Contextual cues from cancer cells govern cancer-associated fibroblast heterogeneity.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.

Dorsoventral polarity directs cell responses to migration track geometries.

How migrating cells differentially adapt and respond to extracellular track geometries remains unknown. Using intravital imaging, we demonstrate that invading cells exhibit dorsoventral (top-to-bottom) polarity in vivo. To investigate the impact of dorsoventral polarity on cell locomotion through different confining geometries, we fabricated microchannels of fixed cross-sectional area, albeit with distinct aspect ratios. Vertical confinement, exerted along the dorsoventral polarity axis, induces myosin II-dependent nuclear stiffening, which results in RhoA hyperactivation at the cell poles and slow bleb-based migration. In lateral confinement, directed perpendicularly to the dorsoventral polarity axis, the absence of perinuclear myosin II fails to increase nuclear stiffness. Hence, cells maintain basal RhoA activity and display faster mesenchymal migration. In summary, by integrating microfabrication, imaging techniques, and intravital microscopy, we demonstrate that dorsoventral polarity, observed in vivo and in vitro, directs cell responses in confinement by spatially tuning RhoA activity, which controls bleb-based versus mesenchymal migration.

Spatiotemporally controlled nano-sized third harmonic generation agents.

Here, we present a new class of third harmonic generation (THG) imaging probes that can be activated with precise spatiotemporal control using non-linear excitation. These probes consist of lipid-coated perfluorocarbon nanodroplets with embedded visible chromophores. The droplets undergo phase transition from liquid to gas upon heating mediated by two-photon absorption of NIR light by the embedded dyes. Resulting microbubbles provide a sharp, local refractive index mismatch, which makes an excellent source of THG signal. Potential applications of these probes include activatable THG agents for biological imaging and "on-demand" delivery of various compounds under THG monitoring.

Real-time motion analysis reveals cell directionality as an indicator of breast cancer progression.

Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.

Phosphoinositides in chemotaxis.

Phosphatidylinositol lipids generated through the action of phosphinositide 3-kinase (PI3K) are key mediators of a wide array of biological responses. In particular, their role in the regulation of cell migration has been extensively studied and extends to amoeboid as well as mesenchymal migration. Through the emergence of fluorescent probes that target PI3K products as well as the use of specific inhibitors and knockout technologies, the spatio-temporal distribution of PI3K products in chemotaxing cells has been shown to represent a key anterior polarity signal that targets downstream effectors to actin polymerization. In addition, through intricate cross-talk networks PI3K products have been shown to regulate signals that control posterior effectors. Yet, in more complex environments or in conditions where chemoattractant gradients are steep, a variety of cell types can still chemotax in the absence of PI3K signals. Indeed, parallel signal transduction pathways have been shown to coordinately regulate cell polarity and directed movement. In this chapter, we will review the current role PI3K products play in the regulation of directed cell migration in various cell types, highlight the importance of mathematical modeling in the study of chemotaxis, and end with a brief overview of other signaling cascades known to also regulate chemotaxis.

Poly(vinylmethylsiloxane) elastomer networks as functional materials for cell adhesion and migration studies.

Cell migration is central to physiological responses to injury and infection and in the design of biomaterial implants. The ability to tune the properties of adhesive materials and relate those properties in a quantitative way to the dynamics of intracellular processes remains a definite challenge in the manipulation of cell migration. Here, we propose the use of poly(vinylmethylsiloxane) (PVMS) networks as novel substrata for cell adhesion and migration. These materials offer the ability to tune independently chemical functionality and elastic modulus. Importantly, PVMS networks are compatible with total internal reflection fluorescence (TIRF) microscopy, which is ideal for interrogating the cell-substratum interface; this latter characteristic presents a distinct advantage over polyacrylamide gels and other materials that swell with water. To demonstrate these capabilities, adhesive peptides containing the arginyl-glycyl-aspartic acid (RGD) tripeptide motif were successfully grafted to the surface of PVMS network using a carboxyl-terminated thiol as a linker. Peptide-specific adhesion, spreading, and random migration of NIH 3T3 mouse fibroblasts were characterized. These experiments show that a peptide containing the synergy sequence of fibronectin (PHSRN) in addition to RGD promotes more productive cell migration without markedly enhancing cell adhesion strength. Using TIRF microscopy, the dynamics of signal transduction through the phosphoinositide 3-kinase pathway were monitored in cells as they migrated on peptide-grafted PVMS surfaces. This approach offers a promising avenue for studies of directed migration and mechanotransduction at the level of intracellular processes.

