Name: Author Spotlight: Shear Assay Protocol for the Determination of Single-Cell Material Properties
Uploaded: 2023-05-19T13:30:00
Description: Watch this Scientific Journal Video about Shear Assay Protocol for the Determination of Single-Cell Material Properties at JoVE.com

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.

1. Preparation for the single-cell shear assay
<ol>
	<li>Cell culture
	<ol>
		<li>Seed approximately 50,000 suspended single cells in a 35 mm x 10 mm Petri dish containing 2 mL of culture media. 
		NOTE: Vortex the suspended cells prior to seeding to break apart cell aggregates.</li>
		<li>Incubate the cells at 37 &#176;C and allow between 10 to 48 h for cell attachment and complete cytoskeletal protein formation. 
		&#8203;NOTE: Consider the duration of cellular attachment, as well as prolif...

Enhancing Targeted Cancer Diagnosis by Quantifying Single-Cell Mechanics

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shear+assay+study+of+single+normal/breast+cancer+cell+deformation+and+detachment+from+poly-di-methyl-siloxane+(PDMS)+surfaces.">A shear assay study of single normal/breast cancer cell deformation and detachment from poly-di-methyl-siloxane (PDMS) surfaces.</a> Journal of the Mechanical Behavior of Biomedical Materials. 91, 76-90 (2019).</li><li>Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+biophysics+of+cancer+cells.">Biomechanics and biophysics of cancer cells.</a> Acta Biomaterialia. 3 (4), 413-438 (2007).</li><li>Cao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+viscoelasticity+of+human+osteosarcoma+cells+using+a+shear+assay+method.">Investigation of the viscoelasticity of human osteosarcoma cells using a shear assay method.</a> Journal of Materials Research. 21 (8), 1922-1930 (2006).</li><li>Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+measurement+of+human+osteosarcoma+cell+elastic+modulus+using+shear+assay+experiments.">On the measurement of human osteosarcoma cell elastic modulus using shear assay experiments.</a> Journal of Materials Science. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropipette+aspiration+of+living+cells.">Micropipette aspiration of living cells.</a> Journal of Biomechanics. 33 (1), 15-22 (2000).</li><li>Evans, E., Yeung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparent+viscosity+and+corticcal+tension+of+blood+granulocytes+determined+by+micropipet+aspiration.">Apparent viscosity and corticcal tension of blood granulocytes determined by micropipet aspiration.</a> Biophysical Journal. 56 (1), 151-160 (1989).</li><li>Van Vliet, K. J., Bao, G., Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biomechanics+toolbox:+experimental+approaches+for+living+cells+and+biomolecules.">The biomechanics toolbox: experimental approaches for living cells and biomolecules.</a> Acta Materialia. 51 (19), 5881-5905 (2003).</li><li>Moeendarbary, E., Harris, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics:+principles,+practices,+and+prospects.">Cell mechanics: principles, practices, and prospects.</a> Wiley Interdisciplinary Reviews. Systems Biology and Medicine. 6 (5), 371-388 (2014).</li><li>Choi, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+shear+stress+promotes+epithelial-mesenchymal+transition+by+downregulating+ERK+and+GSK3beta+activities.">Hydrodynamic shear stress promotes epithelial-mesenchymal transition by downregulating ERK and GSK3beta activities.</a> Breast Cancer Research. 21 (1), 6 (2019).</li><li>Northcott, J. M., Dean, I. S., Mouw, J. K., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeling+stress:+The+mechanics+of+cancer+progression+and+aggression.">Feeling stress: The mechanics of cancer progression and aggression.</a> Frontiers in Cell and Developmental Biology. 6, 17 (2018).</li><li>Onwudiwe, K., Najera, J., Siri, S., Datta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+tumor+mechanical+stresses+promote+cancer+immune+escape.">Do tumor mechanical stresses promote cancer immune escape.</a> Cells. 11 (23), 3840 (2022).</li><li>Heldin, C. H., Rubin, K., Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+interstitial+fluid+pressure+-+an+obstacle+in+cancer+therapy.">High interstitial fluid pressure - an obstacle in cancer therapy.</a> Nature Reviews. Cancer. 4 (10), 806-813 (2004).</li><li>Krog, B. L., Henry, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+the+circulating+tumor+cell+microenvironment.">Biomechanics of the circulating tumor cell microenvironment.</a> Advances in Experimental Medicine and Biology. 1092, 209-233 (2018).</li><li>Moose, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cells+resist+mechanical+destruction+in+circulation+via+RhoA/actomyosin-dependent+mechano-adaptation.">Cancer cells resist mechanical destruction in circulation via RhoA/actomyosin-dependent mechano-adaptation.</a> Cell Reports. 30 (11), 3864-3874 (2020).</li><li>Mao, B. H., Nguyen Thi, K. M., Tang, M. J., Kamm, R. D., Tu, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interface+stiffness+and+topographic+feature+dictate+interfacial+invasiveness+of+cancer+spheroids.">The interface stiffness and topographic feature dictate interfacial invasiveness of cancer spheroids.</a> Biofabrication. 15 (1), (2023).</li><li>Kashani, A. 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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. T...

