Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

Farahat, Waleed A.; Wood, Levi B.; Zervantonakis, Ioannis K.; Schor, Alisha R.; Ong, Lee-Ling Sharon; Neal, Devin; Kamm, Roger D.; Asada, H. Harry

doi:10.1371/journal.pone.0037333

Cited by 112 publications

(126 citation statements)

References 51 publications

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“…mammary adenocarcinoma cells embedded in a collagen type I hydrogel (Fig. 1A) (17,20). By holding one medium channel (upstream) at a higher fluid pressure than the other channel (downstream), a stable and repeatable flow field can be generated through the collagen gel (Movie S1 and SI Appendix, Fig.…”

Section: Significancementioning

confidence: 99%

Mechanotransduction of fluid stresses governs 3D cell migration

Polacheck

German

Mammoto

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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225

215

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Solid tumors are characterized by high interstitial fluid pressure, which drives fluid efflux from the tumor core. Tumor-associated interstitial flow (IF) at a rate of ∼3 μm/s has been shown to induce cell migration in the upstream direction (rheotaxis). However, the molecular biophysical mechanism that underlies upstream cell polarization and rheotaxis remains unclear. We developed a microfluidic platform to investigate the effects of IF fluid stresses imparted on cells embedded within a collagen type I hydrogel, and we demonstrate that IF stresses result in a transcellular gradient in β1-integrin activation with vinculin, focal adhesion kinase (FAK), FAK PY397 , F actin, and paxillindependent protrusion formation localizing to the upstream side of the cell, where matrix adhesions are under maximum tension. This previously unknown mechanism is the result of a force balance between fluid drag on the cell and matrix adhesion tension and is therefore a fundamental, but previously unknown, stimulus for directing cell movement within porous extracellular matrix.mechanobiology | breast cancer | metastasis I ntegrins and associated focal adhesion (FA) proteins form a tension-sensitive mechanical link between the extracellular matrix (ECM) and the cytoskeleton, and serve as key components in the signaling cascade by which cells transduce mechanical signals into biological responses (mechanotransduction) (1, 2). Contractile stresses generated by the cell are balanced by tractions at cell-substrate adhesions, and the FA protein vinculin accumulates at regions of high substrate stress (3, 4). The FA protein paxillin colocalizes with vinculin (4) and mediates β1-integrin FA turnover through interaction with FA kinase (FAK) (5). The FAK-paxillin signaling axis recruits vinculin to β1 integrins at regions of high matrix adhesion tension (6), and paxillin-a key mechanosensor (7)-mediates protrusion formation at regions of high stress on 2D substrates (8), and FAK-paxillin-vinculin signaling is required for mechanosensing and durotaxis (9).The tumor microenvironment imparts mechanical and chemical signals on tumor and stromal cells (10), and advanced breast carcinomas are characterized by high interstitial fluid pressure (11), an indicator of poor prognosis (12). This elevated fluid pressure drives interstitial flow (IF) and alters chemical transport within the tumor (13), and IF influences tumor cell migration through the generation of autocrine chemokine gradients (14). Equally important, although not as well understood, is the physical drag imparted on the ECM and constitutive cells (15) by IF, which is analogous to the FA-activating shear stresses generated on endothelial cells by hemodynamic forces (16). With endothelial cells, shear stress can be the dominant mechanical stimulus that induces FAK activation and cytoskeletal remodeling; however, for cells embedded within a porous matrix scaffold, the ratio of the force due to the pressure drop across the cell to the total shear force is inversely proportional to hydrogel p...

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Section: Significancementioning

confidence: 99%

Mechanotransduction of fluid stresses governs 3D cell migration

Polacheck

German

Mammoto

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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225

215

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“…Spheroids of 40-to 70-μm diameter were selected with sieves as reported (66). Spheroids were seeded in the central region of a microfluidic device using standard soft lithography techniques (94,95). The central region of the device is flanked by two channels.…”

Section: Fgfr4-fiin-2 and Fgfr4 V550l-fiin-3 Crystallization And Strumentioning

confidence: 99%

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

Li¹,

Wang²,

Tanizaki

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

165

140

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The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.drug discovery | cancer drug resistance | kinase inhibitor | structure-based drug design R eceptor tyrosine kinases (RTKs) serve as critical sensors of extracellular cues that activate a myriad of intracellular signaling pathways to regulate cell state. There are 58 receptor tyrosine kinases in the human genome, and many have been demonstrated to be constitutively activated through amplification or mutation in particular cancers. The signals emanating from these RTKs, such as epidermal growth factor receptor (EGFR), FGF receptor (FGFR), platelet-derived growth factor receptor (PDGFR), protein kinase Kit (KIT), and protein kinase c-Met (MET), have been pharmacologically proven to be essential to the survival of cancers expressing mutant forms of these proteins. However, rapid resistance to monotherapy with first-generation RTK inhibitors has been universally observed. Resistance typically arises from the emergence of cancer cells expressing mutant forms of RTKs that are impervious to the action of first-generation drugs or from the activation of by-pass signaling mechanisms. Resistance can be overcome by developing new inhibitors that target the mutant RTK directly or target bypass signaling mechanisms. Indeed this approach has been deployed succes...

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“…In our microfluidic device (Farahat et al, 2012), shown in Figure 1, we inject collagen gel between the two channels. Through one channel,we seed cells, which attach to one side of the collagen and form a monolayer.…”

Section: Cell-matrix Interactions and 2d/3d Imagesmentioning

confidence: 99%

“…A confocal microscope captures fluorescence and transmitted light images of each migration region, including the cell monolayer. Image adapted from (Farahat et al, 2012)). …”

Section: Introductionmentioning

confidence: 99%

A Bayesian filtering approach to incorporate 2D/3D time-lapse confocal images for tracking angiogenic sprouting cells interacting with the gel matrix

Ong

Dauwels

Ang

et al. 2014

Medical Image Analysis

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We present a new approach to incorporating information from heterogeneous images of migrating cells in 3D gel. We study 3D angiogenic sprouting, where cells burrow into the gel matrix, communicate with other cells and create vascular networks. We combine time-lapse fluorescent images of stained cell nuclei and transmitted light images of the background gel to track cell trajectories. The nuclei images are sampled less frequently due to photo toxicity. Hence, 3D cell tracking can be performed more reliably when 2D sprout profiles, extracted from gel matrix images, are effectively incorporated. We employ a Bayesian filtering approach to optimally combine the two heterogeneous images with different sampling rates. We construct stochastic models to predict cell locations and sprout profiles and condition the likelihood of nuclei location by the sprout profile. The conditional distribution is non-Gaussian and the cell dynamics is non-linear. To jointly update cell and sprout estimates, we use a Rao-Blackwell particle filter. Simulation and experimental results show accurate tracking of multiple cells along with sprout formation, demonstrating synergistic effects of incorporating the two types of images.

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Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

Cited by 112 publications

References 51 publications

Mechanotransduction of fluid stresses governs 3D cell migration

Mechanotransduction of fluid stresses governs 3D cell migration

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

A Bayesian filtering approach to incorporate 2D/3D time-lapse confocal images for tracking angiogenic sprouting cells interacting with the gel matrix

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