Experiments on an ascites hepatoma: I. Enzymatic digestion and alkaline degradation of the cementing substance and separation of cells, in tumor islands

Long-term Extensive Expansion of Mouse Hepatic Stem/Progenitor Cells in a Novel Serum-Free Culture System

A Proinflammatory, Antiapoptotic Phenotype Underlies the Susceptibility to Acute Pancreatitis in Cystic Fibrosis Transmembrane Regulator (−/−) Mice

The nature of the intercellular material of adult mammalian tissues

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

Event Abstract Back to Event Development of photodegradable gelatin hydrogels and image analysis technique for automatic optical cell separation system based on the cellular morphology in embedding culture Masato Tamura1, Shinji Sugiura1, Taku Satoh1, Toshiyuki Kanamori1, Shibuta Mayu2, Kei Kanie2, Ryuji Kato2, Hirofumi Matsui3 and Masumi Yanagisawa4 1 National Institute of Advanced Industrial Science and Technology (AIST), Department of Life Science and Biotechnology, Japan 2 Nagoya University, Graduate School of Pharmaceutical Sciences, Japan 3 University of Tsukuba, Faculty of Medicine, Japan 4 Engineering System Co., Ltd., Japan This paper reports novel cell separation strategy and automated cell separation system based on the cellular morphology under 3D culture environment in the photodegradable hydrogel. Recently, we developed gelatin-based photodegradable hydrogels by NHS-activated-ester reaction [1], and applied this hydrogels to optical cell separation [2]. More recently, we developed “click-crosslinkable and photodegradable gelatin hydrogels” [3]. The present study applied this hydrogels to the most promising application, cell separation based on the cellular morphology. The present study includes proof concept of morphology-based cell separation and development of automatic cell separation system. 3D culture environment with a specific extracellular matrix regulates cellular function and phenotype. In addition, cancer cell morphology is alternated depending on its malignancy in 3D culture environment [4]. Recently we developed the predication model of stem cell differentiation by image analysis [5]. In this paper, we introduced this image analysis technique and the features of click-crosslinkable and photodegradable gelatin hydrogels to the automated morphology-based cell separation system in 3D culture environment in the above mentioned photodegradable gelatin hydrogels. Figure 1 shows the schematic procedure for optical cell separation based on the cellular morphology under 3D culture environment in the photodegradable hydrogel. Suspension of cells including heterogeneous population is mixed with pregel solutions and cells are encapsulated in the gelatin-based photodegradable hydrogels (Figure 1a). After the culture in 3D environment (Figure 1b), microscopic images of the cells are captured. The captured images are analyzed to distinguish the target cells from the other cells by using the image analysis algorithm, which we previously developed for analyzing stem cells (Figure 2) [5]. The hydrogels around the target area is irradiated with UV light (Figure 1c). The cells in the irradiated area are collected by automated pipetting system (Figure 1d). We developed automated system for this optical cell separation procedure, including cultivation, image acquisition, image analysis, light irradiation, and pipetting for cell collection (Figure 3). We are currently developing an automated image analysis algorithm to distinguish cancer cells from normal cells under 3D environment. The automated optical cell separation system with image analysis algorithm will be applied to the establishment of novel cancer-cell lines from clinical samples such as biopsy tissue. KAKENHI (14J07186); NEDO (1009004)

Enhanced Self-Renewal Capability in Hepatic Stem/Progenitor Cells Drives Cancer Initiation

Phosphorylation of Ephrin-B1 Regulates Dissemination of Gastric Scirrhous Carcinoma

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Collective cell migration plays an essential role in vertebrate development, yet the extent to which dynamically changing microenvironments influence this phenomenon remains unclear. Observations of the distribution of the extracellular matrix (ECM) component fibronectin during the migration of loosely connected neural crest cells (NCCs) lead us to hypothesize that NCC remodeling of an initially punctate ECM creates a scaffold for trailing cells, enabling them to form robust and coherent stream patterns. We evaluate this idea in a theoretical setting by developing an individual-based computational model that incorporates reciprocal interactions between NCCs and their ECM. ECM remodeling, haptotaxis, contact