PIEZO1-mediated mechanosensing governs NK-cell killing efficiency and infiltration in three-dimensional matrices.

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK-cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK-cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK-cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK-cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK-cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK-cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK-cell functions.

The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

Cell-surface contacts determine volume and mechanical properties of human embryonic kidney 293 T cells.

Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell-cell and cell-matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F-actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F-actin density. Instead, we observe changes in the organization of F-actin indicated by the appearance of blebs in the semi-adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology.

Mobilization of CD11b+/Ly6chi monocytes causes multi organ dysfunction syndrome in acute pancreatitis.

Objective: Acute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with multi-organ dysfunction and high mortality. The pathogenesis of life -threatening organ complications, such as respiratory and renal failure, is unknown. Design: Organ dysfunction was investigated in a mouse model of AP. The influence of monocytes and neutrophils on multi organ dysfunction syndrome (MODS) was investigated in vivo by antibody depletion. Using real-time-fluorescence and deformability-cytometry (RT-DC) analysis we determined the mechanical properties of neutrophils and monocytes during AP. Furthermore, blood samples of pancreatitis patients were used to characterize severity-dependent chemokine profiles according to the revised Atlanta classification. Results: Similar to AP in humans, severe disease in the mouse model associates with organ dysfunction mainly of lung and kidney, which is triggered by a mobilisation of Ly6g-/CD11b+/Ly6c hi monocytes, but not of Ly6g+/CD11b+ neutrophils. Monocyte depletion by anti-CCR2 antibody treatment ameliorated lung function (oxygen consumption) without interfering with the systemic immune response. RT-DC analysis of circulation monocytes showed a significant increase in cell size during SAP, but without a compensatory increase in elasticity. Patient chemokine profiles show a correlation of AP severity with monocyte attracting chemokines like MCP-1 or MIG and with leukocyte mobilisation. Conclusion: In AP, the physical properties of mobilized monocytes, especially their large size, result in an obstruction of the fine capillary systems of the lung and of the kidney glomeruli. A selective depletion of monocytes may represent a treatment strategy for pancreatitis as well as for other inflammation-related disorders.

Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells.

Cerebral cavernous malformations (CCM) are low-flow vascular lesions prone to cause severe hemorrhage-associated neurological complications. Pathogenic germline variants in CCM1, CCM2, or CCM3 can be identified in nearly 100% of CCM patients with a positive family history. In line with the concept that tumor-like mechanisms are involved in CCM formation and growth, we here demonstrate an abnormally increased proliferation rate of CCM3-deficient endothelial cells in co-culture with wild-type cells and in mosaic human iPSC-derived vascular organoids. The observation that NSC59984, an anticancer drug, blocked the abnormal proliferation of mutant endothelial cells further supports this intriguing concept. Fluorescence-activated cell sorting and RNA sequencing revealed that co-culture induces upregulation of proangiogenic chemokine genes in wild-type endothelial cells. Furthermore, genes known to be significantly downregulated in CCM3-/- endothelial cell mono-cultures were upregulated back to normal levels in co-culture with wild-type cells. These results support the hypothesis that wild-type ECs facilitate the formation of a niche that promotes abnormal proliferation of mutant ECs. Thus, targeting the cancer-like features of CCMs is a promising new direction for drug development.

Interpretation of cell mechanical experiments in microfluidic systems depend on the choice of cellular shape descriptors.

The capability to parameterize shapes is of essential importance in biomechanics to identify cells, to track their motion, and to quantify deformation. While various shape descriptors have already been investigated to study the morphology and migration of adherent cells, little is known of how the mathematical definition of a contour impacts the outcome of rheological experiments on cells in suspension. In microfluidic systems, hydrodynamic stress distributions induce time-dependent cell deformation that needs to be quantified to determine viscoelastic properties. Here, we compared nine different shape descriptors to characterize the deformation of suspended cells in an extensional as well as shear flow using dynamic real-time deformability cytometry. While stress relaxation depends on the amplitude and duration of stress, our results demonstrate that steady-state deformation can be predicted from single cell traces even for translocation times shorter than their characteristic time. Implementing an analytical simulation, performing experiments, and testing various data analysis strategies, we compared single cell and ensemble studies to address the question of computational costs vs experimental accuracy. Results indicate that high-throughput viscoelastic measurements of cells in suspension can be performed on an ensemble scale as long as the characteristic time matches the dimensions of the microfluidic system. Finally, we introduced a score to evaluate the shape descriptor-dependent effect size for cell deformation after cytoskeletal modifications. We provide evidence that single cell analysis in an extensional flow provides the highest sensitivity independent of shape parametrization, while inverse Haralick's circularity is mostly applicable to study cells in shear flow.

Inhibition of the NLRP3/IL-1ß axis protects against sepsis-induced cardiomyopathy.

BACKGROUND: Septic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin-1ß (IL-1ß), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL-1ß in sepsis-induced cardiomyopathy and cardiac atrophy. METHODS: Male Nlrp3 knockout (KO) and wild-type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham-treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL-1ß effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real-time deformability cytometry (RT-DC) analysis were used to investigate functional and mechanical effects of IL-1ß on cardiomyocytes. RESULTS: Heart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 µL vs. 24.6 ± 8.7 µL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 µL vs. 29.9 ± 9.9 µL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight-tibia length ratio: WT CLP vs. WT sham: -26.6%, P < 0.05; KO CLP vs. KO sham: -3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL-1ß decreased contractility (cell shortening: IL-1ß: -15.4 ± 2.3%, P < 0.001 vs. vehicle, IL-1RA: -6.1 ± 3.3%, P < 0.05 vs. IL-1ß) and relaxation of adult rat ventricular cardiomyocytes (time-to-50% relengthening: IL-1ß: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL-1RA: 564 ± 247 ms, P < 0.001 vs. IL-1ß), which was attenuated by an IL-1 receptor antagonist (IL-1RA). RT-DC analysis indicated that IL-1ß reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF-κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL-1ß activated NF-κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL-1RA. CONCLUSIONS: IL-1ß causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL-1ß pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.

Microfluidic Fabrication of Click Chemistry-Mediated Hyaluronic Acid Microgels: A Bottom-Up Material Guide to Tailor a Microgel's Physicochemical and Mechanical Properties.

The demand for tailored, micrometer-scaled biomaterials in cell biology and (cell-free) biotechnology has led to the development of tunable microgel systems based on natural polymers, such as hyaluronic acid (HA). To precisely tailor their physicochemical and mechanical properties and thus to address the need for well-defined microgel systems, in this study, a bottom-up material guide is presented that highlights the synergy between highly selective bio-orthogonal click chemistry strategies and the versatility of a droplet microfluidics (MF)-assisted microgel design. By employing MF, microgels based on modified HA-derivates and homobifunctional poly(ethylene glycol) (PEG)-crosslinkers are prepared via three different types of click reaction: Diels-Alder [4 + 2] cycloaddition, strain-promoted azide-alkyne cycloaddition (SPAAC), and UV-initiated thiol-ene reaction. First, chemical modification strategies of HA are screened in-depth. Beyond the microfluidic processing of HA-derivates yielding monodisperse microgels, in an analytical study, we show that their physicochemical and mechanical properties-e.g., permeability, (thermo)stability, and elasticity-can be systematically adapted with respect to the type of click reaction and PEG-crosslinker concentration. In addition, we highlight the versatility of our HA-microgel design by preparing non-spherical microgels and introduce, for the first time, a selective, hetero-trifunctional HA-based microgel system with multiple binding sites. As a result, a holistic material guide is provided to tailor fundamental properties of HA-microgels for their potential application in cell biology and (cell-free) biotechnology.

High-throughput cell and spheroid mechanics in virtual fluidic channels.

Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master mold using benchtop devices, design modifications are time consuming and require sophisticated cleanroom equipment. Here, we introduce virtual fluidic channels as a flexible and robust alternative to microfluidic devices made by soft lithography. Virtual channels are liquid-bound fluidic systems that can be created in glass cuvettes and tailored in three dimensions within seconds for rheological studies on a wide size range of biological samples. We demonstrate that the liquid-liquid interface imposes a hydrodynamic stress on confined samples, and the resulting strain can be used to calculate rheological parameters from simple linear models. In proof-of-principle experiments, we perform high-throughput rheology inside a flow cytometer cuvette and show the Young's modulus of isolated cells exceeds the one of the corresponding tissue by one order of magnitude.

Label-free on chip quality assessment of cellular blood products using real-time deformability cytometry.

Without cellular blood products such as platelet concentrates (PC), red blood cell concentrates (RCC), and hematopoietic stem cells (HPSC) modern treatments in medicine would not be possible. An unresolved challenge is the assessment of their quality with minimal cell manipulation. Minor changes in production, storage conditions, or blood bag composition may impact cell function, which can have important consequences on product integrity. This is especially relevant for personalized medicine, such as autologous T-cell therapy. Today a robust methodology that globally determines cell status directly before transfusion or transplantation is lacking. We demonstrate that measuring viscoelastic characteristics of peripheral blood cells using real-time deformability cytometry (RT-DC) provides comprehensive information on product quality, which is not accessible using conventional quality control tests. In addition, RT-DC requires few cells, a minimal sample volume and has a rapid turnaround time. We compared RT-DC to standard in vitro quality assays assessing: i) PC after storage at 4 °C and room temperature; ii) magnetic nanoparticle labeled platelets; iii) RCC stored in blood bags with different plasticizers; iv) RCC after gamma irradiation; and v) HPSC after cryopreservation with 5% or 10% dimethyl sulfoxide, respectively. Additionally, we evaluated the engraftment time of patients' platelets and leukocytes after transplantation of HPSC products. Our results demonstrate that label-free mechano-phenotyping can be used as a potential biomarker for quality assessment of cell-based pharmaceutical products.

H-Bonding-mediated binding and charge reorganization of proteins on gold nanoparticles.

Once introduced into the human body, nanoparticles often interact with blood proteins, which in turn undergo structural changes upon adsorption. Although protein corona formation is a widely studied phenomenon, the structure of proteins adsorbed on nanoparticles is far less understood. We propose a model to describe the interaction between human serum albumin (HSA) and nanoparticles (NPs) with arbitrary coatings. Our model takes into account the competition between protonated and unprotonated polymer ends and the curvature of the NPs. To this end, we explored the effects of surface ligands (citrate, PEG-OMe, PEG-NH2, PEG-COOH, and glycan) on gold nanoparticles (AuNPs) and the pH of the medium on structural changes in the most abundant protein in blood plasma (HSA), as well as the impact of such changes on cytotoxicity and cellular uptake. We observed a counterintuitive effect on the ζ-potential upon binding of negatively charged HSA, while circular dichroism spectroscopy at various pH values showed an unexpected pattern in the reduction of α-helix content, as a function of surface chemistry and curvature. Our model qualitatively reproduces the decrease in α-helix content, thereby offering a rationale based on particle curvature. The simulations quantitatively reproduce the charge inversion measured experimentally through the ζ-potential of the AuNPs in the presence of HSA. Finally, we found that AuNPs with adsorbed HSA display lower toxicity and slower cell uptake rates, compared to functionalized systems in the absence of protein. Our study allows examining and explaining the conformational dynamics of blood proteins triggered by NPs and corona formation, thereby opening new avenues toward designing safer NPs for drug delivery and nanomedical applications.

High-throughput single-cell rheology in complex samples by dynamic real-time deformability cytometry.

In life sciences, the material properties of suspended cells have attained significance close to that of fluorescent markers but with the advantage of label-free and unbiased sample characterization. Until recently, cell rheological measurements were either limited by acquisition throughput, excessive post processing, or low-throughput real-time analysis. Real-time deformability cytometry expanded the application of mechanical cell assays to fast on-the-fly phenotyping of large sample sizes, but has been restricted to single material parameters as the Young's modulus. Here, we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition, our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties.