bims-ecemfi 2024-05-19 papers

bims-ecemfi

Biomed News

on ECM and fibroblasts

Issue of 2024–05–19
twenty-six papers selected by
Badri Narayanan Narasimhan, University of California, San Diego

Mapping the tumor stress network reveals dynamic shifts in the stromal oxidative stress response.
Tailoring of Physical Properties in Macroporous Poly(isocyanopeptide) Cryogels.
Matrix stiffening promotes perinuclear clustering of mitochondria.
Cell Migration Chip (3DCM-chip): a New Tool Toward the Modelling of 3D Cellular Complex Systems.
Myosin-independent stiffness sensing by fibroblasts is regulated by the viscoelasticity of flowing actin.
Fibroblast alignment and matrix remodeling induced by a stiffness gradient in a skin-derived extracellular matrix hydrogel.
Fibroblasts' secretome from calcified and non-calcified dermis in Pseudoxanthoma elasticum differently contributes to elastin calcification.
Tissue flows are tuned by actomyosin-dependent mechanics in developing embryos.
Spatial confinement modulates endothelial cell behavior and traction force in 3D hydrogel microgrooves.
Mechanistic Characterization of Cancer Associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.
Mechano-metabolism of adherent cells in 2D and 3D microenvironments.
An Open-Source Membrane Stretcher for Simultaneous Mechanical and Structural Characterizations of Soft Materials and Biological Tissues.
Directed biomechanical compressive forces enhance fusion efficiency in model placental trophoblast cultures.
Cervical extracellular matrix hydrogel optimizes tumor heterogeneity of cervical squamous cell carcinoma organoids.
Dual-color FISH analyses of xenogeneic human fibroblast sheets transplanted to repair lung pleural defects in an immunocompromised rat model.
Creating 3D constructs with cranial neural crest-derived cell lines using a bio-3D printer.
Protocol to generate scaffold-free, multicomponent 3D melanoma spheroid models for preclinical drug testing.
The Deep Proteomics Approach Identified Extracellular Vesicular Proteins Correlated to Extracellular Matrix in Type One and Two Endometrial Cancer.
Field Guide to Traction Force Microscopy.
Intracellular complement and immunometabolism: The advantages of compartmentalization.
Unifying model for the rheological behavior of hygroresponsive materials.
Glioblastoma Cells Use an Integrin- and CD44-Mediated Motor-Clutch Mode of Migration in Brain Tissue.
TempEasy 3D Hydrogel Coculture System Provides Mechanistic Insights into Prostate Cancer Bone Metastasis.
Biomechanics of Macrophages on Disordered Surface Nanotopography.
An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells.
Biomimetic Approach of Brain Vasculature Rapidly Characterizes Inter- and Intra-Patient Migratory Diversity of Glioblastoma.

Cell Rep. 2024 May 16. pii: S2211-1247(24)00564-3. [Epub ahead of print]43(5): 114236

Mapping the tumor stress network reveals dynamic shifts in the stromal oxidative stress response.

Chen Lior, Debra Barki, Coral Halperin, Christine A Iacobuzio-Donahue, David Kelsen, Ruth Scherz- Shouval.

  The tumor microenvironment (TME) presents cells with challenges such as variable pH, hypoxia, and free radicals, triggering stress responses that affect cancer progression. In this study, we examine the stress response landscape in four carcinomas-breast, pancreas, ovary, and prostate-across five pathways: heat shock, oxidative stress, hypoxia, DNA damage, and unfolded protein stress. Using a combination of experimental and computational methods, we create an atlas of stress responses across various types of carcinomas. We find that stress responses vary within the TME and are especially active near cancer cells. Focusing on the non-immune stroma we find, across tumor types, that NRF2 and the oxidative stress response are distinctly activated in immune-regulatory cancer-associated fibroblasts and in a unique subset of cancer-associated pericytes. Our study thus provides an interactome of stress responses in cancer, offering ways to intersect survival pathways within the tumor, and advance cancer therapy.

Keywords:  CP: Cancer; NRF2; cancer; cancer-associated fibroblasts; fibroblasts; oxidative stress; pericytes; scRNA-seq; stress responses; stroma; tumor microenvrionemnt

DOI:  https://doi.org/10.1016/j.celrep.2024.114236
Biomacromolecules. 2024 May 14.

Tailoring of Physical Properties in Macroporous Poly(isocyanopeptide) Cryogels.

Lotte Gerrits, Bram Bakker, Lynn D Hendriks, Sjoerd Engels, Roel Hammink, Paul H J Kouwer.

Over the years, synthetic hydrogels have proven remarkably useful as cell culture matrixes to elucidate the role of the extracellular matrix (ECM) on cell behavior. Yet, their lack of interconnected macropores undermines the widespread use of hydrogels in biomedical applications. To overcome this limitation, cryogels, a class of macroporous hydrogels, are rapidly emerging. Here, we introduce a new, highly elastic, and tunable synthetic cryogel, based on poly(isocyanopeptides) (PIC). Introduction of methacrylate groups on PIC facilitated cryopolymerization through free-radical polymerization and afforded cryogels with an interconnected macroporous structure. We investigated which cryogelation parameters can be used to tune the architectural and mechanical properties of the PIC cryogels by systematically altering cryopolymerization temperature, polymer concentration, and polymer molecular weight. We show that for decreasing cryopolymerization temperatures, there is a correlation between cryogel pore size and stiffness. More importantly, we demonstrate that by simply varying the polymer concentration, we can selectively tune the compressive strength of PIC cryogels without affecting their architecture. This unique feature is highly useful for biomedical applications, as it facilitates decoupling of stiffness from other variables such as pore size. As such, PIC cryogels provide an interesting new biomaterial for scientists to unravel the role of the ECM in cellular functions.

DOI: https://doi.org/10.1021/acs.biomac.4c00086
Mol Biol Cell. 2024 May 17. mbcE23040139

Matrix stiffening promotes perinuclear clustering of mitochondria.

Piyush Daga, Basil Thurakkal, Simran Rawal, Tamal Das.

Mechanical cues from the tissue microenvironment, such as the stiffness of the extracellular matrix, modulate cellular forms and functions. As numerous studies have shown, this modulation depends on the stiffness-dependent remodeling of cytoskeletal elements. In contrast, very little is known about how the intracellular organelles such as mitochondria respond to matrix stiffness and whether their form, function, and localization change accordingly. Here, we performed an extensive quantitative characterization of mitochondrial morphology, subcellular localization, dynamics, and membrane tension on soft and stiff matrices. This characterization revealed that while matrix stiffness affected all these aspects, matrix stiffening most distinctively led to an increased perinuclear clustering of mitochondria. Subsequently, we could identify the matrix stiffness-sensitive perinuclear localization of filamin as the key factor dictating this perinuclear clustering. The perinuclear and peripheral mitochondrial populations differed in their motility on soft matrix but surprisingly they did not show any difference on stiff matrix. Finally, perinuclear mitochondrial clustering appeared to be crucial for the nuclear localization of RUNX2 and hence for priming human mesenchymal stem cells towards osteogenesis on a stiff matrix. Taken together, we elucidate a dependence of mitochondrial localization on matrix stiffness, which possibly enables a cell to adapt to its microenvironment. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E23-04-0139
Adv Healthc Mater. 2024 May 13. e2400040

Cell Migration Chip (3DCM-chip): a New Tool Toward the Modelling of 3D Cellular Complex Systems.

Silvia Buonvino, Davide Di Giuseppe, Joanna Filippi, Eugenio Martinelli, Dror Seliktar, Sonia Melino.

  3D hydrogel-based cell cultures provide models for studying cell behavior and can efficiently replicate the physiologic environment. Hydrogels can be tailored to mimic mechanical and biochemical properties of specific tissues and allow to produce gel-in-gel models. In this system, microspheres encapsulating cells are embedded in an outer hydrogel matrix, where cells are able to migrate. To enhance the efficiency of such studies, we design a lab-on-a-chip named 3D Cell Migration-chip (3DCM-chip) that offers substantial advantages over traditional methods. 3DCM-chip facilitates the analysis of biochemical and physical stimuli effects on cell migration/invasion in different cell types, including stem, normal and tumor cells. 3DCM-chip provides a smart platform for developing more complex cell co-cultures systems. Herein we investigate the impact of human fibroblasts on MDA-MB 231 breast cancer cells invasiveness. Moreover, how the presence of different cellular lines, including mesenchymal stem cells (hMSCs), normal human dermal fibroblasts (NHDFs), and human umbilical vein endothelial cells (HUVECs), affects the invasive behavior of cancer cells is investigated using 3DCM-chip and producing predictive tumoroid models with a more complex network of interactions between cells and microenvironment. 3DCM-chip moves us closer to creating in vitro systems that can potentially replicate key aspects of the physiological tumor microenvironment. This article is protected by copyright. All rights reserved.

Keywords:  Breast cancer; CAFs; Co‐cultures; PEG‐fibrinogen; cellular invasion

DOI:  https://doi.org/10.1002/adhm.202400040
Commun Mater. 2024 ;pii: 6. [Epub ahead of print]5

Myosin-independent stiffness sensing by fibroblasts is regulated by the viscoelasticity of flowing actin.

Nikhil Mittal, Etienne B Michels, Andrew E Massey, Yunxiu Qiu, Shaina P Royer-Weeden, Bryan R Smith, Alexander X Cartagena-Rivera, Sangyoon J Han.

The stiffness of the extracellular matrix induces differential tension within integrin-based adhesions, triggering differential mechanoresponses. However, it has been unclear if the stiffness-dependent differential tension is induced solely by myosin activity. Here, we report that in the absence of myosin contractility, 3T3 fibroblasts still transmit stiffness-dependent differential levels of traction. This myosin-independent differential traction is regulated by polymerizing actin assisted by actin nucleators Arp2/3 and formin where formin has a stronger contribution than Arp2/3 to both traction and actin flow. Intriguingly, despite only slight changes in F-actin flow speed observed in cells with the combined inhibition of Arp2/3 and myosin compared to cells with sole myosin inhibition, they show a 4-times reduction in traction than cells with myosin-only inhibition. Our analyses indicate that traditional models based on rigid F-actin are inadequate for capturing such dramatic force reduction with similar actin flow. Instead, incorporating the F-actin network's viscoelastic properties is crucial. Our new model including the F-actin viscoelasticity reveals that Arp2/3 and formin enhance stiffness sensitivity by mechanically reinforcing the F-actin network, thereby facilitating more effective transmission of flow-induced forces. This model is validated by cell stiffness measurement with atomic force microscopy and experimental observation of model-predicted stiffness-dependent actin flow fluctuation.

DOI: https://doi.org/10.1038/s43246-024-00444-0
Acta Biomater. 2024 May 13. pii: S1742-7061(24)00257-5. [Epub ahead of print]

Fibroblast alignment and matrix remodeling induced by a stiffness gradient in a skin-derived extracellular matrix hydrogel.

Fenghua Zhao, Meng Zhang, Mehmet Nizamoglu, Hans Kaper, Linda A Brouwer, Theo Borghuis, Janette K Burgess, Martin C Harmsen, Prashant K Sharma.

  Large skin injuries heal as scars. Stiffness gradually increases from normal skin to scar tissue (20x higher), due to excessive deposition and crosslinking of extracellular matrix (ECM) mostly produced by (myo)fibroblasts. Using a custom mold, skin-derived ECM hydrogels (dECM) were UV crosslinked after diffusion of ruthenium (Ru) to produce a Ru-dECM gradient hydrogel. The Ru diffusion gradient equates to a stiffness gradient and models physiology of the scarred skin. Crosslinking in Ru-dECM hydrogels results in a 23-fold increase in stiffness from a stiffness similar to that of normal skin. Collagen fiber density increases in a stiffness-dependent fashion while stress relaxation also alters, with one additional Maxwell element necessary for characterizing Ru-dECM. Alignment of fibroblasts encapsulated in hydrogels suggests that the stiffness gradient directs fibroblasts to orientate at ∼45 ° in regions below 120 kPa. In areas above 120 kPa, fibroblasts decrease the stiffness prior to adjusting their orientation. Furthermore, fibroblasts remodel their surrounding ECM in a gradient-dependent fashion, with rearrangement of cell-surrounding ECM in high-stiffness areas, and formation of interlaced collagen bundles in low-stiffness areas. Overall, this study shows that fibroblasts remodel their local environment to generate an optimal ECM mechanical and topographical environment. STATEMENT OF SIGNIFICANCE: This study developed a versatile in vitro model with a gradient stiffness using skin-derived ECM hydrogel with unchanged biochemical environment. Using Ruthenium crosslinking, a 20-fold stiffness increase was achieved as observed in fibrotic skin. The interaction between fibroblasts and matrix depends on changes in the matrix stiffness. The stiffness gradient directed the alignment of fibroblasts with ∼45° in regions with≤ 120 kPa. The cells in regions with the higher stiffness decreased stiffness first and then oriented themselves. Furthermore, fibroblasts remodeled surrounding ECM and regulated its mechanics in a gradient-dependent fashion to reach an optimal condition. Our study highlights the dynamic interplay between cells and surrounding matrix, shedding light on potential mechanisms and strategies to target scar formation and remodeling.

Keywords:  collagen; extracellular matrix hydrogel; fibroblasts; gradient stiffness; scarring

DOI:  https://doi.org/10.1016/j.actbio.2024.05.018
Commun Biol. 2024 May 16. 7(1): 577

Fibroblasts' secretome from calcified and non-calcified dermis in Pseudoxanthoma elasticum differently contributes to elastin calcification.

Francesco Demetrio Lofaro, Sonia Costa, Maria Luisa Simone, Daniela Quaglino, Federica Boraldi.

Pseudoxanthoma elasticum (PXE) is a rare disease characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with this genetic metabolic condition, it is not known why elastic fibers in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome are investigated in vitro in CUS and CAS fibroblasts. Here we show that, compared to CUS, CAS fibroblasts exhibit: a) differently distributed and organized focal adhesions and stress fibers; b) modified cell-matrix interactions (i.e., collagen gel retraction); c) imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases; d) differentially expressed pro- and anti-calcifying proteoglycans and elastic-fibers associated glycoproteins. These data emphasize that in the development of pathologic mineral deposition fibroblasts play an active role altering the stability of elastic fibers and of the extracellular matrix milieu creating a local microenvironment guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component. In conclusion, this study contributes to a better understanding of the mechanisms of the mineral deposition that can be also associated with several inherited or age-related diseases (e.g., diabetes, atherosclerosis, chronic kidney diseases).

DOI: https://doi.org/10.1038/s42003-024-06283-6
PRX Life. 2023 Jul-Sep;1(1):pii: 013004. [Epub ahead of print]1(1):

Tissue flows are tuned by actomyosin-dependent mechanics in developing embryos.

R Marisol Herrera-Perez, Christian Cupo, Cole Allan, Alicia B Dagle, Karen E Kasza.

Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, the mechanical properties of embryonic epithelial tissues and the factors that control these properties are not well understood. Actomyosin generates contractile tensions and contributes to the mechanical properties of cells and cytoskeletal networks in vitro, but it remains unclear how the levels and patterns of actomyosin activity contribute to embryonic epithelial tissue mechanics in vivo. To dissect the roles of cell-generated tensions in the mechanics of flowing epithelial tissues, we use optogenetic tools to manipulate actomyosin contractility with spatiotemporal precision in the Drosophila germband epithelium, which rapidly flows during body axis elongation. We find that manipulating actomyosin-dependent tensions by either optogenetic activation or deactivation of actomyosin alters the solid-fluid mechanical properties of the germband epithelium, leading to changes in cell rearrangements and tissue-level flows. Optogenetically activating actomyosin leads to increases in the overall level but decreases in the anisotropy of tension in the tissue, whereas optogenetically deactivating actomyosin leads to decreases in both the level and anisotropy of tension compared to in wild-type embryos. We find that optogenetically activating actomyosin results in more solid-like (less fluid-like) tissue properties, which is associated with reduced cell rearrangements and tissue flow compared to in wild-type embryos. Optogenetically deactivating actomyosin also results in more solid-like properties than in wild-type embryos but less solid-like properties compared to optogenetically activating actomyosin. Together, these findings indicate that increasing the overall tension level is associated with more solid-like properties in tissues that are relatively isotropic, whereas high tension anisotropy fluidizes the tissue. Our results reveal that epithelial tissue flows in developing embryos involve the coordinated actomyosin-dependent regulation of the mechanical properties of tissues and the tensions driving them to flow in order to achieve rapid tissue remodeling.

DOI: https://doi.org/10.1103/prxlife.1.013004
Mater Today Bio. 2024 Jun;26 101074

Spatial confinement modulates endothelial cell behavior and traction force in 3D hydrogel microgrooves.

Wenli Jiang, Xinghong Yao, Jian Zhong, Zhi Ouyang, Junyi Shen, Yan Qiu, Ye Zeng.

  The mechanical environment of vascular endothelial cells (ECs) encompasses a wide range of curvatures due to variations in blood vessel diameters. Integrins, key mediators of cell-matrix interactions, establish connections between the extracellular matrix and the actin cytoskeleton, influencing diverse cellular behaviors. In this study, we explored the impact of spatial confinement on human umbilical vein ECs (HUVECs) cultured within three-dimensional hydrogel microgrooves of varying curvatures and the underlying role of integrins in mediating cellular responses. Employing maskless lithography, we successfully fabricated precise and wall curvatures-controlled hydrogel microgrooves, conferring spatial constraints on the cells. Our investigations revealed substantial alterations in HUVEC behavior within the hydrogel microgrooves with varying sidewall curvatures, marked by reduced cell size, enhanced orientation, and increased apoptosis. Interestingly, microgroove curvature emerged as a crucial factor influencing cell orientation and apoptosis, with rectangular microgrooves eliciting distinct changes in cell orientation, while ring-form microgrooves exhibited higher apoptosis rates. The side-wall effect in the 20 μm region near the microgroove wall had the greatest influence on cell orientation and apoptosis. HUVECs within the microgrooves exhibited elevated integrin expression, and inhibition of αV-integrin by cilengitide significantly curtailed cell apoptosis without affecting proliferation. Additionally, integrin-mediated cell traction force closely correlated with the spatial confinement effect. Cilengitide not only reduced integrin and focal adhesion expression but also attenuated cell traction force and cytoskeletal actin filament alignment. Overall, our findings elucidate the spatial confinement of ECs in hydrogel microgrooves and underscores the pivotal role of integrins, particularly αV-integrin, in mediating cell traction force and apoptosis within this microenvironment.

Keywords:  Cell apoptosis; Cell traction force; Hydrogel microgroove; Micropatterning; Vascular endothelial cell

DOI:  https://doi.org/10.1016/j.mtbio.2024.101074
Cancer Res Commun. 2024 May 15.

Mechanistic Characterization of Cancer Associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

Joseph P Gallant, Hallie M Hintz, Gihan S Gunaratne, Matthew T Breneman, Emma E Recchia, Jayden L West, Kendahl L Ott, Erika Heninger, Abigail E Jackson, Natalie Y Luo, Zachary T Rosenkrans, Reinier Hernandez, Shuang G Zhao, Joshua M Lang, Labros Meimetis, David Kosoff, Aaron M LeBeau.

Cancer-associated fibroblasts (CAFs) are a prominent cell type within the tumor microenvironment where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease Fibroblast Activation Protein (FAP) is expressed on the surface of highly pro-tumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating pro-tumorigenic CAFs will disrupt the crosstalk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate (ADC) using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell co-culture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the pro-inflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of pro-inflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies are through effects on the immune microenvironment and anti-tumor immune response.

DOI: https://doi.org/10.1158/2767-9764.CRC-24-0248
bioRxiv. 2024 May 03. pii: 2024.04.30.591879. [Epub ahead of print]

Mechano-metabolism of adherent cells in 2D and 3D microenvironments.

Anuja Jaganathan, Joshua Toth, Xingyu Chen, Laurent Pieuchot, Yihui Shen, Cynthia Reinhart-King, Vivek B Shenoy.

Cells regulate their shape and metabolic activity in response to the mechano-chemical properties of their microenvironment. To elucidate the impact of matrix stiffness and ligand density on a cell's bioenergetics, we developed a non-equilibrium, active chemo-mechanical model that accounts for mechanical energy of the cell and matrix, chemical energy from ATP hydrolysis, interfacial energy, and mechano-sensitive regulation of stress fiber assembly through signaling. By integrating the kinetics and energetics of these processes we introduce the concept of the metabolic potential of the cell that, when minimized, gives experimentally testable predictions of the cell contractility, shape, and the ATP consumption. Specifically, we show that MDA-MB-231 breast cancer cells in 3D collagen gels follow a spherical to spindle to spherical change in morphology with increasing matrix stiffness consistent with experimental observations. This biphasic transition in cell shape emerges from a competition between increased contractility accompanied by ATP hydrolysis enabled by mechano-sensitive signaling, which lowers the volumetric contribution to the metabolic potential of elongated cells and the interfacial energy which is lower for spherical shapes. On 2D hydrogels, our model predicts a hemispherical to spindle to disc shape transition with increasing gel stiffness. In both cases, we show that increasing matrix stiffness monotonically increases the cell's contractility as well as ATP consumption. Our model also predicts how the increased energy demand in stiffer microenvironments is met by AMPK activation, which is confirmed through experimental measurement of activated AMPK levels as a function of matrix stiffness carried out here in both 2D and 3D micro-environments. Further, model predictions of increased AMPK activation on stiffer micro-environments are found to correlate strongly with experimentally measured upregulation of mitochondrial potential, glucose uptake and ATP levels. The insights from our model can be used to understand mechanosensitive regulation of metabolism in physiological events such as metastasis and tumor progression during which cells experience dynamic changes in their microenvironment and metabolic state.

DOI: https://doi.org/10.1101/2024.04.30.591879
bioRxiv. 2024 May 03. pii: 2024.05.01.592106. [Epub ahead of print]

An Open-Source Membrane Stretcher for Simultaneous Mechanical and Structural Characterizations of Soft Materials and Biological Tissues.

Shannon Li, Alyssa Gee, Nathan Cai, Alexandra Bermudez, Neil Y C Lin.

The ability to simultaneously measure material mechanics and structure is central for understanding their nonlinear relationship that underlies the mechanical properties of materials, such as hysteresis, strain-stiffening and -softening, and plasticity. This experimental capability is also critical in biomechanics and mechanobiology research, as it enables direct characterizations of the intricate interplay between cellular responses and tissue mechanics. Stretching devices developed over the past few decades, however, do not often allow simultaneous measurements of the structural and mechanical responses of the sample. In this work, we introduce an open-source stretching system that can apply uniaxial strain at a submicron resolution, report the tensile force response of the sample, and be mounted on an inverted microscope for real-time imaging. Our system consists of a pair of stepper-based linear motors that stretch the sample symmetrically, a force transducer that records the sample tensile force, and an optically clear sample holder that allows for high-magnification microscopy. Using polymer samples and cellular specimens, we characterized the motion control accuracy, force measurement robustness, and microscopy compatibility of our stretching system. We envision that this uniaxial stretching system will be a valuable tool for characterizing soft and living materials.

DOI: https://doi.org/10.1101/2024.05.01.592106
Sci Rep. 2024 May 17. 14(1): 11312

Directed biomechanical compressive forces enhance fusion efficiency in model placental trophoblast cultures.

Prabu Karthick Parameshwar, Chen Li, Kaline Arnauts, Junqing Jiang, Sabra Rostami, Benjamin E Campbell, Hongyan Lu, Derek Hadar Rosenzweig, Cathy Vaillancourt, Christopher Moraes.

  The syncytiotrophoblast is a multinucleated structure that arises from fusion of mononucleated cytotrophoblasts, to sheath the placental villi and regulate transport across the maternal-fetal interface. Here, we ask whether the dynamic mechanical forces that must arise during villous development might influence fusion, and explore this question using in vitro choriocarcinoma trophoblast models. We demonstrate that mechanical stress patterns arise around sites of localized fusion in cell monolayers, in patterns that match computational predictions of villous morphogenesis. We then externally apply these mechanical stress patterns to cell monolayers and demonstrate that equibiaxial compressive stresses (but not uniaxial or equibiaxial tensile stresses) enhance expression of the syndecan-1 and loss of E-cadherin as markers of fusion. These findings suggest that the mechanical stresses that contribute towards sculpting the placental villi may also impact fusion in the developing tissue. We then extend this concept towards 3D cultures and demonstrate that fusion can be enhanced by applying low isometric compressive stresses to spheroid models, even in the absence of an inducing agent. These results indicate that mechanical stimulation is a potent activator of cellular fusion, suggesting novel avenues to improve experimental reproductive modelling, placental tissue engineering, and understanding disorders of pregnancy development.

Keywords:  Choriocarcinoma; Compression; Fusion; Mechanics; Placenta

DOI:  https://doi.org/10.1038/s41598-024-61747-3
Sci Adv. 2024 May 17. 10(20): eadl3511

Cervical extracellular matrix hydrogel optimizes tumor heterogeneity of cervical squamous cell carcinoma organoids.

Haonan Song, Haoyuan Jiang, Weichu Hu, Yan Hai, Yihuan Cai, Hu Li, Yuru Liao, Yi Huang, Xiaogang Lv, Yefei Zhang, Jiping Zhang, Yan Huang, Xiaomei Liang, Hao Huang, Xinhua Lin, Yifeng Wang, Xiao Yi.

Cervical cancer, primarily squamous cell carcinoma, is the most prevalent gynecologic malignancy. Organoids can mimic tumor development in vitro, but current Matrigel inaccurately replicates the tissue-specific microenvironment. This limitation compromises the accurate representation of tumor heterogeneity. We collected para-cancerous cervical tissues from patients diagnosed with cervical squamous cell carcinoma (CSCC) and prepared uterine cervix extracellular matrix (UCEM) hydrogels. Proteomic analysis of UCEM identified several tissue-specific signaling pathways including human papillomavirus, phosphatidylinositol 3-kinase-AKT, and extracellular matrix receptor. Secreted proteins like FLNA, MYH9, HSPA8, and EEF1A1 were present, indicating UCEM successfully maintained cervical proteins. UCEM provided a tailored microenvironment for CSCC organoids, enabling formation and growth while preserving tumorigenic potential. RNA sequencing showed UCEM-organoids exhibited greater similarity to native CSCC and reflected tumor heterogeneity by exhibiting CSCC-associated signaling pathways including virus protein-cytokine, nuclear factor κB, tumor necrosis factor, and oncogenes EGR1, FPR1, and IFI6. Moreover, UCEM-organoids developed chemotherapy resistance. Our research provides insights into advanced organoid technology through native matrix hydrogels.

DOI: https://doi.org/10.1126/sciadv.adl3511
BMC Res Notes. 2024 May 15. 17(1): 139

Dual-color FISH analyses of xenogeneic human fibroblast sheets transplanted to repair lung pleural defects in an immunocompromised rat model.

Masato Kanzaki, Ryo Takagi, Shota Mitsuboshi, Hiroaki Shidei, Tamami Isaka, Masayuki Yamato.

   BACKGROUND: Pulmonary air leaks (PALs) due to visceral pleura injury during surgery is frequently observed after pulmonary resections and the complication is difficult to avoid in thoracic surgery. The development of postoperative PALs is the most common cause of prolonged hospitalization. Previously, we reported that PALs sealants using autologous dermal fibroblast sheets (DFSs) harvested from temperature-responsive culture dishes successfully closed intraoperative PALs during lung resection.
OBJECTIVE: In this study, we investigated the fate of human DFSs xenogenetically transplanted onto lung surfaces to seal PALs of immunocompromised rat. Dual-color FISH analyses of human fibroblast was employed to detect transplantation human cells on the lung surface.
RESULTS: One month after transplantation, FISH analyses revealed that transplanted human fibroblasts still composed a sheet-structure, and histology also showed that beneath the sheet's angiogenesis migrating into the sheets was observed from the recipient tissues. FISH analyses revealed that even at 3 months after transplantation, the transplanted human fibroblasts still remained in the sheet. Dual-color FISH analyses of the transplanted human fibroblasts were sparsely present as a result of the cells reaching the end of their lifespan, the cells producing extracellular matrix, and remained inside the cell sheet and did not invade the lungs of the host.
CONCLUSIONS: DFS-transplanted human fibroblasts showed that they are retained within cell sheets and do not invade the lungs of the host.

Keywords:  Cell sheet; Extracellular matrix; Fibroblast; Pleural defect; Temperature-responsive culture dish

DOI:  https://doi.org/10.1186/s13104-024-06792-x
J Oral Biosci. 2024 May 13. pii: S1349-0079(24)00088-4. [Epub ahead of print]

Creating 3D constructs with cranial neural crest-derived cell lines using a bio-3D printer.

Masahide Taguchi, Shohei Yoshimoto, Kanako Suyama, Satoko Sumi, Shirabe Ohki, Kayoko Ogata, Ryota Fujimoto, Daiki Murata, Koichi Nakayama, Kyoko Oka.

   OBJECTIVES: The development of bio-three-dimensional (bio-3D) printers has led to significant advances in regenerative medicine. Three-dimensional constructs, including spheroids, are maintained by extracellular matrix proteins secreted by cells so that the cells can be cultured in conditions closer to the physiological environment. This study aimed to create a useful 3D construct as a model of the dentin-pulp complex METHODS: We examined the expression patterns of extracellular matrix proteins and cell proliferation areas in a 3D construct created using O9-1 cells derived from cranial neural crest cells of mice. The 3D construct was created by sticking the spheroid cultures onto a needle array using a bio-3D printer.
RESULTS: Cell proliferation areas along with characteristic expression of tenascin C and DMP1 were evaluated. The expression of tenascin C and DMP1 was significantly enhanced in the spheroids compared to that in two-dimensional cultures. Moreover, cell proliferation regions and tenascin C expression were confirmed in the outer layer of spheroids in the embryonic stem cell medium, with insignificant DMP1 expression being observed. Interestingly, in a 3D construct cultured in calcification-induction medium, DMP1 expression was promoted, and DMP1-positive cells existed in the outermost layer without overlapping with tenascin C expression.
CONCLUSIONS: The extracellular matrix proteins, tenascin C and DMP1, were expressed in a polarized manner in spheroids and 3D constructs, similar to the findings in the dental papilla. Therefore, these 3D constructs show potential as artificial models for studying odontogenesis.

Keywords:  3D constructs; bio-3D printer; spheroid; tenascin C

DOI:  https://doi.org/10.1016/j.job.2024.05.005
STAR Protoc. 2024 May 13. pii: S2666-1667(24)00223-5. [Epub ahead of print]5(2): 103058

Protocol to generate scaffold-free, multicomponent 3D melanoma spheroid models for preclinical drug testing.

Cristian Angeli, Joanna Patrycja Wroblewska, Eliane Klein, Christiane Margue, Stephanie Kreis.

  Three-dimensional (3D) models play an increasingly important role in preclinical drug testing as they faithfully mimic interactions between cancer cells and the tumor microenvironment (TME). Here, we present a protocol for generating scaffold-free 3D multicomponent human melanoma spheroids. We describe steps for characterizing models using live-cell imaging and histology, followed by drug testing and assessment of cell death through various techniques such as imaging, luminescence-based assays, and flow cytometry. Finally, we demonstrate the models' adaptability for co-cultures with immune cells.

Keywords:  Cancer; Cell Biology; High Throughput Screening; Organoids

DOI:  https://doi.org/10.1016/j.xpro.2024.103058
Int J Mol Sci. 2024 Apr 24. pii: 4650. [Epub ahead of print]25(9):

The Deep Proteomics Approach Identified Extracellular Vesicular Proteins Correlated to Extracellular Matrix in Type One and Two Endometrial Cancer.

Valeria Capaci, Feras Kharrat, Andrea Conti, Emanuela Salviati, Manuela Giovanna Basilicata, Pietro Campiglia, Nour Balasan, Danilo Licastro, Federica Caponnetto, Antonio Paolo Beltrami, Lorenzo Monasta, Federico Romano, Giovanni Di Lorenzo, Giuseppe Ricci, Blendi Ura.

  Among gynecological cancers, endometrial cancer is the most common in developed countries. Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that contain proteins involved in immune response and apoptosis. A deep proteomic approach can help to identify dysregulated extracellular matrix (ECM) proteins in EVs correlated to key pathways for tumor development. In this study, we used a proteomics approach correlating the two acquisitions-data-dependent acquisition (DDA) and data-independent acquisition (DIA)-on EVs from the conditioned medium of four cell lines identifying 428 ECM proteins. After protein quantification and statistical analysis, we found significant changes in the abundance (p < 0.05) of 67 proteins. Our bioinformatic analysis identified 26 pathways associated with the ECM. Western blotting analysis on 13 patients with type 1 and type 2 EC and 13 endometrial samples confirmed an altered abundance of MMP2. Our proteomics analysis identified the dysregulated ECM proteins involved in cancer growth. Our data can open the path to other studies for understanding the interaction among cancer cells and the rearrangement of the ECM.

Keywords:  extracellular matrix; mass spectrometry; proteomics

DOI:  https://doi.org/10.3390/ijms25094650
Cell Mol Bioeng. 2024 Apr;17(2): 87-106

Field Guide to Traction Force Microscopy.

Aleksandra K Denisin, Honesty Kim, Ingmar H Riedel-Kruse, Beth L Pruitt.

   Introduction: Traction force microscopy (TFM) is a widely used technique to measure cell contractility on compliant substrates that mimic the stiffness of human tissues. For every step in a TFM workflow, users make choices which impact the quantitative results, yet many times the rationales and consequences for making these decisions are unclear. We have found few papers which show the complete experimental and mathematical steps of TFM, thus obfuscating the full effects of these decisions on the final output.
Methods: Therefore, we present this "Field Guide" with the goal to explain the mathematical basis of common TFM methods to practitioners in an accessible way. We specifically focus on how errors propagate in TFM workflows given specific experimental design and analytical choices.
Results: We cover important assumptions and considerations in TFM substrate manufacturing, substrate mechanical properties, imaging techniques, image processing methods, approaches and parameters used in calculating traction stress, and data-reporting strategies.
Conclusions: By presenting a conceptual review and analysis of TFM-focused research articles published over the last two decades, we provide researchers in the field with a better understanding of their options to make more informed choices when creating TFM workflows depending on the type of cell being studied. With this review, we aim to empower experimentalists to quantify cell contractility with confidence.
Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00801-6.

Keywords:  Cell biomechanics; Mechanobiology; Traction force microscopy

DOI:  https://doi.org/10.1007/s12195-024-00801-6
Eur J Immunol. 2024 May 17. e2350813

Intracellular complement and immunometabolism: The advantages of compartmentalization.

Ben C King, Anna M Blom.

  The complement system is a proteolytic cascade triggered by pathogen and danger-associated molecular patterns, with resultant outcomes of inflammation, cellular activation, and opsonization of material for removal by phagocytosis. While first discovered as an activity in serum, it is now recognized that complement components play important roles at local and individual cell-intrinsic levels. In particular, apart from the extracellular serum activities of complement, it is now believed that complement also acts intracellularly, as part of a cellular signal transduction cascade that can stimulate cellular survival and activation, and individual immune cell phenotypes, via effects on cellular metabolism. This review will describe what is currently known about how complement functions in intracellular signal transduction, and outline the functional advantages of a compartmentalized and intracellular complement system.

Keywords:  Immunometabolism; Inflammation; Intracellular complement

DOI:  https://doi.org/10.1002/eji.202350813
Phys Rev E. 2024 Apr;109(4-1): 044139

Unifying model for the rheological behavior of hygroresponsive materials.

Júlio O Amando de Barros, Falk K Wittel.

Hygroresponsive materials exhibit a complex structure-to-property relationship. The interactions of water within these materials under varying hygric and mechanical loads play a crucial role in their macroscopic deformation and final application. While multiple models are available in literature, many lack a comprehensive physical understanding of these phenomena. In this paper, we introduce a stick-slip fiber bundle model that captures the fundamental behaviors of hygroresponsive materials. We incorporate moisture-dependent elements and rules governing the initiation and relaxation of slip strains as well as failure to the statistical approach offered by fiber bundle models. The additional features are based on well-founded interpretations of the structure-to-property relationship in cellulosic materials. Slip strains are triggered by changes in load and moisture, as well as by creep deformations. When subjected to moisture cycles, the model accumulates slip strains, resulting in mechanosorptive behavior. When the load is removed, slip strains are partially relaxed, and subsequent moisture cycles trigger further relaxation, as expected from observations with mechanosorptive material. Importantly, these slip strains are not considered plastic strains; instead, they are unified, nonlinear frozen strains, activated by various stimuli. Failure of fibers is defined by a critical number of slip events allowing for an integrated simulation from intact, via damaged, failed states. We investigate the transition between these regimes upon changes in the hygric and mechanical loading history for relevant parameter ranges. Our enhanced stick-slip fiber bundle model increases the understanding of the intricate behavior of hygroresponsive materials and contributes to a more robust framework for analyzing and interpreting their properties.

DOI: https://doi.org/10.1103/PhysRevE.109.044139
Cell Mol Bioeng. 2024 Apr;17(2): 121-135

Glioblastoma Cells Use an Integrin- and CD44-Mediated Motor-Clutch Mode of Migration in Brain Tissue.

Sarah M Anderson, Marcus Kelly, David J Odde.

   Purpose: Glioblastoma (GBM) is an aggressive malignant brain tumor with 2 year survival rates of 6.7% (Stupp et al. in J Clin Oncol Off J Am Soc Clin Oncol 25:4127-4136, 2007; Mohammed et al. in Rep Pract Oncol Radiother 27:1026-1036, 2002). One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue (Lefranc et al. in J Clin Oncol Off J Am Soc Clin Oncol 23:2411-2422, 2005; Hoelzinger et al. in J Natl Cancer Inst 21:1583-1593, 2007). To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Several models of cell migration have been proposed, including the motor-clutch, bleb-based motility, and osmotic engine models.
Methods: Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration.
Results: We found that nearly all cell-vasculature interactions reflected pulling, rather than pushing, on vasculature at the cell leading edge, a finding consistent with a motor-clutch mode of migration, and inconsistent with an osmotic engine model or confined bleb-based migration. Reducing myosin motor activity, a key component in the motor-clutch model, was found to decrease migration speed at high doses for all cell types including U251 and 6 low-passage patient-derived xenograft lines (3 proneural and 3 mesenchymal subtypes). Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor-to-clutch ratio. Targeting of molecular clutches including integrins and CD44 slowed migration of U251 cells.
Conclusions: Overall we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch.
Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00799-x.

Keywords:  Cancer; Cell migration; Cell traction; Glioblastoma

DOI:  https://doi.org/10.1007/s12195-024-00799-x
ACS Appl Mater Interfaces. 2024 May 13.

TempEasy 3D Hydrogel Coculture System Provides Mechanistic Insights into Prostate Cancer Bone Metastasis.

Zhaobao Zhang, Wen Chen, Mingchen Sun, Tilly Aalders, Gerald W Verhaegh, Paul H J Kouwer.

  Patients diagnosed with advanced prostate cancer (PCa) often experience incurable bone metastases; however, a lack of relevant experimental models has hampered the study of disease mechanisms and the development of therapeutic strategies. In this study, we employed the recently established Temperature-based Easy-separable (TempEasy) 3D cell coculture system to investigate PCa bone metastasis. Through coculturing PCa and bone cells for 7 days, our results showed a reduction in PCa cell proliferation, an increase in neovascularization, and an enhanced metastasis potential when cocultured with bone cells. Additionally, we observed increased cell proliferation, higher stemness, and decreased bone matrix protein expression in bone cells when cocultured with PCa cells. Furthermore, we demonstrated that the stiffness of the extracellular matrix had a negligible impact on molecular responses in both primary (PCa cells) and distant malignant (bone cells) sites. The TempEasy 3D hydrogel coculture system is an easy-to-use and versatile coculture system that provides valuable insights into the mechanisms of cell-cell communication and interaction in cancer metastasis.

Keywords:  3D cocultures; artificial extracellular matrices; bone metastasis; neovascularization; paracrine cell−cell signaling; polyisocyanide hydrogels; proliferation; prostate cancer

DOI:  https://doi.org/10.1021/acsami.4c03453
ACS Appl Mater Interfaces. 2024 May 15.

Biomechanics of Macrophages on Disordered Surface Nanotopography.

Zixin Huo, Wenjie Yang, Javad Harati, Ajinkya Nene, Francesca Borghi, Claudio Piazzoni, Paolo Milani, Shifeng Guo, Massimiliano Galluzzi, Diana Boraschi.

  Macrophages are involved in every stage of the innate/inflammatory immune responses in the body tissues, including the resolution of the reaction, and they do so in close collaboration with the extracellular matrix (ECM). Simplified substrates with nanotopographical features attempt to mimic the structural properties of the ECM to clarify the functional features of the interaction of the ECM with macrophages. We still have a limited understanding of the macrophage behavior upon interaction with disordered nanotopography, especially with features smaller than 10 nm. Here, we combine atomic force microscopy (AFM), finite element modeling (FEM), and quantitative biochemical approaches in order to understand the mechanotransduction from the nanostructured surface into cellular responses. AFM experiments show a decrease of macrophage stiffness, measured with the Young's modulus, as a biomechanical response to a nanostructured (ns-) ZrOx surface. FEM experiments suggest that ZrOx surfaces with increasing roughness represent weaker mechanical boundary conditions. The mechanical cues from the substrate are transduced into the cell through the formation of integrin-regulated focal adhesions and cytoskeletal reorganization, which, in turn, modulate cell biomechanics by downregulating cell stiffness. Surface nanotopography and consequent biomechanical response impact the overall behavior of macrophages by increasing movement and phagocytic ability without significantly influencing their inflammatory behavior. Our study suggests a strong potential of surface nanotopography for the regulation of macrophage functions, which implies a prospective application relative to coating technology for biomedical devices.

Keywords:  atomic force microscopy (AFM); biomechanics; macrophages; mechanotransduction; nanotopography

DOI:  https://doi.org/10.1021/acsami.4c04330
bioRxiv. 2024 May 02. pii: 2023.09.08.556877. [Epub ahead of print]

An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells.

Bailey N Warder, Kara A Nelson, Justin Sui, Lauren Anllo, Stephen DiNardo.

Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed, and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad, and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and Non-muscle Myosin II (MyoII) towards neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells, and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.

DOI: https://doi.org/10.1101/2023.09.08.556877
Small Methods. 2024 May 15. e2400210

Biomimetic Approach of Brain Vasculature Rapidly Characterizes Inter- and Intra-Patient Migratory Diversity of Glioblastoma.

Michele Crestani, Nikolaos Kakogiannos, Simone Iori, Fabio Iannelli, Tania Dini, Claudio Maderna, Monica Giannotta, Giuliana Pelicci, Paolo Maiuri, Pascale Monzo, Nils C Gauthier.

  Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.

Keywords:  cell migration; cytoskeleton; glioblastoma; micropatterning

DOI:  https://doi.org/10.1002/smtd.202400210