guidance, and cell-cell repulsion are sufficient for cells to establish streams in silico, however, additional mechanisms, such as chemotaxis, are required to consistently guide cells along the correct target corridor. Further model investigations imply that contact guidance and differential cell-cell repulsion between leader and follower cells are key contributors to robust collective cell migration by preventing stream breakage. Global sensitivity analysis and simulated gain- and loss-of-function experiments suggest that long-distance migration without jamming is most likely to occur when leading cells specialize in creating ECM fibers, and trailing cells specialize in responding to environmental cues by upregulating mechanisms such as contact guidance. Editor's evaluation This important study presents predictions from a computational model demonstrating the impact of the extracellular matrix on collective cell migration in the neural crest. The evidence supporting the claims of the authors is solid, and the study is interesting to cell biologists exploring cell migration in different contexts. https://doi.org/10.7554/eLife.83792.sa0 Decision letter eLife's review process Introduction Vertebrate neural crest cells (NCCs) are an important model for collective cell migration. Discrete streams of migratory NCCs distribute throughout the embryo to contribute to nearly every organ (Le Douarin and Kalcheim, 1999; Tang and Bronner, 2020). Consequently, mistakes in NCC migration may result in severe birth defects, termed neurocristopathies (Vega-Lopez et al., 2018). In contrast to well-studied tightly adhered cell cluster models, such as border cell and lateral line migration (Peercy and Starz-Gaiano, 2020; Olson and Nechiporuk, 2021), much less is known about how ‘loosely’ connected cells, such as NCCs, invade through immature extracellular matrix (ECM) and communicate with neighbors to migrate collectively. Moreover, several invasive mesenchymal cancers resemble collective NCC migration, with cells moving away from the tumor mass in chain-like arrays or narrow strands (Friedl et al., 2004; Friedl and Alexander, 2011). Therefore, utilizing the neural crest experimental model to gain a deeper knowledge of the cellular and molecular mechanisms underlying collective cell migration has the potential to improve the repair of human birth defects and inform strategies for controlling cancer cell invasion and metastasis. Time-lapse analyses of NCC migratory behaviors in several embryo model organisms have revealed that leader NCCs in discrete migratory streams in the head (Teddy and Kulesa, 2004; Genuth et al., 2018), gut (Druckenbrod and Epstein, 2007; Young et al., 2014), and trunk (Kasemeier-Kulesa et al., 2005; Li et al., 2019) are highly exploratory. Leading NCCs extend thin filopodial protrusions in many directions, interact with the ECM and other cell types, then select a preferred direction for invasion. Trailing cells extend protrusions to contact leaders and other cells to maintain cell neighbor relationships and move collectively (Kulesa et al., 2008; Ridenour et al., 2014; Richardson et al., 2016). These observations suggest a leader-follower model for NCC migration (McLennan et al., 2012) in which NCCs at the front of migrating collectives read out guidance signals and communicate over long distances with trailing cells. This model challenged other proposals that NCCs respond to a chemical gradient signal and sustain cohesive movement via an interplay of local cell co-attraction and contact inhibition of locomotion (Theveneau and Mayor, 2012; Szabó et al., 2016; Merchant et al., 2018; Merchant and Feng, 2020). The precise cellular mechanisms that underlie leader-follower migration behavior, for example, the involvement with ECM or the nature of communication signals created by leaders, have not been elucidated. One paradigm that has emerged suggests that loosely connected streams of NCCs move in a collective manner by leaders communicating through long-range signals that are interpreted and amplified by follower NCCs. In support of this, chick leader cranial NCCs show enhanced expression of secreted factors, such as angiopoietin-2 (Ang-2) (McLennan et al., 2015a) and fibronectin (FN) (Morrison et al., 2017). Knockdown of Ang-2 reduces the chemokinetic behavior of follower chick NCCs, resulting in disrupted collective cell migration (McKinney et al., 2016). These observations suggest that follower NCC collective movement depends on signals deposited in the microenvironment by leaders or the adjacent mesoderm. Signals in the FN-rich ECM may also provide microenvironmental cues for NCC collective migration. NCCs cultured in FN-rich