-contrast images (numbered in hours upon cell seeding) show the continuous directional migration of individual NIH 3T3 fibroblast from the blunt end of the teardrop island to the tip of an adjacent island. Scale bar: 50 μm. Reproduced and adapted with permission from G. Kumar et al., Adv. Mater. (Weinheim, Ger.) 19, 1084 (2007). Copyright © 2007 by WILEY-VCH Verlag GmbH&Co.