Ratchetaxis in channels: cells move directionally by pushing walls asymmetrically

AbstractCell motility is essential in a variety of biological phenomena ranging from early development to organ homeostasis and diseases. This phenomenon was so far mainly studied and characterized on flat surfaces in vitro whereas this situation is rarely seen in vivo. Recently, cell motion in 3D microfabricated channels was reported to be possible, and it was shown that confined cells push on walls. However, rules setting cell directions in this context were not characterized yet. Here, we show by using assays that ratchetaxis operates in 3D ratchets on fibroblasts and on epithelial cancerous cells. Open ratchets rectify cell motion, whereas closed ratchets impose a direct cell migration along channels set by the cell orientation at the channel entry point. We also show that nuclei are pressed at constrictions zones through mechanisms involving dynamic asymmetries of focal contacts, stress fibers, and intermediate filaments. Interestingly, cells do not pass these constricting zones when defective in the keratin fusion implicated in squamous cancer. By combining ratchetaxis with chemical gradients, we finally report that cells are sensitive to local asymmetries in confinement and that topological and chemical cues may be encoded differently by cells. Altogether our ratchet channels could mimic small blood vessels where cells are confined: cells would probe local asymmetries which would determine their entry into tissues and direction. Our results could shed light on invasions mechanisms in cancer.

Download Full-text

Generation of fluorescent cell-derived-matrix to study 3D cell migration

10.1101/746941 ◽

2019 ◽

Author(s):

Amélie Luise Godeau ◽

Hélène Delanoë-Ayari ◽

Daniel Riveline

Keyword(s):

Cell Migration ◽

Flat Surfaces ◽

Polymeric Networks ◽

Motile Cells ◽

Matrix Deformation ◽

3D Cell Migration ◽

Cell Motion ◽

Cellular Forces

AbstractCell migration is involved in key phenomena in biology, ranging from development to cancer. Fibroblasts move between organs in 3D polymeric networks. So far, motile cells were mainly tracked in vitro on Petri dishes or on coverslips, i.e. 2D flat surfaces, which made the extrapolation to 3D physiological environments difficult. We therefore prepared 3D Cell Derived Matrix (CDM) with specific characteristics with the goal of extracting the main readouts required to measure and characterise cell motion: cell specific matrix deformation through the tracking of fluorescent fibronectin within CDM, focal contacts as the cell anchor and acto-myosin cytoskeleton which applies cellular forces. We report our method for generating this assay of physiological-like gel with relevant readouts together with its potential impact in explaining cell motility in vivo.

Download Full-text

Pan-ERBB kinase inhibition augments CDK4/6 inhibitor efficacy in oesophageal squamous cell carcinoma

Gut ◽

10.1136/gutjnl-2020-323276 ◽

2021 ◽

pp. gutjnl-2020-323276

Author(s):

Jin Zhou ◽

Zhong Wu ◽

Zhouwei Zhang ◽

Louisa Goss ◽

James McFarland ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Large Scale ◽

Cell Cancer ◽

Oesophageal Squamous Cell Carcinoma ◽

Squamous Cancer ◽

Striking Success

ObjectiveOesophageal squamous cell carcinoma (OSCC), like other squamous carcinomas, harbour highly recurrent cell cycle pathway alterations, especially hyperactivation of the CCND1/CDK4/6 axis, raising the potential for use of existing CDK4/6 inhibitors in these cancers. Although CDK4/6 inhibition has shown striking success when combined with endocrine therapy in oestrogen receptor positive breast cancer, CDK4/6 inhibitor palbociclib monotherapy has not revealed evidence of efficacy to date in OSCC clinical studies. Herein, we sought to elucidate the identification of key dependencies in OSCC as a foundation for the selection of targets whose blockade could be combined with CDK4/6 inhibition.DesignWe combined large-scale genomic dependency and pharmaceutical screening datasets with preclinical cell line models, to identified potential combination therapies in squamous cell cancer.ResultsWe identified sensitivity to inhibitors to the ERBB family of receptor kinases, results clearly extending beyond the previously described minority of tumours with EGFR amplification/dependence, specifically finding a subset of OSCCs with dual dependence on ERBB3 and ERBB2. Subsequently. we demonstrated marked efficacy of combined pan-ERBB and CDK4/6 inhibition in vitro and in vivo. Furthermore, we demonstrated that squamous lineage transcription factor KLF5 facilitated activation of ERBBs in OSCC.ConclusionThese results provide clear rationale for development of combined ERBB and CDK4/6 inhibition in these cancers and raises the potential for KLF5 expression as a candidate biomarker to guide the use of these agents. These data suggested that by combining existing Food and Drug Administration (FDA)-approved agents, we have the capacity to improve therapy for OSCC and other squamous cancer.

Download Full-text

Efficient Therapeutic Delivery by a Novel Cell-Penetrating Peptide Derived from Acinus

Cancers ◽

10.3390/cancers12071858 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1858

Author(s):

Justine Habault ◽

Claire Fraser ◽

Ewa Pasquereau-Kotula ◽

Maëlys Born-Bony ◽

Anne Marie-Cardine ◽

...

Keyword(s):

Ex Vivo ◽

Clinical Settings ◽

Aspartic Acid Residue ◽

Circulating Cells ◽

C Terminus ◽

Cell Penetrating ◽

Endocytosis Pathway ◽

Cancerous Cells

In this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an aspartic acid residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (residues 377–399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sézary patients’ circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sézary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings.

Download Full-text

Requirements for the Manufacturing of Scaffold Biomaterial With Features at Multiple Scales

Manufacturing Engineering and Materials Handling, Parts A and B ◽

10.1115/imece2005-82515 ◽

2005 ◽

Author(s):

I. M. Sebastine ◽

D. J. Williams

Keyword(s):

Tissue Engineering ◽

Multiple Scales ◽

Cell Attachment ◽

Structural Parameters ◽

Complex Function ◽

Three Dimensional ◽

Cell Orientation ◽

Length Scales

Tissue engineering aims to restore the complex function of diseased tissue using cells and scaffold materials. Tissue engineering scaffolds are three-dimensional (3D) structures that assist in the tissue engineering process by providing a site for cells to attach, proliferate, differentiate and secrete an extra-cellular matrix, eventually leading cells to form a neo-tissue of predetermined, three-dimensional shape and size. For a scaffold to function effectively, it must possess the optimum structural parameters conducive to the cellular activities that lead to tissue formation; these include cell penetration and migration into the scaffold, cell attachment onto the scaffold substrate, cell spreading and proliferation and cell orientation. In vivo, cells are organized in functional tissue units that repeat on the order of 100 μm. Fine scaffold features have been shown to provide control over attachment, migration and differentiation of cells. In order to design such 3D featured constructs effectively understanding the biological response of cells across length scales from nanometer to millimeter range is crucial. Scaffold biomaterials may need to be tailored at three different length scales: nanostructure (<1μm), microstructure (<20–100μm), and macrostructure (>100μm) to produce biocompatible and biofunctional scaffolds that closely resemble the extracellular matrix (ECM) of the natural tissue environment and promote cell adhesion, attachment, spreading, orientation, rate of movement, and activation. Identification of suitable fabrication techniques for manufacturing scaffolds with the required features at multiple scales is a significant challenge. This review highlights the effect and importance of the features of scaffolds that can influence the behaviour of cells/tissue at different length scales in vitro to increase our understanding of the requirements for the manufacture of functional 3D tissue constructs.

Download Full-text

Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101) and The novel Bcl-2 selective Inhibitor GDC-0199 Results in superior In Vitro and In Vivo Anti-tumor activity in models Of B-Cell Malignancies

Blood ◽

10.1182/blood.v122.21.4412.4412 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4412-4412 ◽

Cited By ~ 4

Author(s):

Deepak Sampath ◽

Sylvia Herter ◽

Frank Herting ◽

Ellen Ingalla ◽

Michelle Nannini ◽

...

Keyword(s):

Cell Death ◽

Single Agent ◽

Employment Equity ◽

Direct Cell ◽

Xenograft Models ◽

Equity Ownership ◽

Anti Cd20 ◽

Agent Treatment

Introduction Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcgRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in CLL, indolent NHL and DLCBL. ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and MM. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl-2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. Experimental Methods The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2, SU-DHL4 DLBCL and Z138 MCL cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. Results GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC-0199 and GA101 induced a greater than additive anti-tumor effects in the SU-DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Moreover, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 model. Conclusions Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to the combination of rituximab with GDC-0199. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively the preclinical data presented here supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently in a phase Ib clinical trial (clinical trial.gov identifier NCT01685892). Disclosures: Sampath: Genentech: Employment, Equity Ownership. Herter:Roche: Employment. Herting:Roche: Employment. Ingalla:Genentech: Employment. Nannini:Genentech: Employment. Bacac:Roche: Employment. Fairbrother:Genentech: Employment, Equity Ownership. Klein:Roche Glycart AG: Employment.

