Cell Stiffness Research Articles

Biomechanics of Macrophages on Disordered Surface Nanotopography.

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.

In Situ Quantitative Imaging of Plasma Membrane Stiffness in Live Cells Using a Genetically Encoded FRET Sensor.

Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

Excessive endometrial PlGF- Rac1 signalling underlies endometrial cell stiffness linked to pre-eclampsia

Cell stiffness is regulated by dynamic interaction between ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1) proteins, besides other biochemical and molecular regulators. In this study, we investigated how the Placental Growth Factor (PlGF) changes endometrial mechanics by modifying the actin cytoskeleton at the maternal interface. We explored the global effects of PlGF in endometrial stromal cells (EnSCs) using the concerted approach of proteomics, atomic force microscopy (AFM), and electrical impedance spectroscopy (EIS). Proteomic analysis shows PlGF upregulated RhoGTPases activating proteins and extracellular matrix organization-associated proteins in EnSCs. Rac1 and PAK1 transcript levels, activity, and actin polymerization were significantly increased with PlGF treatment. AFM further revealed an increase in cell stiffness with PlGF treatment. The additive effect of PlGF on actin polymerization was suppressed with siRNA-mediated inhibition of Rac1, PAK1, and WAVE2. Interestingly, the increase in cell stiffness by PlGF treatment was pharmacologically reversed with pravastatin, resulting in improved trophoblast cell invasion. Taken together, aberrant PlGF levels in the endometrium can contribute to an altered pre-pregnancy maternal microenvironment and offer a unifying explanation for the pathological changes observed in conditions such as pre-eclampsia (PE).

Remodeled Extracellular Matrix Guides Endothelial Cell Phenotype and Transcriptome to Fibrotic Niche

Impairment of endothelial barrier integrity causes vascular injury leading to acute lung injury (ALI), which, if not resolved, can compromise a patient’s lung functions and mortality. While it is known that both endothelial cells (EC) and their underlying extracellular matrix (ECM) are required for preserving lung homeostasis and functions, much of the efforts to understand mechanisms regulating these functions have focused mainly on EC. A fundamental unresolved question is whether lung-remodeled ECM formed due to leaky endothelium impacts EC functions, and if so, how? Herein, using lung ECM derived from a mouse lacking focal adhesion kinase (FAK), specifically in the endothelium (remodeled ECM), we assessed ECM components and their role in regulating EC functions and their relevance in vivo. Previously, we have shown that conditional deletion of FAK in endothelium increases EC permeability and induces vascular injury at homeostasis. Mass spectrometric analysis showed 805 proteins in control ECM (FAKfl/fl mice lung derived ECM) consisting of glycoproteins, matrisome-associated proteins, and secreted factors. However, the remodeled ECM had 722 proteins and was highly enriched with fibrillin 1, fibrinogen, and collagen I. Moreover, the remodeled ECM was two-fold stiffer than the control ECM. Remodeled ECM with its inherent composition recapitulates the 3D microenvironment, encouraging EC morphological alteration, increased cell stiffness, and enhanced focal adhesion (FA) formation compared to control ECM. In addition, RNA sequencing identified increased expression of genes linked with fibrosis, including integrin b8, TGFb2, GDF15, SMAD7, COL1A1, IL33, ID3, BMPR1A, and ADAMTS4 in EC seeded on remodeled ECM. Interestingly, fibrotic signaling observed in EC plated on remodeled ECM mimicked in vivo in EC-FAK−/− mice as they showed increased hydroxyproline content and collagen deposition in the lungs, indicating loss of EC-FAK as the crucial initiator of fibrotic signaling. In summary, studies for the first time show disrupted EC signaling as the instructor of fibrotic niche in the lungs for targeting pulmonary fibrosis. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3115: Mechanics And Genomic Profiles Reveal Aging And Developmental Lineage Heterogeneities In Human Induced Pluripotent Stem Cell-derived Smooth Muscle Cells.

Effective stem cell therapies and disease modeling depend on understanding epigenetic factors of aging. Accurately modeling age-dependent phenotypes such as aortic, and smooth muscle cell stiffening, requires cell models that reflect donor age. Improved knowledge of how donor aging influences stem cell-derived smooth muscle cells is essential for precise disease modeling and creating targeted therapeutics. We hypothesize that traits of vascular aging are retained in human induced pluripotent stem cells (iPSCs) and manifest in differentiated smooth muscle cells (SMCs). We aim to compare iPSC stiffness and gene expression between young (under 40, n=5 lines) and old (over 60, n=4 lines) donors, and examine age-dependent differences in their iPSC-SMCs. We measured cell stiffness using atomic force microscopy (AFM), and analyzed bulk and single-cell RNA gene expression in iPSCs and iPSC-SMCs of neuroectoderm (NE) or paraxial mesoderm (PM) lineages, respectively. AFM revealed no significant age-related differences in iPSC colony stiffness. Bulk RNA expression showed high similarity (>94% Pearson correlation) between young and old donor iPSCs, with principal component analysis indicating low variability and no clear age separation. Single-cell stiffness measurements found old NE-SMCs significantly stiffer than young ones (p=0.032), with no marked difference in PM-SMCs between ages. Old and young NE-SMCs were stiffer than their respective PM-SMC counterparts (old, p=0.006; young, p=0.007). UMAP analysis of single-cell RNA expression displayed distinct groupings for old and young NE-SMCs, but not for PM-SMCs. Cluster analysis revealed three clusters predominantly of old NE-SMCs and one of young NE-SMCs. Our study reveals age-related stiffening in NE lineage iPSC-SMCs, but not in PM lineage or iPSCs, indicating that iPSCs preserve certain donor age-related traits that manifest as lineage-specific phenotypes in SMCs post-differentiation. RNA sequencing showed similar expression patterns in young and old iPSCs, but age-specific differences in NE-SMCs. The integration of AFM and RNA expression data sheds light on the emergence of age-dependent phenotypes in differentiated, sensitive cell types, which are not present in immature cells.

Smooth Muscle Cofilin Confers Elasticity to Coronary Resistance Microvessels in Diabetes

Coronary resistance microvessels (CRMs) from type 2 diabetic (T2D) mice and pigs are less stiff compared to normal, a finding that is dictated by less stiff coronary vascular smooth muscle cells (VSMCs) Cofilin is an endogenous actin regulatory protein that depolymerizes filamentous F-actin, and recent studies have shown that portions of F-actin bound to cofilin are less stiff compared to their unbound F-actin counterparts. In this study, we tested the hypothesis that augmented cofilin confers elasticity to diabetic CRMs. CRMs were isolated and pooled (n=6 pooled CRMs per biological replicate) for proteomic data collection from both normal (n=3) and diabetic (n=5) mice. In separate experiments, we utilized deidentified normal and T2D human primary coronary VSMCs obtained from commercial vendors (Lonza and ATCC; n=4 per group) and experiments were performed on passage 5-8 VSMCs. Using these cells, we determined cofilin expression by Western blot, and cofilin siRNA (Cell Signaling Technology) was used 48 hours prior to experimentation to knock down cofilin expression. Elastic modulus, a measure of cell stiffness, was assessed by atomic force microscopy (AFM). Globular G-actin was labeled with fluorescein-deoxyribonuclease I (DNAse I) and F-actin was labeled with phalloidin. Images were taken in blinded manner and Image J was used to assess expression. In the proteomics screen, we observed zero cofilin spectral hits in the normal CRMs and 3-4 spectral hits in db/db CRMs (0.00 ± 0.0 vs. 3.40 ± 0.25, p<0.0001). Cofilin protein was increased in the coronary VSMCs of diabetic patients compared to normal coronary VSMCs (1.00 ± 0.16 vs. 1.837± 0.35, p=0.052). In human coronary VSMCs, silencing cofilin caused a significant increase in the F/G actin ratio when normal and diabetic data were pooled (scramble 3.029 ± 0.49 vs. siRNA 4.029 ± 0.68, p= 0.018, n=8). This increased F/G ratio was significant in the diabetic coronary VSMCs (scramble 2.636 ± 0.46 vs. siRNA 3.685 ± 0.41, p=0.049, n=4), and trended in the normal cells but did not reach statistical significance (scramble 3.423 ± 0.89 vs. cofilin siRNA 4.373 ± 1.37, p=0.224, n=4). Cofilin knockdown also caused a significant increase in elastic modulus by AFM when normal and diabetic data were pooled (scramble siRNA 1.814 ± 0.25 vs. cofilin siRNA 3.074 ± 0.35, p=0.0057, n=8). This trend persisted in the normal VMSCs (scramble siRNA 2.038 ± 0.31 vs. cofilin siRNA 3.445± 0.30, p=0.075, n=4) and in the diabetic VSMCs (scramble siRNA 1.589 ± 0.40 vs. cofilin siRNA 2.702 ± 0.62, p=0.085, n=4) but did not reach significance due to low available sample number. Overall, we demonstrate increased cofilin in human diabetic coronary VSMCs and in CRMs from diabetic mice, and silencing cofilin increased F/G actin ratio and increased cellular stiffness. These data support the notion that increased cofilin in T2D VSMCs may be the cause of decreased diabetic CRM stiffness. Support: NIH R00 HL116769, R01 HL165124, and S10 OD023438 and Nationwide Children’s Hospital to AJT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3138: Atheroprone Flow, Aging, And Oxysterols Impair Endothelial Cell Nuclear Health Via Disruption Of Autoregulatory Perinuclear Cytoskeletal Dynamics

The vascular hemodynamic environment plays a well-known role with progressive pathological conditions such as atherosclerosis, hypercholesterolemia, and aging. Our lab has previously shown that the exposure of oxysterols to endothelial cells (ECs) causes increased cell stiffening. This stiffening effect is compounded when the hemodynamic environment changes from atheroprotective laminar flow to atheroprone oscillatory flow. The increasing EC stiffening demonstrates that these disruptive effects significantly alter cellular biomechanics. The complexity of events associated with these altered biomechanical states leaves many questions unanswered about spatiotemporal changes in the biomechanical structures of endothelial cells exposed to a varying hemodynamic environment and oxysterols. To address this void, we observed the effects of 7-Ketocholesterol exposure (a major oxysterol) on the primary cytoskeletal/biomechanical components (F-actin, Microtubules, Intermediate Filaments, etc.) of human aortic endothelial cells during various time points of laminar/oscillatory flow conditions through a combination of sophisticated multiplexed 6-color labeling, ultra-high resolution 3D microscopy, 3D computer vision, and machine learning. Additionally, we applied these techniques to extracted aortic tissues from young and aged mice (on low fat diet vs high fat diet) and focused on obtaining information from both the laminar flow areas (descending aorta) and the oscillatory flow areas (aortic arch). Our findings demonstrate that oxysterol exposure and aging cause significant remodeling & alteration of crucial endothelial cytoskeletal/biomechanical components and that these alterations are unique to each combinatorial condition. Notably, we find that cytoskeletal remodeling associated with endothelial stiffening is accompanied by major nuclear distortion, a hallmark of aging and senescence which subsequently causes DNA damage and genomic instability. We also find that this nuclear distortion is driven by dynamic biomechanical changes and mediated by the hemodynamic environment, where atheroprotective flow promotes cytoskeletal configurations protecting the nuclei, and atheroprone flow promotes cytoskeletal configurations that leave the nuclei unprotected.

STAT3 mediates ECM stiffness-dependent progression in ovarian cancer.