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&#160;which vary between tumorigenic and non-tumorigenic cells. Moreover, the altered mechanical features of cancer cells (i.e., they are often &#34;softer&#34; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELL CULTURE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>.25% Trypsin, 2.21 mM EDTA, 1x[-] sodium bicarbonate</td>
			<td>Corning</td>
			<td>25-053-ci</td>
			<td>For cellular detachment from substrate in cell culture</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Falcon by Corning</td>
			<td>05-527-90</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Falcon by Corning</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium (DMEM) 1x</td>
			<td>Corning</td>
			<td>10-013-cv</td>
			<td>Or any other media for culturing cells. DMEM was used for culturing U87 cells</td>
		</tr>
		<tr>
			<td>gloves</td>
			<td>any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>Heracell Vios 160i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51033770</td>
			<td>For Incubation during cell culture</td>
		</tr>
		<tr>
			<td>Hood</td>
			<td>any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>micropipette</td>
			<td>any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>micropipette tips</td>
			<td>any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica/any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline without calcium and magnesium PBS, 1x</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>pipetman</td>
			<td>any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>pipette tips</td>
			<td>any</td>
			<td></td>
			<td>For sterile cell culture</td>
		</tr>
		<tr>
			<td>Precision GP 10 liquid incubator</td>
			<td>Thermo Scientific</td>
			<td>TSGP02</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>SHEAR ASSAY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL beaker</td>
			<td>any</td>
			<td></td>
			<td>For creating DMEM + methyl cellulose viscous shear media</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow chamber + rubber gasket</td>
			<td>Glycotech</td>
			<td>31-001</td>
			<td>Circular Flow chamber Kit ( for 35 mm tissue culture dishes)</td>
		</tr>
		<tr>
			<td>Hybrid Rheometer</td>
			<td>HR-2 Discovery Hybrid Rheometer</td>
			<td></td>
			<td>For determination of shear fluid viscosity</td>
		</tr>
		<tr>
			<td>magnetic stir bar</td>
			<td>any</td>
			<td></td>
			<td>For creating DMEM + methyl cellulose viscous shear media</td>
		</tr>
		<tr>
			<td>magnetic stir plate</td>
			<td>any</td>
			<td></td>
			<td>For creating DMEM + methyl cellulose viscous shear media</td>
		</tr>
		<tr>
			<td>methyl cellulose</td>
			<td>any</td>
			<td></td>
			<td>To increase viscosity of DMEM in flow media</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>KD Scientific Geminin 88 plus</td>
			<td>788088</td>
			<td>For programming fluid infusion and withdrawal</td>
		</tr>
		<tr>
			<td>syringes, tubing, and connectors</td>
			<td></td>
			<td></td>
			<td>For shear apparatus setup</td>
		</tr>
		<tr>
			<td>SOFTWARE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABAQUS software</td>
			<td>Simulia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digitial Image Correlation software</td>
			<td>LaVision, Germany</td>
			<td>DAVIS 10.1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Leica/any microscope software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MATLAB</td>
			<td>MATLAB_R2020B</td>
			<td></td>
		</tr>
	</tbody>
</table>

shear assay protocol for the determination of single-cell material properties

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.

Author Spotlight: Shear Assay Protocol for the Determination of Single-Cell Material Properties  

Shear Assay Protocol for the Determination of Single-Cell Material Properties

The physical hallmarks of cancer are relatively new and exciting topics in cancer research. Our research seeks to specifically characterize the mechanical properties of both cancers and non-cancer cells to better understand the physical mechanisms by which cancer cells survive shear forces in the body and invade the immune system. Recent development in my period of research is the use of microfluidic devices and high-throughput techniques to understudy in a larger scale, cellular deformability and the viscoelastic properties of cells, and also in the identification of novel biomarkers such as the regulation of cytoskeletal proteins and extracellular matrix proteins for early cancer diagnosis.Current experimental challenges involved in this field are the complexity of the tumor microenvironment, scaling throughput, clinical translation, standardization and reproducibility, and integration of multimodal data. So, some of the significant findings I've established in this field from my past research and from my current research is that cancer cells can be distinguished from normal cells based on their responses to mechanical stressors. And the extent to which resist mechanical stressors is dependent on structural integrity, which is dictated to a reasonable extent by the presence of cytoskeletal proteins like the filamentous actin and other structural proteins within the cell.Our protocol offers a simple, repeatable, and non-destructive approach to characterize cultured cells mechanically. This is in contrast to other techniques like AFM and optical tweezers, which require a certain level of technical expertise in order to perform an experiment in a relatively low throughput. Within the use of the shear acid technique, will greatly advance the single cell mechanical characterization, and will also aid in many different cross-sectional areas such as rare disease diagnosis, personalized diagnosis and care, and monitoring single-cell drug interactions.

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-...

Watch this Scientific Journal Video about Shear Assay Protocol for the Determination of Single-Cell Material Properties at JoVE.com

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.