Download Full-text

A Graph-Based Framework for Multiscale Modeling of Physiological Transport

Frontiers in Network Physiology ◽

10.3389/fnetp.2021.802881 ◽

2022 ◽

Vol 1 ◽

Author(s):

M. Deepa Maheshvare ◽

Soumyendu Raha ◽

Debnath Pal

Keyword(s):

Physical Space ◽

Whole Body ◽

Dispersive Transport ◽

Modeling Framework ◽

Biologically Relevant ◽

Transport Dynamics ◽

Biological Phenomena ◽

Function Relationship

Trillions of chemical reactions occur in the human body every second, where the generated products are not only consumed locally but also transported to various locations in a systematic manner to sustain homeostasis. Current solutions to model these biological phenomena are restricted in computability and scalability due to the use of continuum approaches in which it is practically impossible to encapsulate the complexity of the physiological processes occurring at diverse scales. Here, we present a discrete modeling framework defined on an interacting graph that offers the flexibility to model multiscale systems by translating the physical space into a metamodel. We discretize the graph-based metamodel into functional units composed of well-mixed volumes with vascular and cellular subdomains; the operators defined over these volumes define the transport dynamics. We predict glucose drift governed by advective–dispersive transport in the vascular subdomains of an islet vasculature and cross-validate the flow and concentration fields with finite-element–based COMSOL simulations. Vascular and cellular subdomains are coupled to model the nutrient exchange occurring in response to the gradient arising out of reaction and perfusion dynamics. The application of our framework for modeling biologically relevant test systems shows how our approach can assimilate both multi-omics data from in vitro–in vivo studies and vascular topology from imaging studies for examining the structure–function relationship of complex vasculatures. The framework can advance simulation of whole-body networks at user-defined levels and is expected to find major use in personalized medicine and drug discovery.

Download Full-text

Tendon-derived biomimetic surface topographies induce phenotypic maintenance of tenocytes in vitro

10.1101/2020.07.23.217224 ◽

2020 ◽

Author(s):

Aysegul Dede Eren ◽

Aliaksei Vasilevich ◽

E. Deniz Eren ◽

Phanikrishna Sudarsanam ◽

Urandelger Tuvshindorj ◽

...

Keyword(s):

Gene Expression ◽

Cell Shape ◽

Flat Surface ◽

Flat Surfaces ◽

Tissue Culture Polystyrene ◽

Surface Topographies ◽

And Function ◽

Shape Changes

AbstractThe tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shape in vivo and in vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for seven days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype and function of tendon-related cells.

Download Full-text

A High-Throughput Device for Patterned Differentiation of Embryoid Bodies

Journal of Robotics and Mechatronics ◽

10.20965/jrm.2013.p0623 ◽

2013 ◽

Vol 25 (4) ◽

pp. 623-630 ◽

Cited By ~ 2

Author(s):

Xiaoming He ◽

◽

Hiroshi Kimura ◽

Teruo Fujii ◽

◽

...

Keyword(s):

Microfluidic Device ◽

Embryoid Body ◽

Embryoid Bodies ◽

Culture Methods ◽

Theoretical Simulation ◽

Chemical Gradients ◽

Conventional Culture ◽

Differentiation And Proliferation

Although ontogenesis in vivo may proceed in a spatiotemporally heterogeneous environment, in vitro differentiation of an embryoid body (EB) has been carried out in uniform conditions using conventional culture methods at low throughput. In the present study, a microfluidic device with multiple culture chambers for simultaneous patterned differentiation of multiple EBs of pluripotent stem cells is newly developed. Theoretical simulation and experiments using a suspension of fluorescent particles or fluorescent solution show that proper chemical gradients can be formed with almost no flow in the chambers. After multiple EBs are introduced into the device, these EBs move along the flow channel and into trapping cups. The EBs are pushed by air bubbles into the culture chambers. These multiple EBs can be cultured within the culture chambers after flowing culture medium removes the air bubble from the device. In our experiment, differentiation and proliferation of these multiple EBs are studied by exposing them to two different media for 6 days: one to induce differentiation and the other to keep the pluripotent and self-renewing state of the cells. It is shown that patterned differentiation of the multiple EBs is successfully conducted simultaneously in the device when these two media are perfused into the device. The results suggest that differentiation and proliferation of multiple EBs can be analyzed by applying chemical gradients in the present microfluidic device. This will be a helpful tool in a wide variety of experiments involving EBs or spheroids.

Download Full-text

“Microenvironmental contaminations” induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking

Blood ◽

10.1182/blood-2009-05-224030 ◽

2010 ◽

Vol 115 (26) ◽

pp. 5347-5354 ◽

Cited By ~ 84

Author(s):

Francois Lassailly ◽

Emmanuel Griessinger ◽

Dominique Bonnet

Keyword(s):

Gene Transfer ◽

Cell Tracking ◽

Near Infrared ◽

Cross Validation ◽

Cell Types ◽

Optical Tracking ◽

Direct Cell ◽

Marked Cell

Abstract Determining how normal and leukemic stem cells behave in vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer–free labeling technologies. The lipophilic dye 3,3,3′,3′ tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine perchlorate [DiI], DiD, DiR, and PKH26) can give rise to microenvironmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non–genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.

Download Full-text