This protocol outlines the quantification of the mechanical properties of cancerous and non-cancerous cell lines in vitro. Conserved differences in the mechanics of cancerous and normal cells can act as a biomarker that may have implications in prognosis and diagnosis.
Irregular biomechanics are a hallmark of cancer biology subject to extensive study. The mechanical properties of a cell are similar to those of a material. A cell’s resistance to stress and strain, its relaxation time, and its elasticity are all properties that can be derived and compared to other types of cells. Quantifying the mechanical properties of cancerous (malignant) versus normal (non-malignant) cells allows researchers to further uncover the biophysical fundamentals of this disease. While the mechanical properties of cancer cells are known to consistently differ from the mechanical properties of normal cells, a standard experimental procedure to deduce these properties from cells in culture is lacking.
This paper outlines a procedure to quantify the mechanical properties of single cells in vitro using a fluid shear assay. The principle behind this assay involves applying fluid shear stress onto a single cell and optically monitoring the resulting cellular deformation over time. Cell mechanical properties are subsequently characterized using digital image correlation (DIC) analysis and fitting an appropriate viscoelastic model to the experimental data generated from the DIC analysis. Overall, the protocol outlined here aims to provide a more effective and targeted method for the diagnosis of difficult-to-treat cancers.
Studying the biophysical differences between cancerous and non-cancerous cells allows for novel diagnostic and therapeutic opportunities1. Understanding how differences in biomechanics/mechanobiology contribute to tumor progression and treatment resistance will reveal new avenues for targeted therapy and early diagnosis2.
While it is known that cancer cell mechanical properties differ from normal cells (e.g., viscoelasticity of the plasma membrane and nuclear envelope)3,4,5, robust and reproducible methods for measuring these properties in live cells are lacking6. The shear assay method is used to quantify the mechanical properties of cells by subjecting single cells to fluid shear stress and analyzing their individual responses and resistance to the applied stress3,4,5,7,8,9. Although several methods and techniques have been used to characterize the mechanical properties of single cells, these tend to affect cell material properties by i) perforating/damaging the cell membrane due to the indentation depth, complex tip geometries, or substrate stiffening associated with atomic force microscopy (AFM)10,11, ii) inducing cellular photodamage during optical trapping12,13, or iii) inducing complex stress states associated with micropipette aspiration14,15. These external effects are associated with significant uncertainties in the accuracy of cell viscoelasticity measurements6,16,17.
To address these limitations, the shear assay method described here provides a highly controllable and simple approach to simulate physiological flow in the body without affecting cellular material properties in the process. Fluid shear stresses in this assay represent mechanical stresses experienced by cells in the body either by fluids within the tumor interstitium or in the blood during circulation18,19,20. Further, these fluid stresses promote various malignant behaviors in cancer cells, including progression, migration, metastasis, and cell death19,21,22,23 which vary between tumorigenic and non-tumorigenic cells. Moreover, the altered mechanical features of cancer cells (i.e., they are often "softer" than normal cells found within the same organ) allow them to persist in hostile tumor microenvironments, invade surrounding normal tissues, and metastasize to distant sites24,25,26. By creating a pseudo-biological environment where cells experience physiological levels of fluid shear stress, a process that is physiologically relevant and not destructive to the cell is achieved. The cellular responses to these applied fluid shear stresses allow us to characterize cell mechanical properties.
This paper provides a shear assay protocol for the extensive study of the mechanical properties and behavior of cancerous and non-cancerous cells under applied shear stress. Cells respond to external forces in an elastic and viscous manner and can therefore be idealized as a viscoelastic material3. This technique is categorized into: (i) cell culture of dispersed single cells, (ii) controlled application of fluid shear stress, (iii) in situ imaging and observation of cellular behavior (including resistance to stress and deformation), (iv) strain analysis of cells to determine the extent of deformation, and (v) characterization of the viscoelastic properties of single cells. By interrogating these mechanical properties and behaviors, complex cellular mechanobiology can be distilled to quantifiable data. A protocol outlining this method allows for the cataloging of and comparison between various malignant and non-malignant cell types. Quantifying these differences has the potential to establish diagnostic and therapeutic biomarkers.
1. Preparation for the single-cell shear assay
2. Shear assay experiment
3. Data processing
4. Mechanical property characterization
The shear assay protocol coupled with deformation analysis using DIC and a viscoelastic model is successful in quantifying the mechanical properties of a single cell in vitro. This method has been tested on human and murine cell lines, including normal human breast cells (MCF-10A)3,4,9, less metastatic triple-negative breast cancer cells (MDA-MB-468)3, triple-negative breast cancer cells (MDA-MB-231)3, human osteosarcoma cells7,8, and most recently glioblastoma cells (Figure 3). DIC of image frames extracted from shear assay recordings is successful in producing strain-time data compatible with the creep stress response (Figure 3), which is fit to a viscoelastic model (Figure 4). With the use of MATLAB, the viscoelastic model can be used to fit the strain-time data to obtain the viscoelastic properties of the different cells.