ECM move with thin, anchored filopodia through a field that is punctate immediately distal to the migrating front, while ‘pioneer’ NCCs within the front appear to form radially oriented bundles of FN-containing filaments (Rovasio et al., 1983). Later work on the NCC epithelial-to-mesenchymal transition showed that inhibition of ECM-integrin receptors resulted in delamination of NCCs into the neural tube lumen (Kil et al., 1996). Moreover, FN is abundant in the mouse head and neck (Mittal et al., 2010), and there is a dramatic reduction in the number of migrating NCCs that reach the heart in FN null mice (Wang and Astrof, 2016). Thus, FN is critical for NCC to reach their targets, although its precise role in collective cell migration remains unclear. Mathematical modeling of NCC migration can help provide insight into the role of FN and identify the origins of multiscale collective behaviors (Giniūnaitė et al., 2020; Kulesa et al., 2021). Previous models for NCC migration demonstrated that contact guidance, a mechanism by which cells align themselves along ECM fibers, can establish collective behavior and create single-file cell chains (Painter, 2009). Later models incorporating ECM degradation and haptotaxis, a process by which cells migrate up adhesive ECM gradients, demonstrated how matrix heterogeneity and cell-cell interactions could determine cell migratory patterns (Painter et al., 2010; Wynn et al., 2013). Models for other collectively migrating cells, such as fibroblasts, suggest that ECM remodeling by motile cells can enable anisotropic collective movement, with migratory directions dictated by fiber orientation (Dallon et al., 1999; Groh and Louis, 2010; Chauviere et al., 2010; Azimzade et al., 2019; Wershof et al., 2019; Pramanik et al., 2021; Suveges et al., 2021; Metzcar et al., 2022). No models have yet addressed how collective migration arises from cell remodeling of an initially isotropic, immature ECM. In this paper, we develop an agent-based model (ABM; also known as an individual-based model) of chick cranial NCC migration that considers a cell-reinforced migratory cue in which ‘leader’ cells remodel an initially punctate and immature FN matrix to signal ‘follower’ cells. Other processes detailed in the model include cell-cell repulsion, haptotaxis, and contact guidance. This framework incorporates new observations of FN distribution in the chick cranial NCC microenvironment and simulates gain- and loss-of-function of FN. Detailed simulations of the model over parameter space, coupled with global sensitivity analysis of ABM parameters, identifies mechanisms that dominate the formation of migrating streams and other NCC macroscopic behavior. Through this analysis, we find that migration is most efficient when leading NCCs specialize in remodeling FN to steer the collective, as cells otherwise enter a ‘jammed’ state in which migration is greatly reduced and cells are densely packed together (Sadati et al., 2013). The addition of ‘guiding signals’ that direct NCCs along a target corridor limits excessive lateral migration in the ABM but concurrently promotes separation between leader and trailing cells in the stream. We survey ABM parameter combinations that recover robust collective migration without such stream breaks, which underscore the potential importance of contact guidance. Results FN protein expression within the mesoderm is unorganized and punctate prior to cranial NCC emigration, but filamentous after it has been traversed by leader cells We confirmed that FN throughout the head, neck and cardiovascular regions in the chick embryo is distributed in patterns that overlap with NCC migratory pathways (Figure 1; Duband and Thiery, 1982). To assess the in vivo distribution of FN protein at higher spatial resolution, we examined transverse cryosections (Figure 1A; Hamburger and Hamilation, 1951) at developmental stages (HH12–13) midway through cranial NCC migration to reveal that NCCs are in close association with a heterogeneous meshwork of FN (Figure 1B). Punctate FN, not yet fibrils, are found both distal to the invasive NCC migratory front and adjacent to the leader NCC subpopulation (Figure 1C). By contrast, elongated FN fibrils are located behind the leading edge of invasive NCC streams (Figure 1D). FN fibrils proximal to the invasive NCC migratory front did not reveal a preferred directional orientation. Similar punctate FN was also visible in regions outside the NCC migratory pathway, subjacent to the surface ectoderm (Figure 1C). Figure 1 with 1 supplement see all Download asset Open asset In vivo observations of fibronectin (FN) and results from gain/loss-of-function experiments. (A) Schematic of a typical neural