The treatment of ovarian cancer remains a medical challenge and its malignant progression is connected with obvious changes in both tissue and cell stiffness. However, the accurate mechanical-responsive molecules and mechanism remains unclear in ovarian cancer. Based on our previous results combined with the crucial regulatory role of STAT3 in the malignant progression of various cancer types, we want to investigate the relationship between STAT3 and matrix stiffness in ovarian cancer and further explore the potential mechanisms. Collagen-coated polyacrylamide gels (1, 6, and 60 kPa) were prepared to mimic soft or hard matrix stiffness. Western blotting, qRT-PCR, flow cytometry, IHC, EdU assays, and TEM were used to evaluate the effect of STAT3 in vitro under different matrix stiffnesses. Furthermore, a BALB/c nude mouse model was established to assess the relationship in vivo. Our results confirmed the differential expression of STAT3/p-STAT3 not only in normal and malignant ovarian tissues but also under different matrix stiffnesses. Furthermore, we verified that STAT3 was a mechanically responsive gene both in vitro and in vivo, and the mechanical response was carried out by altering the migration-related molecules (TNFAIP1) and adhesion-related molecules (LPXN, CNN3). The novel findings suggest that STAT3, a potential therapeutic target for clinical diagnosis and treatment, is a mechanically responsive gene that responds to matrix stiffness, particularly regulation in migration and adhesion in the progression of ovarian cancer.

Anisotropic power-law viscoelasticity of living cells is dominated by cytoskeletal network structure

During physiological and pathological processes, cells experience significant morphological alterations with the re-arrangement of cytoskeletal filaments, resulting in anisotropic viscoelasticity. Here, a structure-based cell model is proposed to study the anisotropic viscoelastic mechanical behaviors of living cells. We investigate how cell shape affects its creep responses in longitudinal and perpendicular directions. It is shown that cells exhibit power-law rheological behavior in both longitudinal and perpendicular directions under step stress, with a more solid-like behavior along the longitudinal direction. We reveal that the cell volume and cytoskeletal filament orientation, which have been neglected in most existing models, play a critical role in regulating cellular anisotropic viscoelasticity. The stiffness of the cell in both directions increases linearly with increasing its aspect ratio, due to the decrease of cell volume. Moreover, the increase in the cell's aspect ratio produces the aggregation of cytoskeletal filaments along the longitudinal direction, resulting in higher stiffness in this direction. It is also shown that the increase in cell's aspect ratio corresponds to a process of cellular ordering, which can be quantitatively characterized by the orientational entropy of cytoskeletal filaments. In addition, we present a simple yet robust method to establish the relationship between cell's aspect ratio and cell volume, thus providing a theoretical framework to capture the anisotropic viscoelastic behavior of cells. This study suggests that the structure-based cell models may be further developed to investigate cellular rheological responses to external mechanical stimuli and may be extended to the tissue scale. Statement of significanceThe viscoelastic behaviors of cells hold significant importance in comprehending the roles of mechanical forces in embryo development, invasion, and metastasis of cancer cells. Here, a structure-based cell model is proposed to study the anisotropic viscoelastic mechanical behaviors of living cells. Our study highlights the crucial role of previously neglected factors, such as cell volume and cytoskeletal filament orientation, in regulating cellular anisotropic viscoelasticity. We further propose an orientational entropy of cytoskeletal filaments to quantitatively characterize the ordering process of cells with increasing aspect ratios. Moreover, we derived the analytical interrelationships between cell aspect ratio, cell stiffness, cell volume, and cytoskeletal fiber orientation. This study provides a theoretical framework to describe the anisotropic viscoelastic mechanical behavior of cells.

Determining the Young's Modulus of the Bacterial Cell Envelope.

Bacteria experience substantial physical forces in their natural environment, including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young's modulus of the cell envelope of Escherichia coli range from 2 to 18 MPa. We developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here, we extend this technique to determine Young's modulus of the cell envelope of E. coli and of the pathogens Vibrio cholerae and Staphylococcus aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Young's modulus from observed deformations. The Young's modulus values of the cell envelope were 2.06 ± 0.04 MPa for E. coli, 0.84 ± 0.02 MPa for E. coli treated with a chemical (A22) known to reduce cell stiffness, 0.12 ± 0.03 MPa for V. cholerae, and 1.52 ± 0.06 MPa for S. aureus (mean ± SD). The microfluidic approach allows examination of hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes behind differences in cell envelope Young's modulus among bacterial species and strains.

Study on the Equivalent Stiffness of a Local Resonance Metamaterial Concrete Unit Cell

This paper addresses the pressing scientific problem of accurately predicting the equivalent stiffness of local resonance metamaterial concrete unit cells. Existing theoretical models often fail to capture the nuanced dynamics of these complex systems, resulting in suboptimal predictions and hindering advancements in engineering applications. To address this deficit, this paper proposes a novel two-dimensional theoretical vibration model that incorporates shear stiffness, a crucial yet often overlooked parameter in previous formulations. Motivated by the need for improved predictive accuracy, this paper rigorously validates a new theoretical model through numerical simulations, considering variations in material parameters and geometric dimensions. The analysis reveals several key findings: firstly, the equivalent stiffness increases with elastic modulus while the error rate decreases, holding geometric parameters and Poisson’s ratio constant. Secondly, under fixed geometric parameters and coating elastic modulus, the equivalent stiffness rises with an increasing Poisson’s ratio, accompanied by a decrease in error rate. Importantly, this paper demonstrates that the proposed model exhibits the lowest error rate across all parameter conditions, facilitating superior prediction of equivalent stiffness. This advancement holds significant implications for the design and optimization of metamaterial structures in various engineering applications for vibration isolation, with promising enhancements of performance and efficiency.

Oligomeric Amyloid-β and Tau Alter Cell Adhesion Properties and Induce Inflammatory Responses in Cerebral Endothelial Cells Through the RhoA/ROCK Pathway.

Dysfunction of cerebral endothelial cells (CECs) has been implicated in the pathology of Alzheimer's disease (AD). Despite evidence showing cytotoxic effects of oligomeric amyloid-β (oAβ) and Tau (oTau) in the central nervous system, their direct effects on CECs have not been fully investigated. In this study, we examined the direct effects of oAβ, oTau, and their combination on cell adhesion properties and inflammatory responses in CECs. We found that both oAβ and oTau increased cell stiffness, as well as the p-selectin/Sialyl-LewisX (sLeX) bonding-mediated membrane tether force and probability of adhesion in CECs. Consistent with these biomechanical alterations, treatments with oAβ or oTau also increased actin polymerization and the expression of p-selectin at the cell surface. These toxic oligomeric peptides also triggered inflammatory responses, including upregulations of p-NF-kB p65, IL-1β, and TNF-α. In addition, they rapidly activated the RhoA/ROCK pathway. These biochemical and biomechanical changes were further enhanced by the treatment with the combination of oAβ and oTau, which were significantly suppressed by Fasudil, a specific inhibitor for the RhoA/ROCK pathway. In conclusion, our data suggest that oAβ, oTau, and their combination triggered subcellular mechanical alterations and inflammatory responses in CECs through the RhoA/ROCK pathway.

Local intracellular stiffening of ascidian embryo in cleavage developmental stage observed by atomic force microscopy

During the cleavage stage in ascidian embryos, cells undergo repeated and regular cell division in the animal hemisphere. We investigated how the intracellular stiffness, that is, the apparent Young’s modulus of cells in the animal hemisphere changes in the cleavage stages from the 16-cell to 76-cell stages using atomic force microscopy (AFM). The AFM force mapping experiments showed that a pair of dot-like stiffening clearly appeared in the intracellular region, which involves the cortical and cytoplasmic regions indented by the AFM probe, in a period of the interphase after cell division. The dot-like structures disappeared before beginning to increase the whole cell stiffening towards the proceeding mitotic phase. We found that the positioning of a pair of dots was located so as to intersect the daughter cells formed in the proceeding cell division. These results suggest that the local intracellular stiffening observed in the interphase may play an important role in controlling the orientation of the mitotic spindle that determines the direction of cell division.

Relating Chemo-Mechanical Hysteresis and Formation Protocols for Anode-Free Lithium Metal Batteries

Cell formation is an energy and time-intensive empirically-guided process crucial to manufacturing secondary lithium-ion batteries. As the rechargeable battery industry moves towards manufacturing lithium metal batteries—where a metallic lithium negative electrode is used instead of a porous graphite composite—the cell formation process may need reconsidering. The effects of formation rate and cycling protocol on lithium metal battery performance are poorly understood. In this work, we used operando acoustic transmission to measure physical changes during the formation cycles and the effect of formation cycling protocols on the long-term cycling of anode-free lithium metal pouch cells—where all the lithium inventory comes from the positive electrode and is deposited as metallic lithium on copper foil during initial charge. We show that a faster C/3 formation protocol results in comparable cycling performance and cell stiffness change to a slower C/10 formation step. Variations in acoustic metrics across different electrolytes tested are attributed to differences in gas formation, cell swelling, and lithium deposition morphology. NMC811 cathodes paired with a high-concentration ether electrolyte are shown to be particularly prone to gas formation, which is mitigated by using a localized high-concentration ether electrolyte and single-crystal NMC532. The results highlight differences in formation behavior between anode-free lithium metal cells and lithium-ion cells. These are important to consider when bringing new manufacturing plants online for lithium metal batteries.

Active Tunable Elastic Metasurface for Abnormal Flexural Wave Transmission

An active elastic metasurface has more flexibility than a passively modulated elastic metasurface, owing to the manipulation of the phase gradient that can be realized without changing the geometrical configuration. In this study, a negative proportional feedback control system was employed to provide positive active control stiffness for adaptive unit cells, with the aim of achieving the active modulation of the phase gradient. The relationship between the control gain and the phase velocity of the flexural wave was derived, and the transfer coefficients and phase shifts of the flexural wave through the adaptive unit cells were resolved using the transfer matrix method. Finite element simulations for wave propagations in the adaptive unit cells were conducted, and they verified the analytic solutions. Based on this theoretical and numerical work, we designed active elastic metasurfaces with adaptive unit cells with sub-wavelength thicknesses according to the generalized Snell’s law. These metasurfaces show flexibility in achieving abnormal functions for transmitted waves, including negative refraction and wave focusing, and transforming guided waves at different operating frequencies by manipulating the control gain. Therefore, the proposed active metasurface has great potential in the fields of the tunable manipulation of elastic waves and the design of smart devices.

Abstract 2748: Single cell biomechanical profiling of the metastatic potential of MDA-MB-231 cells

Abstract Cancer metastasis is a multistep complex process accompanied by changes in the mechanical properties of cancer cells, such as adhesion, viscoelasticity, deformability, and motility. Connexin 43 (Cx43), a gap junction protein with a putative tumor suppressor function, plays a pivotal role in Epithelial to Mesenchymal transition (EMT). It affects cell motility indicators and promotes cytoskeleton reorganization and, ultimately, metastasis when downregulated, corroborating resistance to chemotherapy. The lack of universal markers that can predict the metastatic ability of cancer cells necessitates the evaluation of parameters, such as cell adhesion, viscoelasticity, and stiffness, to serve as unique biophysical metrics that predict cancer cell metastatic potential and elucidate how metastasis and cell mechanics are coupled. Therefore, we utilized fluidic-based single-cell force spectroscopy (SCFS) and Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) to measure the mechanical properties of single cancer cells with different metastatic potential. To explore the relationship between cell mechanics and metastatic cell behavior, we quantitatively demonstrate the progression of cancer cell biomechanics across different metastatic states using MDA-MB-231 breast cancer cells with varying connexin 43 (Cx43) expression as a model of cells with varying metastatic potential. We measured, at a single-cell level and in real-time, the adhesion and viscoelastic properties of MDA-MB-231 cells and their response to chemotherapy. Our data show that cells with higher metastatic potential (down-regulated Cx43 expression) exhibited a softer, less adherent, and more viscoelastic phenotype, consistent with their metastatic state when in clusters compared to cells at the lower spectrum of metastatic potential (up-regulated Cx43 expression). These mechanical biomarkers are significantly absent in single cells outside of a cluster. Of particular interest, we also observed significant cellular stiffening upon treatment with docetaxel (DTX), a chemotherapeutic agent that targets microtubules, with a rapid and profound response of highly aggressive cells when compared to cells with attenuated metastatic potential. Such treatment schemes could render cancer cells less deformable, potentially impeding their ability to invade and metastasize to other tissues. Our study demonstrates a unique quantitative metric that unfolds biomechanical markers for discerning the metastatic state of cancer cells. Our data represents a stepping stone to understanding how mechanical changes in cancer cells may determine how cancer progresses and responds to chemotherapeutic agents and may provide novel modalities to inhibit metastasis. Citation Format: Zeina Habli, Ahmad Zantout, Nadine Al Hajj, Raya Saab, Marwan El-Sabban, Massoud L. Khraiche. Single cell biomechanical profiling of the metastatic potential of MDA-MB-231 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2748.