Figure 5 describes the range of properties that have been previously studied using the shear assay technique. Additional mechanical properties can be characterized using this method, depending on the parameter of interest. The results of these studies using the shear assay technique showed that cancer cells were generally more mechanically compliant and less viscous than normal cells (Figure 6)3,4,7,8. Figure 6A-C describes the mechanical properties of normal breast cells (MCF-10A) and triple-negative breast cancer cells (less metastatic MDA-MB-468 and highly metastatic MDA-MB-231). In Figure 6A,B, the stiffness and viscosities of the cells can be observed to decrease with increased cancer progression, from a normal cell state, to a slightly metastatic state, and to a highly metastatic state. Statistically significant variations were found between the normal MCF-10A cells and highly metastatic MDA-MB-231 cells, and between the less metastatic MDA-MB-468 cells and the highly metastatic MDA-MB-231 cells. There were no significant variations between the normal MCF-10A and the less metastatic MDA-MB-468 cells. Figure 6C shows the relaxation time for these cell types, which showed no significant variations; this may vary with other types of cells.
Figure 1: Shear assay experimental setup. The method involves the use of (A) a programmable syringe pump to both infuse and withdraw viscous flow media into and out of (B) a flow chamber at a constant shear rate. (C) A rubber gasket containing a rectangular window (dimensions: 20.5 mm x 2.5 mm x 0.254 mm) and vacuum suction is fit in a 35 mm x 10 mm Petri dish containing cultured cells of interest to establish a flow field. (D) The cells are subjected to flow within the established field and are deformed by fluid shear stress, which is derived via (E) an equation for wall shear stress. (F) A cell of interest within the flow field is imaged using a microscope at 63x magnification, and real-time deformation is captured and recorded using the microscope software on the monitor. The induced fluid shear stress is utilized along with the imaged deformation to quantify the mechanical properties of the single cell. This figure is modified from Hu et al.3. Please click here to view a larger version of this figure.
Figure 2: Strain analysis using digital image correlation. Images extracted from a recording of the shear assay show the maximum shear strain experienced by individual cells throughout the experiment. DIC tracks changes in the naturally patterned structure of the cells by locating and tracking a block of pixels subset within each frame or image. (A) The image of the cell before the DIC analysis. (B) Masking of the cells, selecting the region of interest, and seeding the cells to aid deformation tracking. (C) The onset of deformation. (D) Deformation build-up. (E) Increasing deformation over time. (F) Application of strain gauges to quantify strain during deformation. Abbreviation: DIC = digital image correlation. This figure is modified from Hu et al.3. Please click here to view a larger version of this figure.
Figure 3: Representative sample of strain versus time data of glioblastoma cells and mathematical modeling of the viscoelastic model. (A) The strain versus time data obtained by DIC show different creep regimes (primary, secondary, and tertiary) of the cell. (B) The strain versus time data is exported to MATLAB for quantification of the mechanical properties of the individual cell. Abbreviation: DIC = digital image correlation. This figure is modified from Hu et al.3. Please click here to view a larger version of this figure.
Figure 4: Viscoelastic model for the determination of viscoelastic properties. A three-parameter generalized Maxwell model, which comprises Kelvin (dashpot) and Voigt (dashpot + spring) models connected in series, for characterization of the viscoelastic behavior of cells. The result of the viscoelastic modeling using the shear technique is the cell's elasticity, viscosity, and relaxation time. This figure is modified from Hu et al.3. Please click here to view a larger version of this figure.
Figure 5: Graphical depiction of the types of material properties of cells that can be extracted from the shear assay technique, including stiffness, viscosity, creep, and relaxation time. Please click here to view a larger version of this figure.
Figure 6: Material properties of cells obtained from the shear assay technique. Cell stiffness was significantly different between the nucleus and cytoplasm of normal breast cells (MCF-10A) and highly metastatic breast cells (MDA-MB-231), and between less metastatic cells (MDA-MB-468) and highly metastatic cells (MDA-MB-231) (A). However, there was no significant difference between the stiffness of the nucleus and cytoplasm of normal cells (MCF-10A) and less metastatic cells (MDA-MB-468). Similar trends were also found in nuclear and cytoplastic viscosities (B). The relaxation times of these cells did not exhibit significant mechanical differences (C), though other cell types may. This figure is modified from Hu et al.3. Please click here to view a larger version of this figure.
The shear assay method, which includes setting up an pseudo-mechanobiological environment to simulate the interaction of cells with the surrounding mechanical microenvironment and their responses to mechanical stresses, has produced a catalog of cellular mechanical properties, whose patterns show conserved physical atypia among cancerous cell lines3,4,5,7,8. This method combines an understanding of basic fluid mechanics and physics to characterize unique mechanical properties of cells and provides potential material or mechanical biomarkers for the detection of several difficult-to-treat cancers3,4.
