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 proliferation and growth rates, to ensure adequate cellular growth and attachment while avoiding cell aggregation. These parameters vary with cell type.</li>
	</ol>
	</li>
</ol>
2. Shear assay experiment
<ol>
	<li>Preparation of shear assay viscous flow media
	<ol>
		<li>To ensure a slightly viscous flow media (0.015-0.02 Pa&#183;s), measure and add 0.05 wt% of non-toxic and non-allergenic methylcellulose (4 &#160;Pa&#183;s) to the culture media.</li>
		<li>To ensure a homogeneous mix, preheat the base culture media for ~10-20 min at a temperature of ~60-70 &#176;C with a magnetic stirrer/hot plate. While continually stirring the media, gently add the methylcellulose such that it quickly disperses, to avoid coagulation of the methylcellulose particles. Allow this process to continue for ~15-24 h to ensure a clear solution of media + cellulose. 
		NOTE: Avoid excessive heating of the solution.</li>
		<li>To measure the viscosity of the flow media, test ~0.5-1 mL of the representative flow media using a rheometer. From the readout, determine the fluid viscosity and utilize this value to represent the viscosity of the shear fluid medium (&#956;) to compute shear stress using equation (2).</li>
	</ol>
	</li>
	<li>Shear apparatus setup
	<ol>
		<li>Set up the shear assay system of dual syringes (60 mL or 100 mL) connected to a programmable syringe pump for infusion and withdrawal of the viscous culture media (Figure 1).</li>
		<li>Attach both syringes to the flow chamber via 1/16 inch tubing and tubing connectors.</li>
		<li>Fasten a rubber gasket to provide a controlled, uniform flow on single cells along the flow path (Figure 1). The rubber gasket comes in different sizes depending on the flow profile to be attained (laminar or turbulent) and the desired area of observation (e.g., length of 22.5 mm, width of 2.5 mm, and height of 0.254 mm) (Figure 1).</li>
		<li>Program the pump to infuse and withdraw a certain volume of fluid (e.g., 60 mL) at a designated rate (e.g., 1 mL/min) and select the corresponding syringes (e.g., 60 mL). 
		NOTE: Account for the maximum infusion and withdrawal volume presets to avoid jamming or malfunction. Use equation (2) for the calculation of the required pump shear rate (assuming the required stress and viscosity are known).</li>
	</ol>
	</li>
	<li>Pre-shear setup
	<ol>
		<li>Fill the syringe with the prepared viscous flow media.</li>
		<li>Attach the syringe, filled with 60 or 100 mL (or as needed) of viscous flow media, and an empty 60 mL syringe to their respective locations on the programmable syringe pump. Via tubing and tubing connectors, connect both syringes to the flow chamber.</li>
		<li>To ensure easy identification of single cells and a fastened connection between the flow chamber and the Petri dish, attach the rubber gasket to the flow chamber.</li>
		<li>Aspirate the cell culture media from the Petri dish containing the cells of interest.</li>
		<li>Wash off dead cells and loosely attached cells using phosphate-buffered saline (PBS).</li>
		<li>Aspirate the PBS.</li>
		<li>Insert and fix the flow chamber and rubber gasket (~34 mm x 9 mm) onto the Petri dish (35 mm x 10 mm) containing the attached cells.</li>
		<li>Place the fitted microfluidic flow chamber + cells on a cultured dish onto an inverted microscope, with a microscope objective high enough to obtain high-quality images with high pixel values (usually between 40x and 63x magnification) and a display monitor.</li>
		<li>Select the live image (time-lapse on some software) option from the microscope software on the display monitor. Ensure the microscope software on the PC has either a t (time-lapse) functionality or can take video recordings.</li>
		<li>Focus the microscope objective, ensuring adequate contrast and distinct cell edges. This is necessary for the image analysis post-shear. Move the microscope stage to ensure the cells are clearly visible on the display monitor and are live images.</li>
		<li>Select a cell or multiple distinct cells within the imaging/flow path of the fitted flow chamber + Petri dish (the area/path created by the fitting of the gasket to the flow chamber).</li>
	</ol>
	</li>
	<li>Shear and imaging
	<ol>
		<li>To maintain a continuous uniform flow, select similar infusion and withdrawal rates and ensure laminar flow of the fluid, usually between 1 mL/min and 5 mL/min. For low laminar flow regimes, ensure a Reynold&#39;s number of Re &#60; 100.</li>
		<li>Click on Run on the shear pump to inject and withdraw the shear fluid (prepared viscous media) at a continuous rate. Ensure that there are no bubbles during fluid infusion, as this might introduce external unaccounted stress on the cells.</li>
		<li>Begin recording a video by clicking record on the microscope software before the infused shear fluid makes contact with the cell(s) of interest under the microscope.</li>
		<li>Continue to record for 7 min, or for the desired duration of stress exposure, or until the cell(s) of interest shears off the bottom of the dish. Click stop recording on the microscope software when the run is completed as desired.</li>
		<li>Save the recording and extract as .tiff files. Preferably, extract images at 1 frame per second for facile analysis.</li>
	</ol>
	</li>
</ol>
3. Data processing
