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      Characterizing cellular mechanical phenotypes with mechano-node-pore sensing

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          Abstract

          The mechanical properties of cells change with their differentiation, chronological age, and malignant progression. Consequently, these properties may be useful label-free biomarkers of various functional or clinically relevant cell states. Here, we demonstrate mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell-analysis method that utilizes a four-terminal measurement of the current across a microfluidic channel to quantify simultaneously cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We define a new parameter, the whole-cell deformability index (wCDI), which provides a quantitative mechanical metric of the resistance to compressive deformation that can be used to discriminate among different cell types. The wCDI and the transverse deformation under constant strain show malignant MCF-7 and A549 cell lines are mechanically distinct from non-malignant, MCF-10A and BEAS-2B cell lines, and distinguishes between cells treated or untreated with cytoskeleton-perturbing small molecules. We categorize cell recovery time, Δ T r, as instantaneous (Δ T r~0 ms), transient (Δ T r⩽40 ms), or prolonged (Δ T r>40 ms), and show that the composition of recovery types, which is a consequence of changes in cytoskeletal organization, correlates with cellular transformation. Through the wCDI and cell-recovery time, mechano-NPS discriminates between sub-lineages of normal primary human mammary epithelial cells with accuracy comparable to flow cytometry, but without antibody labeling. Mechano-NPS identifies mechanical phenotypes that distinguishes lineage, chronological age, and stage of malignant progression in human epithelial cells.

          Supplementary information

          The online version of this article (doi:10.1038/micronano.2017.91) contains supplementary material, which is available to authorized users.

          Sensors: Microfluidic-based cell measurements could aid clinical diagnosis

          A simple and innovative technique for measuring the mechanical properties of cells could lead to a versatile clinical diagnostic tool. The ability to measure differences in the mechanical properties of cells can be used to detect changes in cells that are caused by disease, aging, or environmental interactions. Present technologies for performing such measurements, however, can analyse only a few cells each hour. This led Lydia Sohn at the University of California, Berkeley, in the United States, and colleagues to use a microfluidic platform that integrates node-pore sensors with a contraction channel to measure mechanical differences in populations of cells efficiently. The team's device, which measures the current across the microfluidic channel and quantifies four biophysical properties of a single cell simultaneously, has broad applications for understanding biomechanical properties of cells, clinical diagnostics, and therapeutics.

          Supplementary information

          The online version of this article (doi:10.1038/micronano.2017.91) contains supplementary material, which is available to authorized users.

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          Most cited references63

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          Micropipette aspiration of living cells

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            Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines.

            Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here, we applied a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacologic inhibition of myosin II increases invasiveness, whereas increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power-law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.
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              Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.

              Mature podocytes are among the most complex differentiated cells and possess a highly branched array of foot processes that are essential to glomerular filtration in the kidney. Such differentiated podocytes are unable to replicate and culturing of primary podocytes results in rapid growth arrest. Therefore, conditionally immortalized mouse podocyte clones (MPC) were established, which are highly proliferative when cultured under permissive conditions. Nonpermissive conditions render the majority of MPC cells growth arrested within 6 days and induce many characteristics of differentiated podocytes. Both proliferating and differentiating MPC cells express the WT-1 protein and an ordered array of actin fibers and microtubules extends into the forming cellular processes during differentiation, reminiscent of podocyte processes in vivo. These cytoskeletal rearrangements and process formation are accompanied by the onset of synaptopodin synthesis, an actin-associated protein marking specifically differentiated podocytes. In addition, focal contacts are rearranged into an ordered pattern in differentiating MPC cells. Most importantly, electrophysiological studies demonstrate that differentiated MPC cells respond to the vasoactive peptide bradykinin by changes in intracellular calcium concentration. These results suggest a regulatory role of podocytes in glomerular filtration. Taken together, these studies establish that conditionally immortalized MPC cells retain a differentiation potential similar to podocytes in vivo. Therefore, the determinative steps of podocyte differentiation and process formation are studied for the first time using an inducible in vitro model.
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                Author and article information

                Contributors
                sohn@berkeley.edu
                Journal
                Microsyst Nanoeng
                Microsyst Nanoeng
                Microsystems & Nanoengineering
                Nature Publishing Group UK (London )
                2096-1030
                2055-7434
                12 March 2018
                12 March 2018
                2018
                : 4
                : 17091
                Affiliations
                [1 ]GRID grid.47840.3f, ISNI 0000 0001 2181 7878, Department of Mechanical Engineering, , University of California at Berkeley, ; Berkeley, 94720-1740 CA USA
                [2 ]GRID grid.47840.3f, ISNI 0000 0001 2181 7878, Department of Bioengineering, , University of California at Berkeley, ; Berkeley, 94720-1762 CA USA
                [3 ]GRID grid.410425.6, ISNI 0000 0004 0421 8357, Department of Population Sciences, , Beckman Research Institute, City of Hope, ; Duarte, 91010 CA USA
                [4 ]GRID grid.266102.1, ISNI 0000 0001 2297 6811, Department of Pharmaceutical Chemistry, , University of California, San Francisco, ; San Francisco, 94143 CA USA
                [5 ]GRID grid.184769.5, ISNI 0000 0001 2231 4551, Biological Systems and Engineering Division, , Lawrence Berkeley National Laboratory, ; CA, 94720 USA
                [6 ]GRID grid.47840.3f, ISNI 0000 0001 2181 7878, Graduate Program in Bioengineering, University of California, Berkeley, and University of California, San Francisco, ; Berkeley, 94720 CA USA
                Article
                BFmicronano201791
                10.1038/micronano.2017.91
                5958920
                29780657
                e6846be2-0eb2-4b21-aee0-83feeb39ef8a
                © The Author(s) 2018

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 15 July 2017
                : 16 September 2017
                : 16 October 2017
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                © The Author(s) 2018

                engineering,microfluidics
                engineering, microfluidics

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