bims-ecemfi 2024-08-11 papers

bims-ecemfi

Biomed News

on ECM and fibroblasts

Issue of 2024–08–11
eleven papers selected by
Badri Narayanan Narasimhan, University of California, San Diego

Structurally decoupled hyaluronic acid hydrogels for studying matrix metalloproteinase-mediated invasion of metastatic breast cancer cells.
Nucleocytoplasmic transport senses mechanics independently of cell density in cell monolayers.
Focal adhesion-mediated directional cell migration guided by gradient-stretched substrate.
On the generation of force required for actin-based motility.
PyFMLab: Open-source software for atomic force microscopy microrheology data analysis.
Adiponectin suppresses stiffness-dependent, pro-fibrotic activation of lung fibroblasts.
Exploring doxorubicin transport in 2D and 3D models of MDA-MB-231 sublines: impact of hypoxia and cellular heterogeneity on doxorubicin accumulation in cells.
Cell shape and orientation control galvanotactic accuracy.
Engineered Nanofibrillar Collagen with Tunable Biophysical Properties for Myogenic, Endothelial, and Osteogenic Cell Guidance.
Single-cell transcriptome profiles the heterogeneity of tumor cells and microenvironments for different pathological endometrial cancer and identifies specific sensitive drugs.
Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.

Int J Biol Macromol. 2024 Aug 05. pii: S0141-8130(24)05298-X. [Epub ahead of print] 134493

Structurally decoupled hyaluronic acid hydrogels for studying matrix metalloproteinase-mediated invasion of metastatic breast cancer cells.

Kasra Goodarzi, Shreyas S Rao.

  In recent years, polymeric hydrogels have been employed to investigate cancer cell-extracellular matrix (ECM) interactions in vitro. In the context of breast cancer, cancer cells are known to degrade the ECM using matrix-metalloproteinases (MMPs) to support invasion resulting in disease progression. Polymeric hydrogels incorporating MMP-cleavable peptides have been employed to study cancer cell invasion, however, the approaches employed to incorporate these peptides often change other hydrogel properties. This underscores the need for decoupling hydrogel properties while incorporating MMP-cleavable peptides. Herein, we report structurally decoupled hyaluronic acid (HA) hydrogels formulated using varying ratios of a biologically sensitive MMP-cleavable peptide and an insensitive counterpart (Dithiothreitol (DTT) or polyethylene glycol dithiol (PEGDT)) to study MMP-mediated metastatic breast cancer cell invasion. Rheological, swelling ratio, estimated mesh size, and permeability measurements showed similar mechanical and physical properties for hydrogels crosslinked with different DTT (or PEGDT)/MMP ratios. However, their degradation rate in the presence of collagenase correlated with the ratio of MMP-cleavable peptide. Encapsulated metastatic breast cancer spheroids in HA hydrogels with MMP sensitivity exhibited increased invasiveness compared to those without MMP sensitivity after 14 days of culture. Overall, such structurally decoupled HA hydrogels provide a platform to study MMP-mediated breast cancer cell invasion in vitro.

Keywords:  Breast cancer metastasis; Crosslinking; Hyaluronic acid; Hydrogel; Invasion; Spheroids

DOI:  https://doi.org/10.1016/j.ijbiomac.2024.134493
J Cell Sci. 2024 Aug 09. pii: jcs.262363. [Epub ahead of print]

Nucleocytoplasmic transport senses mechanics independently of cell density in cell monolayers.

Ignasi Granero-Moya, Valeria Venturini, Guillaume Belthier, Bart Groenen, Marc Molina-Jordán, Miguel González-Martín, Xavier Trepat, Jacco van Rheenen, Ion Andreu, Pere Roca-Cusachs.

  Cells sense and respond to mechanical forces through mechanotransduction, which regulates processes in health and disease. In single adhesive cells, mechanotransduction involves the transmission of force from the extracellular matrix to the cell nucleus, where it affects nucleocytoplasmic transport (NCT) and the subsequent nuclear localization of transcriptional regulators such as YAP. However, if and how NCT is mechanosensitive in multicellular systems is unclear. Here, we characterize and use a fluorescent sensor of nucleocytoplasmic transport (Sencyt) and demonstrate that nucleocytoplasmic transport responds to mechanics but not cell density in cell monolayers. Using monolayers of both epithelial and mesenchymal phenotype, we show that NCT is altered in response both to osmotic shocks, and to the inhibition of cell contractility. Further, NCT correlates with the degree of nuclear deformation measured through nuclear solidity, a shape parameter related to nuclear envelope tension. In contrast, YAP but NCT is sensitive to cell density, showing that YAP response to cell-cell contacts is not via a mere mechanical effect of NCT. Our results demonstrate the generality of the mechanical regulation of NCT.

Keywords:  Cell nucleus; Mechanobiology; Mechanotransduction; Sensor

DOI:  https://doi.org/10.1242/jcs.262363
iScience. 2024 Aug 16. 27(8): 110446

Focal adhesion-mediated directional cell migration guided by gradient-stretched substrate.

Zijia Chen, Xiaoning Han, Bo Che, Huiping Feng, Yue Zhou, Linhong Deng, Xiang Wang.

  Soft tissues experience strain under mechanical stresses, storing energy as residual stresses and strain energy. However, the specific impact of such strain on cell migration and its molecular mechanisms remains unclear. In this study, we investigated this by using polydimethylsiloxane (PDMS) membranes with varying prestrain levels but constant stiffness to mimic tissue-like conditions. Results showed that higher prestrain levels enhanced 3T3 fibroblast adhesion and reduced filopodia formation. Elevated prestrain also increased integrin and vinculin expression, which was associated with lower cell migration rates. Notably, both 3T3 fibroblasts and primary rat airway smooth muscle cells migrated faster toward higher prestrain areas on substrates with strain gradients. Knockdown of integrin or vinculin inhibited 3T3 cell migration directionality, highlighting their critical role. This research reveals a mechanobiological pathway where strain gradients direct cell migration, providing insight into a common mechanotransduction pathway influencing cellular responses to both stiffness and strain-related mechanical cues.

Keywords:  Haematology; Molecular biology; Transcriptomics

DOI:  https://doi.org/10.1016/j.isci.2024.110446
Sci Rep. 2024 Aug 08. 14(1): 18384

On the generation of force required for actin-based motility.

Alberto Salvadori, Claudia Bonanno, Mattia Serpelloni, Robert M McMeeking.

  The fundamental question of how forces are generated in a motile cell, a lamellipodium, and a comet tail is the subject of this note. It is now well established that cellular motility results from the polymerization of actin, the most abundant protein in eukaryotic cells, into an interconnected set of filaments. We portray this process in a continuum mechanics framework, claiming that polymerization promotes a mechanical swelling in a narrow zone around the nucleation loci, which ultimately results in cellular or bacterial motility. To this aim, a new paradigm in continuum multi-physics has been designed, departing from the well-known theory of Larché-Cahn chemo-transport-mechanics. In this note, we set up the theory of network growth and compare the outcomes of numerical simulations with experimental evidence.

Keywords:  Actin-based motility; Chemo-transport-mechanics; Continuum mechanics; Finite elements; High performance computing

DOI:  https://doi.org/10.1038/s41598-024-69422-3
Open Res Eur. 2023 ;3 187

PyFMLab: Open-source software for atomic force microscopy microrheology data analysis.

Javier López-Alonso, Mar Eroles, Sébastien Janel, Massimiliano Berardi, Jean-Luc Pellequer, Vincent Dupres, Frank Lafont, Felix Rico.

   Background: Atomic force microscopy (AFM) is one of the main techniques used to characterize the mechanical properties of soft biological samples and biomaterials at the nanoscale. Despite efforts made by the AFM community to promote open-source data analysis tools, standardization continues to be a significant concern in a field that requires common analysis procedures. AFM-based mechanical measurements involve applying a controlled force to the sample and measure the resulting deformation in the so-called force-distance curves. These may include simple approach and retract or oscillatory cycles at various frequencies (microrheology). To extract quantitative parameters, such as the elastic modulus, from these measurements, AFM measurements are processed using data analysis software. Although open tools exist and allow obtaining the mechanical properties of the sample, most of them only include standard elastic models and do not allow the processing of microrheology data. In this work, we have developed an open-source software package (called PyFMLab, as of python force microscopy laboratory) capable of determining the viscoelastic properties of samples from both conventional force-distance curves and microrheology measurements.
Methods: PyFMLab has been written in Python, which provides an accessible syntax and sufficient computational efficiency. The software features were divided into separate, self-contained libraries to enhance code organization and modularity and to improve readability, maintainability, testability, and reusability. To validate PyFMLab, two AFM datasets, one composed of simple force curves and another including oscillatory measurements, were collected on HeLa cells.
Results: The viscoelastic parameters obtained on the two datasets analysed using PyFMLab were validated against data processing proprietary software and against validated MATLAB routines developed before obtaining equivalent results.
Conclusions: Its open-source nature and versatility makes PyFMLab an open-source solution that paves the way for standardized viscoelastic characterization of biological samples from both force-distance curves and microrheology measurements.

Keywords:  Biological Samples; Elasticity; Viscoelasticity; Young’s modulus; cell mechanics; force spectroscopy; soft matter; tissue mechanics

DOI:  https://doi.org/10.12688/openreseurope.16550.1
Am J Physiol Lung Cell Mol Physiol. 2024 Aug 06.

Adiponectin suppresses stiffness-dependent, pro-fibrotic activation of lung fibroblasts.

Julia Nemeth, Wioletta Skronska-Wasek, Sophie Keppler, Annika Schundner, Alexander Groß, Tanja Schönberger, Karsten Quast, Karim C El Kasmi, Clemens Ruppert, Andreas Günther, Manfred Frick.

  Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible respiratory disease with limited therapeutic options. A hallmark of IPF is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We performed an unbiased, next generation sequencing (NGS) screen to identify signaling pathways involved in stiffness-dependent lung fibroblast activation. Adipocytokine signaling was downregulated in primary lung fibroblasts (PFs) cultured on stiff matrices. Re-activating adipocytokine signaling with adiponectin suppressed stiffness-dependent activation of human PFs. Adiponectin signaling depended on CDH13 expression and p38 mitogen-activated protein kinase gamma (p38MAPKγ) activation. CDH13 expression and p38MAPKγ activation were strongly reduced in lungs from IPF donors. Our data suggest that adiponectin-signaling via CDH13 and p38MAPKγ activation suppresses pro-fibrotic activation of fibroblasts in the lung. Targeting of the adiponectin signaling cascade may provide therapeutic benefits in IPF.

Keywords:  Idiopathic pulmonary fibrosis; T-cadherin; adiponectin; mechano-signaling; p38 MAPK

DOI:  https://doi.org/10.1152/ajplung.00037.2024
Am J Cancer Res. 2024 ;14(7): 3584-3599

Exploring doxorubicin transport in 2D and 3D models of MDA-MB-231 sublines: impact of hypoxia and cellular heterogeneity on doxorubicin accumulation in cells.

Indrė Januškevičienė, Vilma Petrikaitė.

  Triple-negative breast cancer (TNBC) treatment is challenging due to its aggressive nature and heterogeneity of this type of cancer, characterized by various subtypes and intratumoral diversity. Doxorubicin (DOX) plays a crucial role in TNBC chemotherapy reducing the tumor size and improving patient survival. However, decreased drug uptake and increased resistance in specific cell subpopulations reduce the effectiveness of the treatment. This study explored the differences in DOX transport in MDA-MB-231 phenotypic sublines in cell monolayer (2D model) and cell spheroids (3D cultures). Cell spheroids were formed using magnetic 3D Bioprinting method. DOX transport into cells and spheroids was evaluated using fluorescence microscopy after different incubation durations with DOX in normoxia and hypoxia. In hypoxia, DOX transport into cells was 2.5 to 5-fold lower than in normoxia. The subline F5 monolayer-cultured cells exhibited the highest DOX uptake, while subline H2 cells showed the lowest uptake in normoxia and hypoxia. In 3D cultures, DOX transport was up to 2-fold lower in spheroids formed from subline H2 cells. Spheroids from subline D8 and MDA-MB-231 parent cells had the highest DOX uptake. A correlation was observed between the characteristics of the cells and their resistance to anticancer drugs. The results indicate that different cancer cell subpopulations in tumours due to differences in drug uptake could significantly impact treatment efficacy.

Keywords:  Heterogeneity; cell sublines; doxorubicin transport; drug resistance; hypoxia; tumor spheroids

DOI:  https://doi.org/10.62347/VNWH9165
ArXiv. 2024 Jul 26. pii: arXiv:2407.17420v2. [Epub ahead of print]

Cell shape and orientation control galvanotactic accuracy.

Ifunanya Nwogbaga, Brian A Camley.

Galvanotaxis is believed to be driven by the redistribution of transmembrane proteins and other molecules, referred to as "sensors", through electrophoresis and electroosmosis. Here, we update our previous model of the limits of galvanotaxis due to stochasticity of sensor movements to account for cell shape and orientation. Computing the Fisher information, we find that cells in principle possess more information about the electric field direction when their long axis is parallel to the field, but that for weak fields maximum-likelihood estimators of the field direction may actually have lower variability when the cell's long axis is perpendicular to the field. In an alternate possibility, we find that if cells instead estimate the field direction by taking the average of all the sensor locations as its directional cue ("vector sum"), this introduces a bias towards the short axis, an effect not present for isotropic cells. We also explore the possibility that cell elongation arises downstream of sensor redistribution. We argue that if sensors migrate to the cell's rear, the cell will expand perpendicular the field - as is more commonly observed - but if sensors migrate to the front, the cell will elongate parallel to the field.
Acta Biomater. 2024 Aug 06. pii: S1742-7061(24)00444-6. [Epub ahead of print]

Engineered Nanofibrillar Collagen with Tunable Biophysical Properties for Myogenic, Endothelial, and Osteogenic Cell Guidance.

Yong How Tan, Krista M Habing, Jessica L Riesterer, Erin S Stempinksi, Steven H Lewis, Carmem S Pfeifer, Sanjay V Malhotra, Karina H Nakayama.

  A goal of regenerative engineering is the rational design of materials to restore the structure-function relationships that drive reparative programs in damaged tissues. Despite the widespread use of extracellular matrices for engineering tissues, their application has been limited by a narrow range of tunable features. The primary objective of this study is to develop a versatile platform for evaluating tissue-specific cellular interactions using Type I collagen scaffolds with highly tunable biophysical properties. The kinetics of collagen fibrillogenesis were modulated through a combination of varied shear rate and pH, during neutralization, to achieve a broad range of fibril anisotropy, porosity, diameter, and storage modulus. The role that each of these properties play in guiding muscle, bone, and vascular cell types was comprehensively identified and informed the in vitro generation of three distinct musculoskeletal engineered constructs. Myogenesis was highly regulated by smaller fibrils and larger storage moduli, endothelial inflammatory phenotype was predominantly guided by fibril anisotropy, and osteogenesis was enhanced by highly porous collagen with larger fibrils. This study introduces a novel approach for dynamically modulating Type I collagen materials and provides a robust platform for investigating cell-material interactions, offering insights for future rational design of tissue-specific regenerative biomaterials. STATEMENT OF SIGNIFICANCE: The biophysical properties of regenerative materials facilitate key cell-substrate interactions that can guide the morphology, phenotype, and biological response of cells. In this study, we describe the fabrication of an engineered collagen hydrogel that can be modified to exhibit control over a wide range of biophysical features including fibril organization and size, nanoscale porosity, and mechanics. We identified the unique combination of collagen features that optimally promote regenerative muscle, bone, and vascular cell types while also delineating the properties that hinder these same cellular responses. This study presents a highly accessible method to control the biophysical properties of collagen hydrogels that can be adapted for a broad range of tissue engineering and regenerative applications.

Keywords:  biophysical modulation; collagen nanopatterning; endothelial cell inflammatory phenotype; myogenesis; osteogenesis

DOI:  https://doi.org/10.1016/j.actbio.2024.08.002
Cell Death Dis. 2024 Aug 07. 15(8): 571

Single-cell transcriptome profiles the heterogeneity of tumor cells and microenvironments for different pathological endometrial cancer and identifies specific sensitive drugs.

Fang Ren, Lingfang Wang, Yuyouye Wang, Jiaxuan Wang, Yuanpei Wang, Xiaole Song, Gong Zhang, Fangfang Nie, Shitong Lin.

Endometrial cancer (EC) is a highly heterogeneous malignancy characterized by varied pathology and prognoses, and the heterogeneity of its cancer cells and the tumor microenvironment (TME) remains poorly understood. We conducted single-cell RNA sequencing (scRNA-seq) on 18 EC samples, encompassing various pathological types to delineate their specific unique transcriptional landscapes. Cancer cells from diverse pathological sources displayed distinct hallmarks labeled as immune-modulating, proliferation-modulating, and metabolism-modulating cancer cells in uterine clear cell carcinomas (UCCC), well-differentiated endometrioid endometrial carcinomas (EEC-I), and uterine serous carcinomas (USC), respectively. Cancer cells from the UCCC exhibited the greatest heterogeneity. We also identified potential effective drugs and confirmed their effectiveness using patient-derived EC organoids for each pathological group. Regarding the TME, we observed that prognostically favorable CD8+ Tcyto and NK cells were prominent in normal endometrium, whereas CD4+ Treg, CD4+ Tex, and CD8+ Tex cells dominated the tumors. CXCL3+ macrophages associated with M2 signature and angiogenesis were exclusively found in tumors. Prognostically relevant epithelium-specific cancer-associated fibroblasts (eCAFs) and SOD2+ inflammatory CAFs (iCAFs) predominated in EEC-I and UCCC groups, respectively. We also validated the oncogenic effects of SOD2+ iCAFs in vitro. Our comprehensive study has yielded deeper insights into the pathogenesis of EC, potentially facilitating personalized treatments for its varied pathological types.

DOI: https://doi.org/10.1038/s41419-024-06960-8
Nat Commun. 2024 Aug 08. 15(1): 6777

Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.

Soeun Kang, Maciek R Antoniewicz, Nissim Hay.

Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.

DOI: https://doi.org/10.1038/s41467-024-51117-y