The shear assay technique has certain limitations. Ensuring a single-cell culture can be challenging, owing to the quick growth and proliferation of certain cell types, making it difficult to visualize and analyze single cells. Stress tolerance and the cellular response can be heterogeneous from one cell to another depending on the cell line, and as such requires many cells and troubleshooting, to determine the average critical stress required to structurally deform the cells while maintaining their viability to analyze their material properties as live cells.
Troubleshooting for this technique includes determining i) the growth and proliferation rates of cell lines of interest and ii) the time needed for cells to adhere to the substrate prior to shear stress. Researchers using this protocol may choose to assess the expression of key cytoskeletal and adherent proteins, such as actin, cadherins, and other focal adhesion proteins. It is also necessary to determine the critical stress magnitudes required to induce the optimal cell deformation for the strain analysis software (DIC).
The shear assay technique can serve as an effective technique to explore the unique mechanical behaviors and inherent mechanical properties of different types of cells, potentially aiding in specific and sensitive identification and diagnoses that are independent of cell surface markers typically used in current cancer cell diagnostic methods. It also provides significant improvements over existing mechanical characterization methods that rupture the cell membrane prior to testing.
The unique shear assay technique has been extensively explored for over two decades for characterization of the behavior of biological cells and the determination of their mechanical properties. This experimental setup closely mimics the effects of fluid shear stresses in the body by applying controlled fluid shear stress on cells in vitro. Normal and cancerous cells are observed to respond to these stresses differently, and thus this technique provides insights to their structural and mechanical properties and behaviors. This technique has been explored in several prior studies to determine the viscoelastic properties of cells, and interestingly, significant cellular mechanical differences were observed in healthy normal cells and cancerous cells3,4,9. Further, significant intracellular mechanical differences were observed between the nucleus and cytoplasm of cells. These unique mechanical properties can therefore be explored as potential mechanical biomarkers for the detection of different cancers in clinical settings.
The authors have nothing to disclose.
The authors thank previous researchers from the Soboyejo group at Worcester Polytechnic Institute who first pioneered this technique: Drs. Yifang Cao, Jingjie Hu, and Vanessa Uzonwanne. This work was supported by the National Cancer Institute (NIH/NCI K22 CA258410 to M.D.). Figures were created with BioRender.com.
CELL CULTURE | |||
.25% Trypsin, 2.21 mM EDTA, 1x[-] sodium bicarbonate | Corning | 25-053-ci | For cellular detachment from substrate in cell culture |
15 mL centrifuge tubes | Falcon by Corning | 05-527-90 | |
35 mm Petri dishes | Corning | 430165 | |
50 mL centrifuge tubes | Falcon by Corning | 14-432-22 | |
centrifuge | any | For sterile cell culture | |
Dulbecco's Modification of Eagle's Medium (DMEM) 1x | Corning | 10-013-cv | Or any other media for culturing cells. DMEM was used for culturing U87 cells |
gloves | any | For sterile cell culture | |
Heracell Vios 160i CO2 Incubator | Thermo Scientific | 51033770 | For Incubation during cell culture |
Hood | any | For sterile cell culture | |
micropipette | any | For sterile cell culture | |
micropipette tips | any | For sterile cell culture | |
Microscope | Leica/any | For sterile cell culture | |
Phosphate Buffered Saline without calcium and magnesium PBS, 1x | Corning | 21-040-CM | |
pipetman | any | For sterile cell culture | |
pipette tips | any | For sterile cell culture | |
Precision GP 10 liquid incubator | Thermo Scientific | TSGP02 | |
T25 flask | Corning | 430639 | |
T75 flask | Corning | 430641U | |
SHEAR ASSAY | |||
100 mL beaker | any | For creating DMEM + methyl cellulose viscous shear media | |
DMEM | Corning | ||
Flow chamber + rubber gasket | Glycotech | 31-001 | Circular Flow chamber Kit ( for 35 mm tissue culture dishes) |
Hybrid Rheometer | HR-2 Discovery Hybrid Rheometer | For determination of shear fluid viscosity | |
magnetic stir bar | any | For creating DMEM + methyl cellulose viscous shear media | |
magnetic stir plate | any | For creating DMEM + methyl cellulose viscous shear media | |
methyl cellulose | any | To increase viscosity of DMEM in flow media | |
Syringe Pump | KD Scientific Geminin 88 plus | 788088 | For programming fluid infusion and withdrawal |
syringes, tubing, and connectors | For shear apparatus setup | ||
SOFTWARE | |||
ABAQUS software | Simulia | ||
Digitial Image Correlation software | LaVision, Germany | DAVIS 10.1.2 | |
Imaging software | Leica/any microscope software | ||
MATLAB | MATLAB | MATLAB_R2020B |