Search

The mechanical phenotype of a cell determines its ability to deform under force and is therefore relevant to cellular functions that require changes in cell shape, such as migration or circulation through the microvasculature. On the practical level, the mechanical phenotype can be used as a global readout of the cell's functional state, a marker for disease diagnostics, or an input for tissue modeling. We focus our review on the current knowledge of structural components that contribute to the determination of the cellular mechanical properties and highlight the physiological processes in which the mechanical phenotype of the cells is of critical relevance. The ongoing efforts to understand how to efficiently measure and control the mechanical properties of cells will define the progress in the field and drive mechanical phenotyping toward clinical applications. [ABSTRACT FROM AUTHOR]

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of five ChAc patients compared to healthy controls during up to 1-year individual off-label treatment with the tyrosine kinase inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results showed that blood cell deformability–including both RBCs and leukocytes - in general was altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observed alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hinted at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc. [ABSTRACT FROM AUTHOR]

Despite fluorescent cell-labelling being widely employed in biomedical studies, some of its drawbacks are inevitable, with unsuitable fluorescent probes or probes inducing a functional change being the main limitations. Consequently, the demand for and development of label-free methodologies to classify cells is strong and its impact on precision medicine is relevant. Towards this end, high-throughput techniques for cell mechanical phenotyping have been proposed to get a multidimensional biophysical characterization of single cells. With this motivation, our goal here is to investigate the extent to which an unsupervised machine learning methodology, which is applied exclusively on morpho-rheological markers obtained by real-time deformability and fluorescence cytometry (RT-FDC), can address the difficult task of providing label-free discrimination of reticulocytes from mature red blood cells. We focused on this problem, since the characterization of reticulocytes (their percentage and cellular features) in the blood is vital in multiple human disease conditions, especially bone-marrow disorders such as anemia and leukemia. Our approach reports promising label-free results in the classification of reticulocytes from mature red blood cells, and it represents a step forward in the development of high-throughput morpho-rheological-based methodologies for the computational categorization of single cells. Besides, our methodology can be an alternative but also a complementary method to integrate with existing cell-labelling techniques. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are professional antigen-presenting cells of the immune system. Upon sensing pathogenic material in their environment, DCs start to mature, which includes cellular processes, such as antigen uptake, processing and presentation, as well as upregulation of costimulatory molecules and cytokine secretion. During maturation, DCs detach from peripheral tissues, migrate to the nearest lymph node, and find their way into the correct position in the net of the lymph node microenvironment to meet and interact with the respective T cells. We hypothesize that the maturation of DCs is well prepared and optimized leading to processes that alter various cellular characteristics from mechanics and metabolism to membrane properties. Here, we investigated the mechanical properties of monocyte-derived dendritic cells (moDCs) using real-time deformability cytometry to measure cytoskeletal changes and found that mature moDCs were stiffer compared to immature moDCs. These cellular changes likely play an important role in the processes of cell migration and T cell activation. As lipids constitute the building blocks of the plasma membrane, which, during maturation, need to adapt to the environment for migration and DC-T cell interaction, we performed an unbiased high-throughput lipidomics screening to identify the lipidome of moDCs. These analyses revealed that the overall lipid composition was significantly changed during moDC maturation, even implying an increase of storage lipids and differences of the relative abundance of membrane lipids upon maturation. Further, metadata analyses demonstrated that lipid changes were associated with the serum low-density lipoprotein (LDL) and cholesterol levels in the blood of the donors. Finally, using lipid packing imaging we found that the membrane of mature moDCs revealed a higher fluidity compared to immature moDCs. This comprehensive and quantitative characterization of maturation associated changes in moDCs sets the stage for improving their use in clinical application. [ABSTRACT FROM AUTHOR]

Many molecular and cellular pathogenic mechanisms of neurodegenerative diseases have been revealed. However, it is unclear what role a putatively impaired neuronal transport with respect to altered mechanical properties of neurons play in the initiation and progression of such diseases. The biochemical aspects of intracellular axonal transport, which is important for molecular movements through the cytoplasm, e.g., mitochondrial movement, has already been studied. Interestingly, transport deficiencies are associated with the emergence of the affliction and potentially linked to disease transmission. Transport along the axon depends on the normal function of the neuronal cytoskeleton, which is also a major contributor to neuronal mechanical properties. By contrast, little attention has been paid to the mechanical properties of neurons and axons impaired by neurodegeneration, and of membraneless, phase-separated organelles such as stress granules (SGs) within neurons. Mechanical changes may indicate cytoskeleton reorganization and function, and thus give information about the transport and other system impairment. Nowadays, several techniques to investigate cellular mechanical properties are available. In this review, we discuss how select biophysical methods to probe material properties could contribute to the general understanding of mechanisms underlying neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Little is known about how cancer cells adapt their mechanical properties in complex 3D microenvironments. Here we generated different types of tumor spheroids within compliant or stiff hydrogels. We then quantitatively mapped the mechanical properties of these spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiffer hydrogels showed elevated Brillouin shifts, hence spheroids became "stiffer" compared to the ones cultured within compliant gels. The spheroid's mechanical properties were modulated by various microenvironment properties including matrix stiffness and degradability and the resultant compressive stress but also depending on whether single cells or cell aggregates were analyzed. Moreover, spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels became stiffer and at the same time, less invasive compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and their microenvironment, which is relevant to better understand cancer progression. Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids' cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids' mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression. [ABSTRACT FROM AUTHOR]

The mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using 2D substrates, much less is known about it in a physiologically more relevant 3D context. Here it is demonstrated that breast cancer tumor spheroids, growing in 3D polyethylene glycol‐heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa build up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy reveals that tumor spheroid stiffness increases with hydrogel stiffness. Also, constituent cell stiffness increases in a Rho associated kinase (ROCK)‐ and F‐actin‐dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase, and elevated levels of the cyclin‐dependent kinase inhibitor p21. Drug‐mediated ROCK inhibition not only reverses cell stiffening upon culture in stiff hydrogels but also increases tumor spheroid growth. Taken together, a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment is revealed here. Thus, the findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments. [ABSTRACT FROM AUTHOR]

Human bone marrow mesenchymal stromal cells (MSCs) are used in clinical trials for the treatment of systemic inflammatory diseases due to their regenerative and immunomodulatory properties. However, intravenous administration of MSCs is hampered by cell trapping within the pulmonary capillary networks. Here, it is hypothesized that traditional 2D plastic‐adherent cell expansion fails to result in appropriate morphorheological properties required for successful cell circulation. To address this issue, a method to culture MSCs in nonadherent 3D spheroids (mesenspheres) is adapted. The biological properties of mesensphere‐cultured MSCs remain identical to conventional 2D cultures. However, morphorheological analyses reveal a smaller size and lower stiffness of mesensphere‐derived MSCs compared to plastic‐adherent MSCs, measured using real‐time deformability cytometry and atomic force microscopy. These properties result in an increased ability to pass through microconstrictions in an ex vivo microcirculation assay. This ability is confirmed in vivo by comparison of cell accumulation in various organ capillary networks after intravenous injection of both types of MSCs in mouse. The findings generally identify cellular morphorheological properties as attractive targets for improving microcirculation and specifically suggest mesensphere culture as a promising approach for optimized MSC‐based therapies. [ABSTRACT FROM AUTHOR]

Although most cancer drugs target the proliferation of cancer cells, it is metastasis, the complex process by which cancer cells spread from the primary tumor to other tissues and organs of the body where they form new tumors, that leads to over 90% of all cancer deaths. Thus, there is an urgent need for anti-metastasis therapy. Surprisingly, emerging evidence suggests that certain anti-cancer drugs such as paclitaxel and doxorubicin can actually promote metastasis, but the mechanism(s) behind their pro-metastatic effects are still unclear. Here, we use a microfluidic microcirculation mimetic (MMM) platform which mimics the capillary constrictions of the pulmonary and peripheral microcirculation, to determine if in-vivo -like mechanical stimuli can evoke different responses from cells subjected to various cancer drugs. In particular, we show that leukemic cancer cells treated with doxorubicin and daunorubicin, commonly used anti-cancer drugs, have over 100% longer transit times through the device, compared to untreated leukemic cells. Such delays in the microcirculation are known to promote extravasation of cells, a key step in the metastatic cascade. Furthermore, we report a significant (p < 0.01) increase in the chemotactic migration of the doxorubicin treated leukemic cells. Both enhanced retention in the microcirculation and enhanced migration following chemotherapy, are pro-metastatic effects which can serve as new targets for anti-metastatic drugs. [ABSTRACT FROM AUTHOR]

Abstract: Whilst rigid, planar surfaces are often used to study cell migration, a physiological scenario requires three-dimensional (3D) scaffolds with tissue-like stiffness. This paper presents a method for fabricating periodic hydrogel scaffolds with a 3D honeycomb-like structure from colloidal crystal templates. The scaffolds, made of hydrogel-walled cavities interconnected by pores, have separately tuneable internal dimensions and adjustable gel stiffness down to that of soft tissues. In conjunction with confocal microscopy, these scaffolds were used to study the importance of cell compliance on invasive potential. Acute promyelocytic leukaemia (APL) cells were differentiated with all-trans retinoic acid (ATRA) and treated with paclitaxel. Their migration ability into the scaffolds'' size-restricted pores, enabled by cell softening during ATRA differentiation, was significantly reduced by paclitaxel treatment, which interferes with cell shape recovery. These findings demonstrate the usability of the scaffolds for investigating factors that affect cell migration, and potentially other cell functions, in a realistic 3D tissue model. [Copyright &y& Elsevier]

To divide in a tissue, both normal and cancer cells become spherical and mechanically stiffen as they enter mitosis. We investigated the effect of oncogene activation on this process in normal epithelial cells. We found that short-term induction of oncogenic RasV12 activates downstream mitogen-activated protein kinase (MEK-ERK) signaling to alter cell mechanics and enhance mitotic rounding, so that RasV12-expressing cells are softer in interphase but stiffen more upon entry into mitosis. These RasV12-dependent changes allow cells to round up and divide faithfully when confined underneath a stiff hydrogel, conditions in which normal cells and cells with reduced levels of Ras-ERK signaling suffer multiple spindle assembly and chromosome segregation errors. Thus, by promoting cell rounding and stiffening in mitosis, oncogenic RasV12 enables cells to proliferate under conditions of mechanical confinement like those experienced by cells in crowded tumors. • Ras-ERK signaling controls cell shape in mitosis • Activation of Ras alters cell mechanics to enhance mitotic stiffening • Cells expressing Ras are better able to round up to divide under stiff gels • Ras limits DNA segregation errors normally observed in confined cell division Matthews et al. show that activation of the oncogenic Ras-ERK signaling pathway changes the shape and mechanical properties of normal epithelial cells in mitosis. This enables Ras-activated cells to round up and accurately segregate chromosomes when dividing in conditions of mechanical confinement. [ABSTRACT FROM AUTHOR]