<ol>
	<li>Digital image correlation procedure (image analysis)
	<ol>
		<li>If the recording from the microscope was extracted as a video file, convert it to image frames (preferably .tiff file format).</li>
		<li>Import the images derived from the shear assay recording to Davis 10.1.2 software (DIC software) to track the movement of the naturally patterned structures of the cell by locating each block of pixel (subset) of the reference image in the respective new images (deformed images) (Figure 2).</li>
		<li>For an optimized correlation, utilize a subset size of 31 x 31 pixels, a step size (deformation distance of each subset) of 20&#160;pixels, and the sum of the differential track option, which tracks the deformation of a new image with respect to the last image. The result of this correlation is a strain-time plot (Figure 3) that can be exported as a .csv file for further analysis in MATLAB.</li>
		<li>Map out the region of interest for a chosen single cell. Select arbitrary points within the mapped cell to track deformation. For an irregular shape, such as the cell, use a polygon mask to map out the cell&#39;s geometry.</li>
		<li>After mapping, choose specific points on the cell (nucleus or cytoplasm) to be analyzed by clicking add strain gauge and drawing out individual strain gauges at points within the defined cellular boundary.</li>
		<li>Click on Run to begin the strain processing and obtain strain versus time data.</li>
		<li>Double-click (or right-click) on the generated strain-time plot and select export data as a spreadsheet.</li>
	</ol>
	</li>
</ol>
4. Mechanical property characterization
<ol>
	<li>Viscoelastic property characterization
	<ol>
		<li>Save the .csv file containing the strain-time data from the DIC software in a separate folder for easy readability by MATLAB.</li>
		<li>Run MATLAB and click on the editor tab to open an editor page to write a code that reads the spreadsheet, cell by cell.</li>
		<li>Change the MATLAB path (the folder path that accesses the file of interest) to access the folder containing the data to be analyzed, for example, Users/Username/Desktop/data.</li>
		<li>On the MATLAB editor page, access the spreadsheet data using the customized code. For example: a1= xlsread(&#39;data&#39;,&#39;run1&#39;,&#39;A4:A183&#39;), where a1 represents the identifier, xlsread is the MATLAB function that reads the .csv file (in this case, as a spreadsheet), data is the file name, run1 is the sheet name, and A4:A183 is the range of data of interest in cell A of the spreadsheet data to be analyzed. For a complete fit, analyze x and y (time and strain, respectively). For example: 
		a1=xlsread(&#39;data&#39;,&#39;run1&#39;,&#39;A4:A183&#39;); 
		b1=xlsread(&#39;data&#39;,&#39;run1&#39;,&#39;B4:B183&#39;); 
		a1 = x (time), and b1 = y (strain).</li>
		<li>In MATLAB, click Apps | Curve Fitter | Custom Equation. Clear the representative custom equation and input the viscoelastic model equation [equation (1)], where&#160;&#949; represents the x variable and t represents the y variable. 
		<img alt="Equation 1" src="/files/ftp_upload/65333/65333eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1) 
		Here,&#160;&#949; represents the strain, &#963; represents the shear stress,&#160;&#951;1 represents the viscosity, E represents the elasticity, t represents the time, and&#160;&#964; represents the relaxation time, which characterizes the maximum time required for the cell to return to its original shape after primary deformation. It is expressed as &#964; = <img alt="Equation 2" src="/files/ftp_upload/65333/65333eq02.jpg" style="margin: auto;" />, where&#160;&#951;2 is the secondary viscosity term for the second dashpot (Figure 4).</li>
		<li>Reassign new variables to the viscoelastic parameters within the custom equation interface. (&#949;, &#951;1, E, &#963;, t, and &#964; = <img alt="Equation 2" src="/files/ftp_upload/65333/65333eq02.jpg" style="margin: auto;" />) will represent (x, a, b, K, y, and c), respectively. Here, x and y are the independent and dependent variables, respectively. Shear stress (&#963;) can be determined using equation (2): 
		<img alt="Equation 3" src="/files/ftp_upload/65333/65333eq03.jpg" style="margin: auto;" />&#160; &#160; &#160;(2) 
		&#8203;Here,&#160;&#956; represents the viscosity of the shear fluid medium, Q is the pump flow rate, and w and h are the width and height of the flow channel shown in Figure 1C, respectively.</li>
		<li>Click on Select Data to select the Time (a1) and Strain (b1) for each set of data.</li>
		<li>Ensure the Auto Fit box option is checked. This runs the fit automatically when data (x and y) are selected.</li>
		<li>Select the fitting methods to tighten the boundary conditions. Click on Advanced Options under the Methods category and select Nonlinear Least Squares. Under Robust, select Off, and under Algorithm, select Trust-Region. Leave every other parameter as it is.</li>
		<li>The new variables post-fitting (a, b, and c) represent the viscoelastic properties, viscosity, elasticity, and relaxation time (&#951;1, E, EQUAT), of the cell, respectively (Figure 5).</li>
		<li>Look for a high R-square value of the fit (R2 &#62; 80%) to ensure that the data output can be considered a true fit of the viscoelastic model (Figure 3)</li>
	</ol>
	</li>
</ol>