Abstract 5423: Selective inhibition of glucose transporter 3 (GLUT3) suppresses triple-negative breast cancer cell migration by modulating actin cytoskeleton and cell mechanics

Abstract Studies have reported clear clinical observations indicating that obesity and diabetes, which often accompany hyperglycemia, are linked to a poor prognosis across various types of cancer. Specifically, multiple clinical studies involving breast cancer patients have demonstrated that hyperglycemia (>7 mM) is associated with an unfavorable prognosis. Despite the evident correlation between elevated glucose levels and cancer metastasis, the causality remains unclear, and the underlying molecular mechanisms are largely unknown. In both normal and diseased physiology, a broad range of tissue glucose levels has been documented, ranging from normal blood circulation (5.5 mM) to normal tissues (1 - 3 mM) and to tumor tissues (<0.5 mM). Consequently, during the early stages of metastasis, cancer cells undergo a significant increase in extracellular glucose levels. The capacity of cancer cells to deform and exert contractile force, collectively known as cell mechanics, plays a crucial role in various stages of metastasis. In our previous work, we demonstrated that elevated extracellular glucose levels boost glycolysis and activate the RhoA signaling pathway. This leads to heightened cell stiffness and increased contractility in human triple-negative breast cancer (TNBC) cells. Furthermore, the modified cell mechanics are associated with enhanced cell migration and invasion. In this study, we present findings indicating that the inhibition of glucose transporter 3 (GLUT3) significantly diminishes the motility of human TNBC cells, including MDA-MB-231 and its highly metastatic variant, MDA-MB-231/HM cells. Mechanistic investigations reveal that GLUT3 inhibition, facilitated by a well-characterized potent GLUT3-selective inhibitor (G3iA), results in reduced glucose uptake and glycolysis. The diminished glycolysis, induced by GLUT3 inhibition, activates 5' AMP-activated protein kinase (AMPK) and enhances the phosphorylation of Yes-associated protein (YAP). Moreover, GLUT3 inhibition leads to a decrease in non-muscle myosin II activity and induces alterations in F-actin rearrangement by influencing actin-binding proteins such as VASP and cofilin. These changes are associated with a reduction in cell migration. In summary, our results suggest that inhibiting GLUT3 modifies the mechanics of TNBC cells, leading to a decrease in cell motility. Given that GLUT3 is notably elevated in TNBC and is linked to metastatic progression and a poor prognosis in breast cancer patients, our findings underscore the need for further in vivo studies to validate G3iA as a potential anti-metastatic drug in hyperglycemic TNBC animal models. Citation Format: Tae-Hyung Kim, Aadil Qadir Bhat, Cristina V. Iancu, Jun-Yong Choe. Selective inhibition of glucose transporter 3 (GLUT3) suppresses triple-negative breast cancer cell migration by modulating actin cytoskeleton and cell mechanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5423.

Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells.

Gastric cancer has a high rate of recurrence, and as such, immunotherapy strategies are being investigated as a potential therapeutic strategy. Although the involvement of immune checkpoints in immunotherapy is well studied, biomechanical cues, such as target cell stiffness, have not yet been subject to the same level of investigation. Changes in the cholesterol content of the cell membrane directly influence tumor cell stiffness. Here, we investigated the effect of cholesterol on NK cell-mediated killing of gastric cancer stem-like cells. We report that surviving tumor cells with stem-like properties elevated cholesterol metabolism to evade NK cell cytotoxicity. Inhibition of cholesterol metabolism enhances NK cell-mediated killing of gastric cancer stem-like cells, highlighting a potential avenue for improving immunotherapy efficacy. This study suggests a possible effect of cancer cell stiffness on immune evasion and offers insights into enhancing immunotherapeutic strategies against tumors.

Bandgap enhancement of a piezoelectric metamaterial beam shunted with circuits incorporating fractional and cubic nonlinearities

Nonlinear metamaterials have been widely studied for their potential to broaden the bandgap for vibration suppression. However, the bandgap widening effect induced by nonlinearity vanishes when the excitation amplitude is insufficient to trigger the nonlinear behavior. In order to broaden the applicable range of excitation amplitude for nonlinear metamaterials, a novel design of a piezoelectric metamaterial beam shunted with combined nonlinear circuits is presented in this work, which incorporates fractional and cubic nonlinearities in circuits to achieve bandgap enhancement under both low and high excitation amplitudes. Theoretical and numerical models are established for predicting the nonlinear dynamics behaviors of the piezoelectric metamaterial beam, and their computational results are in good agreement. The amplitude-dependent bandgap of the metamaterial beam induced by combined nonlinearity is analyzed through the effective bending stiffness of a unit cell and validated through the frequency response of a piezoelectric metamaterial beam. It is found that based on the complementary nature of fractional and cubic nonlinearities in the amplitude-dependent characteristics, combined nonlinearity of the circuits can effectively improve the bandgap width of the beam under both low and high excitation amplitudes. In addition, the effects of the two nonlinearities on the amplitude-dependent characteristics of the bandgap are studied, and the interplay between the two nonlinearities within the combination is analyzed. The results show that the amplitude-dependent characteristics of the combined nonlinear design can be precisely tuned by adjusting the nonlinear coefficients of the circuits. Overall, a piezoelectric metamaterial beam with both fractional and cubic nonlinearities in circuits possesses superior vibration control performance than those with single nonlinear counterparts in circuits.

Epithelial and mesenchymal phenotypes determine the dynamics of circulating breast tumor cells in microfluidic capillaries under chemotherapy-induced stress.

Circulating tumor cells (CTCs) with different epithelial and mesenchymal phenotypes play distinct roles in the metastatic cascade. However, the influence of their phenotypic traits and chemotherapy on their transit and retention within capillaries remains unclear. To explore this, we developed a microfluidic device comprising 216 microchannels of different widths from 5 to 16 μm to mimic capillaries. This platform allowed us to study the behaviors of human breast cancer epithelial MCF-7 and mesenchymal MDA-MB-231 cells through microchannels under chemotherapy-induced stress. Our results revealed that when the cell diameter to microchannel width ratio exceeded 1.2, MCF-7 cells exhibited higher transit percentages than MDA-MB-231 cells under a flow rate of 0.13 mm/s. Tamoxifen (250 nM) reduced the transit percentage of MCF-7 cells, whereas 100 nM paclitaxel decreased transit percentages for both cell types. These differential responses were partially due to altered cell stiffness following drug treatments. When cells were entrapped at microchannel entrances, tamoxifen, paclitaxel, and high-flow stress (0.5 mm/s) induced a reduction in mitochondrial membrane potential (MMP) in MCF-7 cells. Tamoxifen treatment also elevated reactive oxygen species (ROS) levels in MCF-7 cells. Conversely, MMP and ROS levels in entrapped MDA-MB-231 cells remained unaffected. Consequently, the viability and proliferation of entrapped MCF-7 cells declined under these chemical and physical stress conditions. Our findings emphasize that phenotypically distinct CTCs may undergo selective filtration and exhibit varied responses to chemotherapy in capillaries, thereby impacting cancer metastasis outcomes. This highlights the importance of considering both cell phenotype and drug response to improve treatment strategies.