Quantification of the binding affinity of a specific hydroxyapatite binding peptide.

The genesis of bone and teeth involves highly coordinated processes, which involve multiple cell types and proteins that direct the nucleation and crystallization of inorganic hydroxyapatite (HA). Recent studies have shown that peptides mediate the nucleation process, control HA microstructure or even inhibit HA mineralization. Using phage display technology, a short peptide was identified that binds to crystalline HA and to HA-containing domains of human teeth with chemical and morphological specificity. However, the binding affinity and specific amino acids that significantly contribute to this interaction require further investigation. In this study, we employ a microfluidic chip based surface plasmon resonance imaging (SPRi) technique to quantitatively measure peptide affinity by fabricating a novel 4 layer HA SPR sensor. We find the peptide (SVSVGMKPSPRPGGGK) binds with relatively high affinity (K(D) = 14.1 microM +/- 3.8 microM) to HA. The independently measured amino acid fragment SVSV seems to impart a significant contribution to this interaction while the MKPSP fragment may provide a conformational dependent component that enhances the peptides affinity but by itself shows little specificity in the current context. These data show that together, the two moieties promote a stronger synergistic binding interaction to HA than the simple combination of the individual components.

Characterization of non-equilibrium nanoparticle adsorption on a model biological substrate.

The kinetics of nanoparticle (NP) adsorption on a model biological interface (collagen) is measured in microfluidic channels using surface plasmon resonance (SPR) imaging over a range of CdSe/ZnS quantum dot concentrations to investigate the underlying binding process. Spherical CdSe/ZnS core-shell NP, derivatized with 3-mercaptopropionic acid (3-MPA), were considered to be model NPs because of their widespread use in biological applications and their relatively monodisperse size. The kinetic adsorption data suggests that the binding between the NP and the collagen substrate is irreversible at room temperature (pH approximately 7.4), and this type of adsorption process was further characterized in the context of a surface absorption model. Specifically, diffusion-limited adsorption was found to predominate the adsorption process at lower concentrations (<0.4 micromol/L), and NP adsorption was reaction-limited at higher concentration (>0.4 micromol/L). A limited pH study of our system indicates that NPs desorb from collagen under acidic conditions (pH 5.5); no significant desorption was observed under neutral and basic pH conditions. These observations are consistent with electrostatic interactions being the dominant force governing NP desorption from collagen substrates. Our present methodology for characterizing the seemingly irreversible NP adsorption complements our earlier study where NP adsorption onto weakly adsorbing surfaces (self-assembled monolayers) was characterized by Langmuir NP adsorption measurements.

Directional persistence of cell migration coincides with stability of asymmetric intracellular signaling.

It has long been appreciated that spatiotemporal dynamics of cell migration are under the control of intracellular signaling pathways, which are mediated by adhesion receptors and other transducers of extracellular cues. Further, there is ample evidence that aspects of cell migration are stochastic: how else could it exhibit directional persistence over timescales much longer than typical signal transduction processes, punctuated by abrupt changes in direction? Yet the mechanisms by which signaling processes affect those behaviors remain unclear. We have developed analytical methods for relating parallel live-cell microscopy measurements of cell migration dynamics to the intracellular signaling processes that govern them. In this analysis of phosphoinositide 3-kinase signaling in randomly migrating fibroblasts, we observe that hot spots of intense signaling coincide with localized cell protrusion and endure with characteristic lifetimes that correspond to those of cell migration persistence. We further show that distant hot spots are dynamically and stochastically coupled. These results are indicative of a mechanism by which changes in a cell's direction of migration are determined by a fragile balance of relatively rapid intracellular signaling processes.

Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts.

During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3'-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3'-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts.