bims-ecemfi 2024-09-01 papers

Front Cell Dev Biol. 2024 ;12 1444827

From stress fiber to focal adhesion: a role of actin crosslinkers in force transmission.

Hiroki Katsuta, Masahiro Sokabe, Hiroaki Hirata.

The contractile apparatus, stress fiber (SF), is connected to the cell adhesion machinery, focal adhesion (FA), at the termini of SF. The SF-FA complex is essential for various mechanical activities of cells, including cell adhesion to the extracellular matrix (ECM), ECM rigidity sensing, and cell migration. This mini-review highlights the importance of SF mechanics in these cellular activities. Actin-crosslinking proteins solidify SFs by attenuating myosin-driven flows of actin and myosin filaments within the SF. In the solidified SFs, viscous slippage between actin filaments in SFs and between the filaments and the surrounding cytosol is reduced, leading to efficient transmission of myosin-generated contractile force along the SFs. Hence, SF solidification via actin crosslinking ensures exertion of a large force to FAs, enabling FA maturation, ECM rigidity sensing and cell migration. We further discuss intracellular mechanisms for tuning crosslinker-modulated SF mechanics and the potential relationship between the aberrance of SF mechanics and pathology including cancer.

Keywords: cell migration; crosslinking protein; extracellular matrix; focal adhesion; mechanotransduction; rigidity sensing; stress fiber; viscoelasticity

DOI: https://doi.org/10.3389/fcell.2024.1444827

ACS Biomater Sci Eng. 2024 Aug 27.

Engineered Biomaterials and Model Systems to Study YAP/TAZ in Cancer.

Emma Villares, Sharon Gerecht.

The transcriptional coactivators yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are master regulators involved in a multitude of cancer types and a wide range of tumorigenic events, including cancer stem cell renewal, invasion, metastasis, tumor precursor emergence, and drug resistance. YAP/TAZ are known to be regulated by several external cues and stimuli, such as extracellular matrix stiffness, cell spreading, cell geometry, and shear stress. Therefore, there is a need in the field of cancer research to develop and design relevant in vitro models that can accurately reflect the complex biochemical and biophysical cues of the tumor microenvironment central to the YAP/TAZ signaling nexus. While much progress has been made, this remains a major roadblock to advancing research in this field. In this review, we highlight the current engineered biomaterials and in vitro model systems that can be used to advance our understanding of how YAP/TAZ shapes several aspects of cancer. We begin by discussing current 2D and 3D hydrogel systems that model the YAP/TAZ response to ECM stiffness. We then examine the current trends in organoid culture systems and the use of microfluidics to model the effects of cellular density and shear stress on YAP/TAZ. Finally, we analyze the ongoing pitfalls of the present models used and important future directions in engineering systems that will advance our current knowledge of YAP/TAZ in cancer.

Keywords: hydrogel; matrix stiffness; microfluidics; organoid

DOI: https://doi.org/10.1021/acsbiomaterials.4c01170

Sci Rep. 2024 Aug 28. 14(1): 20013

Cell spheroid viscoelasticity is deformation-dependent.

Ruben C Boot, Anouk van der Net, Christos Gogou, Pranav Mehta, Dimphna H Meijer, Gijsje H Koenderink, Pouyan E Boukany.

Tissue surface tension influences cell sorting and tissue fusion. Earlier mechanical studies suggest that multicellular spheroids actively reinforce their surface tension with applied force. Here we study this open question through high-throughput microfluidic micropipette aspiration measurements on cell spheroids to identify the role of force duration and spheroid deformability. In particular, we aspirate spheroid protrusions of mice fibroblast NIH3T3 and human embryonic HEK293T homogeneous cell spheroids into micron-sized capillaries for different pressures and monitor their viscoelastic creep behavior. We find that larger spheroid deformations lead to faster cellular retraction once the pressure is released, regardless of the applied force. Additionally, less deformable NIH3T3 cell spheroids with an increased expression level of alpha-smooth muscle actin, a cytoskeletal protein upregulating cellular contractility, also demonstrate slower cellular retraction after pressure release for smaller spheroid deformations. Moreover, HEK293T cell spheroids only display cellular retraction at larger pressures with larger spheroid deformations, despite an additional increase in viscosity at these larger pressures. These new insights demonstrate that spheroid viscoelasticity is deformation-dependent and challenge whether surface tension truly reinforces at larger aspiration pressures.

DOI: https://doi.org/10.1038/s41598-024-70759-y

Acta Biomater. 2024 Aug 23. pii: S1742-7061(24)00470-7. [Epub ahead of print]

Multiphoton excited polymerized biomimetic models of collagen fiber morphology to study single cell and collective migration dynamics in pancreatic cancer.

Sophie Mancha, Meghan Horan, Ojaswi Pasachhe, Adib Keikhosravi, Kevin W Eliceiri, Kristina A Matkowskyj, Jacob Notbohm, Melissa C Skala, Paul J Campagnola.

The respective roles of aligned collagen fiber morphology found in the extracellular matrix (ECM) of pancreatic cancer patients and cellular migration dynamics have been gaining attention because of their connection with increased aggressive phenotypes and poor prognosis. To better understand how collagen fiber morphology influences cell-matrix interactions associated with metastasis, we used Second Harmonic Generation (SHG) images from patient biopsies with Pancreatic ductal adenocarcinoma (PDAC) as models to fabricate collagen scaffolds to investigate processes associated with motility. Using the PDAC BxPC-3 metastatic cell line, we investigated single and collective cell dynamics on scaffolds of varying collagen alignment. Collective or clustered cells grown on the scaffolds with the highest collagen fiber alignment had increased E-cadherin expression and larger focal adhesion sites compared to single cells, consistent with metastatic behavior. Analysis of single cell motility revealed that the dynamics were characterized by random walk on all substrates. However, examining collective motility over different time points showed that the migration was super-diffusive and enhanced on highly aligned fibers, whereas it was hindered and sub-diffusive on un-patterned substrates. This was further supported by the more elongated morphology observed in collectively migrating cells on aligned collagen fibers. Overall, this approach allows the decoupling of single and collective cell behavior as a function of collagen alignment and shows the relative importance of collective cell behavior as well as fiber morphology in PDAC metastasis. We suggest these scaffolds can be used for further investigations of PDAC cell biology. STATEMENT OF SIGNIFICANCE: Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate, where aligned collagen has been associated with poor prognosis. Biomimetic models representing this architecture are needed to understand complex cellular interactions. The SHG image-based models based on stromal collagen from human biopsies afford the measurements of cell morphology, cadherin and focal adhesion expression as well as detailed motility dynamics. Using a metastatic cell line, we decoupled the roles of single cell and collective cell behavior as well as that arising from aligned collagen. Our data suggests that metastatic characteristics are enhanced by increased collagen alignment and that collective cell behavior is more relevant to metastatic processes. These scaffolds provide new insight in this disease and can be a platform for further experiments such as testing drug efficacy.

Keywords: Pancreatic cancer; collagen fibers, collagen remodeling; collective migration; multiphoton excited fabrication; single cell migration

DOI: https://doi.org/10.1016/j.actbio.2024.08.026

Mol Biol Cell. 2024 Aug 28. mbcE24010008

mDia2 is an important mediator of MRTF-A-dependent regulation of breast cancer cell migration.

Ian Eder, Virginia Yu, Jacob Antonello, Fangyuan Chen, David Gau, Pooja Chawla, Marion Joy, Peter C Lucas, David Boone, Adrian V Lee, Partha Roy.

Dysregulated actin cytoskeleton gives rise to aberrant cell motility and metastatic spread of tumor cells. This study evaluates the effect of overexpression of wild-type vs functional mutants of MRTF-A on migration and invasion of breast cancer (BC) cells. Our studies indicate that SRF's interaction is critical for MRTF-A-induced promotion of both 2D and 3D cell migration, while the SAP-domain function is important selectively for 3D cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction of MRTF. We demonstrate formin-family protein mDia2 as an important mediator of MRTF-stimulated actin polymerization at the leading edge and cell migration. Multiplexed quantitative immunohistochemistry and transcriptome analyses of clinical BC specimens further demonstrate a positive correlation between nuclear localization of MRTF with malignant traits of cancer cells and enrichment of MRTF-SRF gene signature in pair-matched distant metastases vs primary tumors. In conclusion, this study establishes a novel mechanism of MRTF-dependent regulation of cell migration and provides evidence for the association between MRTF activity and increased malignancy in human breast cancer, justifying future development of specific small molecule inhibitors of the MRTF-SRF transcriptional complex as potential therapeutic agents in breast cancer.

DOI: https://doi.org/10.1091/mbc.E24-01-0008

J Vis Exp. 2024 Aug 09.

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures.

Natalie Ap Rodrigues Fernandes, Camyla Rodrigues Nascimento, Laura Andrea González Maldonado, Carlos Rossa Junior.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 µm intervals, covering a depth of 90 µm in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

DOI: https://doi.org/10.3791/67114