Enhancing Targeted Cancer Diagnosis by Quantifying Single-Cell Mechanics

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+the+viscoelastic+properties+and+the+actin+cytoskeletal+structure+of+triple+negative+breast+cancer+cells.">An investigation of the viscoelastic properties and the actin cytoskeletal structure of triple negative breast cancer cells.</a> Journal of the Mechanical Behavior of Biomedical Materials. 86, 1-13 (2018).</li><li>Onwudiwe, K., 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+creep+properties+and+the+cytoskeletal+structures+of+non-tumorigenic+breast+cells+and+triple-negative+breast+cancer+cells.">Investigation of creep properties and the cytoskeletal structures of non-tumorigenic breast cells and triple-negative breast cancer cells.</a> Journal of Biomedical Materials Research. 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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. 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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 authors have no competing financial interests to disclose.

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.

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

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

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.

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 ...

shear-assay-protocol-for-determination-single-cell-material

Research

JoVE Journal

Cancer Research

1.6K Views.  University of Notre Dame. 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. 

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

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. Here, we describe a quick and cost-effective one-step assay for simultaneously assessing the major modes of clonogenic cell death induced by IR, i.e., apoptosis, mitotic catastrophe, and cellular senescence. In this method, cells grown on a cover slip are irradiated with X-rays and stained with 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are identified based on the characteristic morphologies of the DAPI-stained nuclei. Apoptosis is determined by the presence of apoptotic bodies (i.e., condensed and fragmented nuclei). Mitotic catastrophe is determined by the presence of nuclei that exhibit two or more distinct lobes and micronuclei. Cellular senescence is determined by the presence of senescence-associated heterochromatic foci (i.e., nuclear DNA containing 30-50 bright, dense foci). This approach allows the experimenter to easily screen for clonogenic cell death modes using various cell lines, treatment settings, and/or time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence microscopy.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a &#39;molecular timeline&#39; since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC).

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

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

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Chapters in this video

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8⁺ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes