Electrodeformation-Based Biomechanical Chip for Quantifying Global Viscoelasticity of Cancer Cells Regulated by Cell Cycle

Mechanical phenotypes of cells are found to hold vital clues to reveal cellular functions and behaviors, which not only has great physiological significance but also is crucial for disease diagnosis. To this end, we developed a set of electrodeformation-based biomechanical microchip assays to quantify mechanical phenotypes on the single-cell level. By investigating the spatiotemporal dynamics of cancer cells driven by dielectrophoresis forces, we captured the key global viscoelastic indexes including cellular elasticity, viscosity, and transition time that was defined as the ratio of the transient viscosity and elasticity, simultaneously, and thus explored their intrinsic correlation with cell cycle progression. Our results showed that both global elasticity and viscosity have a significant periodic variation with cell cycle progression, but the transition time remained unchanged in the process, indicating that it might be an intrinsic property of cancer cells that is independent of the cell cycle and the type of cell in the experiments. Further, we investigated the molecular mechanism regulating cellular viscoelastic phenotypes on the biomechanical chips through intracellular cytoskeletal perturbation assays. These findings, together with the electrodeformation-based microchip technique, not only reveal the relation between mechanical phenotypes of cancer cells and cell cycle progression but also provide a platform for implementing multi-index mechanical phenotype assays associated with cancer cell cycles in the clinic

Substrate Stiffness Coupling TGF-β1 Modulates Migration and Traction Force of MDA-MB-231 Human Breast Cancer Cells in Vitro

Cancer cell migration is the hallmark of tumor metastasis; however, the mechanisms of cancer cell migration have not been fully understood. Considering the fact that biophysical and biochemical properties of the tumor microenvironment are altered during tumor progression, it is instinctive to think about whether the changed microenvironment can regulate cancer cell migration. Herein, we cultured human breast cancer cells (MDA-MB-231) on polyacrylamide gel substrates with different stiffnesses (1, 5, 10, and 20 kPa) with and without transforming growth factor-β1 (TGF-β1, 2 ng/mL) treatment to evaluate the effects of the altered tumor microenvironment on cancer cell migration in addition to the response of traction force generation and cytoskeleton remodeling. The results demonstrated that MDA-MB-231 migration increased with increasing substrate stiffness and was further enhanced with TGF-β1 addition. Traction forces and cytoskeleton remodeling were also found to be enhanced in response to TGF-β1 treatment. Furthermore, inhibiting myosin IIA-mediated contraction by blebbistatin decreased TGF-β1-enhanced traction force but increased TGF-β1-enhanced migration. These results imply that both biophysical (like stiffness) and biochemical (like TGF-β1) factors could orthogonally regulate cancer cell migration

Improved-Throughput Traction Microscopy Based on Fluorescence Micropattern for Manual Microscopy

<div><p>Traction force microscopy (TFM) is a quantitative technique for measuring cellular traction force, which is important in understanding cellular mechanotransduction processes. Traditional TFM has a significant limitation in that it has a low measurement throughput, commonly one per TFM dish, due to a lack of cell position information. To obtain enough cellular traction force data, an onerous workload is required including numerous TFM dish preparations and heavy cell-seeding activities, creating further difficulty in achieving identical experimental conditions among batches. In this paper, we present an improved-throughput TFM method using the well-developed microcontact printing technique and chemical modifications of linking microbeads to the gel surface to address these limitations. Chemically linking the microbeads to the gel surface has no significant influence on cell proliferation, morphology, cytoskeleton, and adhesion. Multiple pairs of force loaded and null force fluorescence images can be easily acquired by means of manual microscope with the aid of a fluorescence micropattern made by microcontact printing. Furthermore, keeping the micropattern separate from cells by using gels effectively eliminates the potential negative effect of the micropattern on the cells. This novel design greatly improves the analysis throughput of traditional TFM from one to at least twenty cells per petri dish without losing unique advantages, including a high spatial resolution of traction measurements. This newly developed method will boost the investigation of cell-matrix mechanical interactions.</p></div

Experimental validation of beads on surface as an indicator of substrate deformation.