crest cell (NCC) migratory stream in the vertebrate head of the chick embryo, at developmental stage HH12–13 (Hamburger and Hamilation, 1951) at the axial level of the second branchial arch (ba2). (B) Transverse section through the NCC migratory stream at the axial level of rhombomere 4 (r4) triple-labeled for FN (green), nuclei (DAPI-blue) and migrating NCCs (HNK1-red). NCCs migrate subjacent to the surface ectoderm after emerging from the dorsal neural tube (NT). The arrowhead points to the surface ectoderm. The yellow boxes highlight the tissue subregions that contain the leader NCCs (box 1) and are distal to the leader NCCs (box 2), marking distinct shapes of the FN in each box with an asterisk (box 1) and open circle (box 2). (C) FN only. (D) The fibrous (box 1-asterisk) and punctate (box 2-open circle) appearance of FN in the NCC microenvironment. (E) NCC distance migrated in FN morpholino-injected embryos and (F) percentage area of the NCC migratory stream after microinjection of soluble FN into the r4 paraxial mesoderm prior to NCC emigration. NT = neural tube. The scale bars are 50 µm (B–C) and 10 µm (D). Gain- or loss-of-function of FN leads to reduced NCC migration Functional analysis confirms FN is required for normal NCC migration. Knockdown of FN expression introduced into premigratory NCCs within the neural tube led to a significant reduction (nearly 30%) in the total distance migrated by NCCs enroute to BA2 (Figure 1E). Microinjection of soluble FN into the cranial NCC migratory domain, for example, into the paraxial mesoderm adjacent to rhombomere 4 (r4) prior to NCC delamination, also led to a dramatic reduction (by 70%) in NCC migration as indicated by a decrease in the area typically covered by the invading NCCs (Figure 1F). Thus, decreasing the expression of FN – presumably by decreasing the rate at which FN is secreted by motile NCCs – or increasing the FN density in the microenvironment led to significant changes in the NCC migration pattern. We conclude that a balance of FN within the migratory microenvironment is required to promote proper migration. An individual-based model of NCC migration and ECM remodeling produces collectively migrating streams in silico Our observations led us to hypothesize that migrating NCCs remodel punctate FN into a fibrous scaffold for trailing cells. We evaluated this idea in a theoretical setting by constructing a mathematical model in which NCCs are represented as discrete off-lattice point masses, that is, agents, that freely move in a two-dimensional (2D) plane (Figure 2—figure supplement 1). Each agent responds to, and influences, the remodeling of an initially punctate ECM (Figure 2; Figure 2—figure supplement 2). Their velocities are determined using an overdamped version of Newton’s second law. The three types of forces that alter agent trajectories arise from friction (which is proportional to the cell velocity), cell-ECM interactions (specifically, those from haptotaxis and contact guidance), and cell-cell repulsion. Neighboring NCCs, FN puncta, and FN fibers generate the latter two forces and affect the motion of an agent only when they are within a user-specified distance, Rfilo , of the agent center (this distance represents the length of cell filopodia protrusions). Figure 2 with 13 supplements see all Download asset Open asset Integration of extracellular matrix (ECM) within an agent-based model (ABM) of neural crest cell (NCC) migration. Individual snapshots (A) of an example ABM realization reveals that the model can generate a single anisotropically migrating stream over a simulated time of 12 hr. Different realizations generated using the same parameter values but different random seeds (B), however, may produce streams that exhibit the formation of multiple branches and demonstrate the range of possible model behaviors. Black circles denote leader cells, which can secrete new FN, red cells signify follower non-secretory cells, and blue squares correspond to FN puncta. Arrows denote cell velocities or fiber orientations. The extra column of FN at the right boundary is an artifact of visualization. Sobol indices (C) and scatter plots (D) of the horizontal distance traveled by NCCs indicate that this metric is most sensitive to the haptotaxis-contact guidance weight, χ, and the filopodial length, Rfilo , but is less dependent on parameters related to cell-cell repulsion (ci). Statistical significance in (C) is determined from a two-sample t-test that compares the Sobol indices produced by the parameter of interest to those obtained from a dummy parameter (red asterisks indicate significant first-order indices, black asterisks indicate significant total order indices, p<0.01). Each data point in (D) represents the average of 20 ABM realizations. The cell-repulsion (resp. cell-ECM) force accounts for the collective interactions that an agent experiences from neighboring NCCs (resp. FN puncta and fibers). The magnitude of the total cell-cell repulsion force is determined from the sum of radially oriented forces from all neighboring NCCs, whose strength, modulated by a user-defined constant, ci , decreases quadratically with respect to the distance from the agent center of mass. To represent the finding that chick cranial NCCs do not always repel each other upon contact (Kulesa and Fraser, 1998; Kulesa et al., 2004), we adjust the direction of the resultant force by stochastically sampling from a von Mises distribution (Mardia and Jupp, 1999). The parameters of the distribution depend on the number and location of NCCs sensed by the agent. They ensure it is uniform when few NCCs are present but cause it to resemble a periodic normal distribution biased in the direction of lowest NCC density when many cells are sensed (for details, see the Materials and methods section). Thus, when cells sense each other at longer ranges, they may align. When cells are close enough to be overlapping, however, they are more likely to repel each other along the direction of contact, similarly to other implementations of cell-cell repulsion (Colombi et al., 2020). We have verified that increasing the magnitude of the cell-cell repulsion forces increases the average nearest neighbor distance between cells in the ABM (Figure 2—figure supplement 5; Figure 3—figure supplement 4). The magnitude of the total cell-ECM force is similarly determined by summing radially oriented forces originating from neighboring FN puncta and fibers, with the strengths of such forces decreasing quadratically with respect to distance from the agent center. The resultant cell-ECM force direction is determined by a linear combination of signals arising from haptotaxis and contact guidance, which are weighted by a parameter, χ. The haptotaxis and contact guidance cues are both represented as unit vectors. Haptotaxis biases the total cell-ECM force toward increasing FN densities and its cue is sampled from a von Mises distribution whose parameters depend on the number and location of neighboring FN puncta and fibers sensed by the cell. The distribution is biased such that the cell is likely to travel toward the greatest FN concentration sampled. Contact guidance aligns the cell-ECM force along the direction of FN fibers. The unit vector corresponding to this cue is computed from the average orientation of FN fibers sensed by the agent. Cells migrate through an initially isotropic, equi-spaced, square lattice of FN puncta that approximates the matrix distribution prior to NCC migration. Leader cells are represented by a fixed number of ‘secretory’ cells that generate new FN puncta at their centers according to times drawn from an exponential distribution with user-specified mean. The cells start at the left-hand boundary of the lattice, which we take to represent the section of the neural tube located along r4. Non-secretory cells, which cannot create new FN puncta, enter the domain at later times, provided sufficient space is available. We note that the terms ‘secretory’ and ‘non-secretory’ refer only to the ability of cells to secrete new FN puncta, as both cell types can create and align fibers emanating from puncta they pass over. Observations of individual ABM simulations indicate that ECM remodeling, haptotaxis, contact guidance, and cell-cell repulsion can generate stream-like patterns, with secretory cells able to maintain their positions at the front of the stream (Figure 2A; Figure 2—video 1). Cells can migrate the FN lattice is isotropic, and trailing cell velocities are oriented toward leader cells (for the average that the follower cell vector with the horizontal is or for the in Figure patterns most ECM fibers to the horizontal that is, the direction of the target corridor (Figure 2—figure supplements Figure 2—video NCCs do not always maintain a single mass as they migrate collectively. In ABM streams into two or more that regions along the (Figure Figure 2 and 4). there is signal NCCs in a direction to the horizontal Thus, in cells may sense and travel toward FN puncta located to the target corridor. We conclude that additional signals are required to NCC stream In later we how such signals affect the of patterns by NCCs. Global sensitivity analysis suggests a key role for contact guidance in long-distance NCC migration To determine how collective migration depends on ABM parameters, we sensitivity et al., to for collective migration. This analysis Sobol indices (Figure which the by which the of a is to changes in a parameter of interest values indicate that the is more sensitive to the The analysis produces two types of Sobol first-order indices, which the of to a parameter of and indices, which include additional that arise when other parameters are The resulting Sobol indices indicate that the distance NCCs travel in the horizontal direction along the target is most sensitive to χ, the parameter the to which NCC directional migration in response to the FN matrix is by haptotaxis contact guidance or a linear combination of the the of this parameter decreases the distance cells travel The cell filopodial Rfilo , the Sobol indices and a increasing with the This as increasing the number of FN puncta and fibers that the cell be to generate more and migration in the The parameter for the cell-cell repulsion force strength, ci , presents a but significant first-order analysis of its scatter (Figure reveals that when contact guidance the cell-ECM force increasing the cell-cell repulsion decreases the distance that the streams migrate When haptotaxis the direction of the force this is but remains decreasing the distance traveled by NCCs is most sensitive to the mechanism by which cells respond to the with contact guidance longer while haptotaxis and cell-cell repulsion are with the distance that NCCs We results for a the extent of the migrating stream in directions to the target that the lateral of cells in the ABM from the target corridor (Figure 2—figure supplement we refer to this as the The cell-ECM weight, χ, the Sobol indices for this and a that lateral migration increases with of contact guidance. By contrast, scatter plots suggest this is with the filopodial and cell-cell repulsion of Sobol indices for the average distance between neighboring cells reveals the importance of haptotaxis and cell-cell repulsion on cell The cell-ECM and the cell-cell repulsion parameter both significant first-order Sobol indices (Figure 2—figure supplement with the latter parameter the Sobol is significant for the filopodial which that this parameter cell-cell separation via its interactions with the other two This that the cell-ECM and cell-cell repulsion forces are by changing the number of FN puncta and NCCs plots (Figure 2—figure supplement suggest a decreasing between the nearest neighbor distance and the cell-ECM weight, a increasing with the cell-cell repulsion parameter, ci and for the filopodial Leading NCCs collective migration by and remodeling ECM To identify combinations of parameters that collective migration, we simulated experimental that affect cell of new FN (Figure see Materials and while cell of fibers from puncta When leader secretory cells secrete FN puncta more we find that the stream by By contrast, cell new FN puncta, then the average distance the stream decreases by about migration cells can respond to both cell types secrete FN, such that there is between cell then the average distance migrated by cells decreases from the of at the rate or is Similar results for the lateral of NCCs (Figure 3—figure supplement 1). Figure with 4 supplements see all Download asset Open asset distance traveled by neural crest cells (NCCs) after 12 hr. plots for experiments in which (A) fibronectin (FN) is (B) and fiber are and (C) cells invade a FN lattice suggest that the extracellular matrix (ECM) plays an important role in NCC migration. experiments decrease the average over which a cell new FN from to 10 Contact guidance and cell-cell repulsion in (C) correspond to and indicate are different from that of the parameter using a agent-based model (ABM) realizations are to generate each We how cells alter the FN by both FN and fiber in leader follower cells (Figure When leading secretory cells both cell are to remodel the the average distance traveled by the NCC stream decreases by (resp. When follower ‘non-secretory’ cells are from and FN fibers, however, the is with only a reduction in distance Similar results are for the nearest neighbor distance and the lateral of NCCs (Figure 3—figure supplement 2). These results demonstrate within the FN remodeling by leading NCCs plays a key role in long-distance collective migration. By contrast, are when trailing cells cannot remodel the Our suggest that collective migration may be more when leading and trailing NCCs with leading cells remodeling the ECM. The FN matrix distribution is to long-distance collective cell migration ECM remodeling can direct collective cell migration, but the to which the ECM cell trajectories remains unclear. the lattice between FN puncta from 20 to µm collective migration in the decreasing the distance that the stream by The lateral of NCCs is similarly reduced within (Figure 3—figure supplement We conclude that NCCs are less likely to sense FN in their local then they are less likely to migrate as the nearest neighbor distance between cells decreases (Figure 3—figure supplement an cell density and state a in which cells are tightly to movement, and a (Sadati et al., 2013). These lead us to cells in the ABM as in a ‘jammed’ state when they collectively travel less µm over 12 as we have found a but significant between the nearest neighbor distance and the distance traveled in the horizontal direction (Figure 2—figure supplement Figure 3—figure supplement 4). Cells however, mechanisms to for FN and long-distance collective migration (Figure contact guidance is in the ABM the decreases from to then the NCC migration distance in FN increases by The distance that obtained in the FN lattice by the cell-cell repulsion or the rate at which secretory cells produce FN also cause NCCs to travel distances within the FN matrix (by and but changes do not migration to distances to those in These results highlight the importance of contact guidance, ECM remodeling, cell-cell repulsion in long-distance migration, when cells are in a state migration is otherwise directional guidance cells along their target but the model sensitive to stream and cell separation mechanisms that generate collective migration do not that cells travel along a single

Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform

The present study includes proof concept of morphology-based cell separation and development of automatic cell separation system. 3D culture environment with a specific extracellular matrix regulates cellular function and phenotype. In addition, cancer cell morphology changes depending on its malignancy in 3D culture environment. The cell separation system in 3D culture environment should need to obtain the cells according to its morphology, which includes cell phenotypes. Recently, we developed gelatin-based photodegradable hydrogels, and applied this hydrogels to optical cell separation. The target cells in the photodegradable hydrogels were successfully separated by the optical cell separation, the separated cells was growth on another dish. On the other hand, we recently developed the predication model of stem cell differentiation by image analysis. The image analysis technique and the photodegradable gelatin hydrogels are included in the automated morphology-based cell separation system in the 3D culture environment. For forming cell encapsulated-photodegradable hydrogels, suspension of cells including heterogeneous population is mixed with pregel solutions and cells are encapsulated in the gelatin-based photodegradable hydrogels. After the culture in 3D environment, microscopic images of the cells are captured. The captured images are analyzed to distinguish the target cells from the other cells by using the image analysis algorithm, which we previously developed for analyzing stem cells. The hydrogels around the target area is irradiated with light (365nm). The cells in the irradiated area are collected by automated pipetting system. We developed automated system for this optical cell separation procedure, including cultivation, image acquisition, image analysis, light irradiation, and pipetting for cell collection. We demonstrated automated optical cell separation using the model culture system. Normal gastric mucosal cells were cultured in the photodegradable hydrogels. After cultivation for 1 week, the cells were irradiated the light for 5 to 20 min. The cells in the irradiated area were collected by automated pipetting and transferred into a collection dish. The collected cells were viable and attached in the collection dish after collection. We are currently developing an automated image analysis algorithm to distinguish cancer cells from normal cells under 3D environment. The automated optical cell separation system with image analysis algorithm will be applied to the establishment of novel cancer-cell lines from clinical samples such as biopsy tissue. Citation Format: Hirofumi Matsui, Shinji Sugiura, Masato Tamura, Toshiyuki Kanamori, Toshiyuki Takagi, Taku Satou, Ryuji Kato, Kei Kanie, Mayu Shibuta. Development of cellular morphology-based separation system for three-dimensional culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5788. doi:10.1158/1538-7445.AM2017-5788

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.

A Key Role for CC Chemokine Receptor 1 in T-Cell-Mediated Respiratory Inflammation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Resident dendritic cells are the predominant TNF-secreting cell in early renal ischemia–reperfusion injury