<p><b>A)</b> Phase-contrast image of HeLa cell on the PAA gels (beads were both mixed inside and linked on surface of the gels, as illustrated in the inset). <b>B)</b> Scatter plot of RMSD computed for 22 cells utilizing fluorescence images of the beads on surface (vertical axis) and beads inside (horizontal axis). <b>C)</b> Fluorescence image of beads on surface. <b>D)</b> Fluorescence image of beads inside gels. <b>E)</b> Displacement field was calculated using the fluorescence image of beads on surface before and after cell removal by NaOH solution. <b>F)</b> Displacement field was calculated using the fluorescence image of beads inside before and after cell removal by NaOH solution. The solid white line stood for the cell outline in the both <b>E)</b> and <b>F)</b>.</p

Multiple pairs of NF and FL fluorescence images.

<p><b>A)</b> Many FL images had been captured before cell detachment while the NF image of only the last cell was captured after detaching all cells, as other cells could not be found in the traditional TFM. <b>B)</b> Utilizing coordinate system, multiple pairs of NF and FL images were captured in sequence by going back to the original position in the improved-throughput TFM. The circle stands for the PAA substrate. The small rectangle in the circle represents the view field using the 40× objective.</p

Immunostaining of cells on substrate of different topography.

<p><b>A)</b> Representative immunofluorescence confocal microscopic images of the F-actin (red) of cells on substrate with beads inside. <b>B)</b> Representative immunofluorescence confocal microscopic images of the F-actin (red) of cells on substrate with beads on surface. <b>C)</b> Statistical quantification of the mean fluorescence intensity of actin within the HeLa cells on substrate with different positioned beads (n = 28 for beads inside, n = 34 for beads on surface). <b>D)</b> Representative immunofluorescence confocal microscopic images of the vinculin (green) with beads inside. <b>E)</b> Representative immunofluorescence confocal microscopic images of the vinculin (green) with beads on surface. <b>F)</b> Comparison of total vinculin area on PAA gels with beads inside and beads on the gel surface (n = 22 for the former, n = 20 for the latter). Bars represent mean ± standard deviation. Two-tailed t-test was performed for statistical analysis in both <b>C)</b> and <b>F)</b>.</p

The results of improved-throughput measurements.

<p>Each panel was composed of a colorimetric bar, traction force field and phase-contrast image. The recovered traction fields of <b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>, <b>E</b>, <b>F</b>, <b>G</b> and <b>H</b> were only a part of the total traction force fields in one petri dish.</p

Results of MTT assay on substrate with beads inside and on surface.

<p>The optical density at wavelength of 570 nm of each sample at 24 h, 48 h, and 72 h was detected. Bars represent mean ± standard deviation. Two-tailed t-test was performed for statistical comparisons (n = 3, *represents <i>p</i>>>0.05).</p

Eliminating the stage shift in the improved-throughput TFM.

<p><b>A)</b> Apparent stage shift in the original displacement field. <b>B)</b> The actual displacements caused by HeLa cells after correction by the image processing algorithm.</p

Quantitative Analyses of Dynamic Features of Fibroblasts on Different Protein-Coated Compliant Substrates

Cell response to substrate rigidity, closely related to extracellular matrix protein composition, requires actomyosin-generated contractility. By introducing coefficients describing cell spreading and traction dynamics, and a revised high-resolution traction force microscopy, we analyzed the static and dynamic features of fibroblasts on fibronectin- or collagen- coated stiff or soft substrates. Large cell spreading area and branchlike morphology were more favorable on fibronectin than collagen. Cell spreading on fibronectin-coated substrates was more sensitive to rigidity compared with collagen. Low concentration fibronectin-coated substrate induced more dynamic lamellipodia movement than other conditions. Interestingly, the static average cell traction on high concentration fibronectin-coated stiff and soft substrates showed no difference. However, the lamellipodium traction dynamics was sensitive to rigidity on fibronectin. Particularly, lamellipodia on fibronectin-coated soft substrate performed much higher local traction dynamics compared with other groups. Together, dynamics of cell adhesion and traction are regulated by extracellular matrix protein composition, coupled with substrate rigidity