The authors have nothing to disclose.

1. Preparation of Glass Coverslips for PAAs
<ol>
	<li>Clean 12 mm coverslips with low lint wipes.</li>
	<li>Flame the 12 mm coverslips and the 14 mm coverslip in the microwell of each 35 mm glass bottom dish by passing them through a Bunsen burner flame using tweezers.</li>
	<li>Treat the microwells with 200 &#181;l of 0.1 N NaOH for 5 min at room temperature.</li>
	<li>Aspirate and air dry the microwells for 30 min.</li>
	<li>Treat the microwells with 50-100 &#181;l of 3-aminopropyltrimethoxysilane for 10 min at room temperature in the fume hood. This chemical reacts with plastic; therefore, use glass pipettes and do not fill the microwells completely to avoid contact with the dish plastic.</li>
	<li>Wash the microwells with ultrapure water for approximately 10 min until the 3-aminopropyltrimethoxysilane becomes clear.</li>
	<li>Rinse the microwells with ultrapure water twice using a squeeze bottle.</li>
	<li>Wash the microwells with 2 ml of ultrapure water at room temperature on a rocker set at a medium speed (~1 Hz) for 10 min.</li>
	<li>Aspirate and air dry the microwells for 30 min.</li>
	<li>Treat the microwells with 2 ml of 0.5% glutaraldehyde solution at room temperature on a rocker set at a medium speed (~1 Hz) for 30 min.</li>
	<li>Wash the microwells with 2 ml of ultrapure water at room temperature on a rocker set at medium speed (~1 Hz) for 10 min. Repeat two more times for a total of 30 min.</li>
	<li>Dry microwells at a steep angle (60&#186; or greater) for 30 min. 
	Note: Dishes can be stored for 2 months in a dessicator.</li>
</ol>
2. Preparation of PAAs for Invadopodia Assays
<ol>
	<li>For a 1 ml solution of the soft PAA (8% acrylamide and 0.05% BIS), combine 200 &#181;l of 40% acrylamide, 25 &#181;l of 2% BIS, and 574 &#181;l of ultrapure water (Table 1).</li>
	<li>For a 1 ml solution of the hard PAA (8% acrylamide and 0.35% BIS), combine 200 &#181;l of 40% acrylamide, 175 &#181;l of 2% BIS, and 409 &#181;l of ultrapure water (Table 1).</li>
	<li>For a 1 ml solution of the rigid PAA (12% acrylamide and 0.60% BIS), combine 300 &#181;l of 40% acrylamide, 300 &#181;l of 2% BIS, and 169 &#181;l of ultrapure water (Table 1).</li>
	<li>Degas the solutions for 15 min. Add 200, 215, and 230 &#181;l of 1 mg/ml human plasma fibronectin in ultrapure water to the soft, hard, and rigid PAA solutions, respectively. For all solutions, add 1 &#181;l of 10 mg/ml acrylic acid N-hydroxysuccinimide (NHS) ester, 5 &#181;l of a 100 mg/ml ammonium persulfate, and 2 &#181;l of N,N,N&#8242;,N&#8242;-tetramethylethylenediamine (TEMED; Table 1). Mix with gentle pipetting and avoid creating bubbles in solutions.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&#160; 
			PAA 
			(1 ml)</td>
			<td>40% 
			AA 
			(&#956;l)</td>
			<td>2% 
			BIS 
			(&#956;l)</td>
			<td>Ultrapure 
			Water 
			(&#956;l)</td>
			<td>1 mg/ml 
			FN 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">10 mg/ml 
			NHS Ester 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">100 mg/ml 
			APS 
			(&#956;l)</td>
			<td>&#160; 
			TEMED 
			(&#956;l)</td>
			<td>Elastic 
			Modulus 
			(Pa)</td>
		</tr>
		<tr>
			<td>Soft</td>
			<td>200</td>
			<td>25</td>
			<td>574</td>
			<td>200</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>1023</td>
		</tr>
		<tr>
			<td>Hard</td>
			<td>200</td>
			<td>175</td>
			<td>409</td>
			<td>215</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>7307</td>
		</tr>
		<tr>
			<td>Rigid</td>
			<td>300</td>
			<td>300</td>
			<td>169</td>
			<td>230</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>22692</td>
		</tr>
	</tbody>
</table>
Table 1: Ingredient volumes and resulting elastic moduli for soft, hard, and rigid PAAs used in the invadopodia assays. AA = acrylamide, FN = fibronectin, APS = ammonium persulfate.
<ol start="5">
	<li>Pipette 8.48 &#181;l of the PAA solution on to the center of each microwell. 
	Note: This volume theoretically yields a PAA of 75 &#181;m in thickness.</li>
	<li>Gently lower the flamed side of the 12 mm coverslip on to the droplet in the microwell using tweezers.</li>
	<li>Allow the sandwiched PAA solution to polymerize for 15-30 min. Verify polymerization by checking the leftover solution.</li>
	<li>Add 2 ml of 1x PBS to each dish and remove the 12 mm coverslips with tweezers. 
	Note: 10x PBS is made in the laboratory and composed of 0.011 M KH2PO4, 1.54 M NaCl, and 0.056 M Na2HPO4.</li>
	<li>Wash the microwells with 2 ml of 1x PBS at room temperature for 5 min. Repeat two more times for a total of 15 min.</li>
</ol>
3. Preparation of PAAs for Traction Force Assays
<ol>
	<li>For a 1 ml solution of the soft PAA (8% acrylamide and 0.05% BIS), combine 200 &#181;l of 40% acrylamide, 25 &#181;l of 2% BIS, and 566 &#181;l of ultrapure water (Table 2).</li>
	<li>For a 1 ml solution of the hard PAA (8% acrylamide and 0.35% BIS), combine 200 &#181;l of 40% acrylamide, 175 &#181;l of 2% BIS, and 401 &#181;l of ultrapure water (Table 2).</li>
	<li>For a 1 ml solution of the rigid PAA (12% acrylamide and 0.60% BIS), combine 300 &#181;l of 40% acrylamide, 300 &#181;l of 2% BIS, and 161 &#181;l of ultrapure water (Table 2).</li>
	<li>Degas the solutions for 15 min. Add 200, 215, and 230 &#181;l of 1 mg/ml human plasma fibronectin to the soft, hard, and rigid PAA solutions, respectively. Sonicate 8 &#181;l of 200 nm red fluorescent microspheres (excitation/emission of 580/605 nm) for 30 sec. For each solution, add 8 &#181;l of 200 nm red fluorescent microspheres, 1 &#181;l of 10 mg/ml acrylic acid NHS ester, 5 &#181;l of a 100 mg/ml ammonium persulfate, and 2 &#181;l of TEMED (Table 2). Mix with gentle pipetting and avoid creating bubbles in solutions.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td style="width:40px">&#160; 
			PAA 
			(1 ml)</td>
			<td>40% 
			AA 
			(&#956;l)</td>
			<td>2% 
			BIS 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">Ultrapure 
			Water 
			(&#956;l)</td>
			<td>1 mg/ml 
			FN 
			(&#956;l)</td>
			<td>&#160; 
			Micro-spheres 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">10 mg/ml 
			NHS Ester 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">100 mg/ml 
			APS 
			(&#956;l)</td>
			<td>&#160; 
			TEMED 
			(&#956;l)</td>
			<td>Elastic 
			Modulus 
			(Pa)</td>
		</tr>
		<tr>
			<td>Soft</td>
			<td>200</td>
			<td>25</td>
			<td>566</td>
			<td>200</td>
			<td>8</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>1023</td>
		</tr>
		<tr>
			<td>Hard</td>
			<td>200</td>
			<td>175</td>
			<td>401</td>
			<td>215</td>
			<td>8</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>7307</td>
		</tr>
		<tr>
			<td>Rigid</td>
			<td>300</td>
			<td>300</td>
			<td>161</td>
			<td>230</td>
			<td>8</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>22692</td>
		</tr>
	</tbody>
</table>
Table 2: Ingredient volumes and resulting elastic moduli for soft, hard, and rigid PAAs used in the traction force assays. AA = acrylamide, FN = fibronectin, APS = ammonium persulfate.
<ol start="5">
	<li>Pipette 8.48 &#181;l of the PAA solution on to the center of each microwell. 
	Note: This volume theoretically yields a PAA of 75 &#181;m in thickness.</li>
	<li>Gently lower the flamed side of the 12 mm coverslip on to the droplet in the microwell using tweezers.</li>
	<li>Allow the sandwiched PAA solution to polymerize for 15-30 min. Verify polymerization by checking leftover solution.</li>
	<li>Add 2 ml of 1x PBS to each dish and remove the 12 mm coverslips with tweezers.</li>
	<li>Wash the microwells with 2 ml of 1x PBS at room temperature for 5 min. Repeat two more times for a total of 15 min.</li>
</ol>
4. Preparation of ECM for Invadopodia Assays
<ol>
	<li>Heat the gelatin solution (1% gelatin and 1% sucrose) to 37 &#176;C.</li>
	<li>Treat the PAAs with 150 &#181;l of the gelatin solution for 1 min then carefully aspirate from the bottom of the microwells when holding the glass bottom dishes at a 45&#176; angle.</li>
	<li>Dry the remaining thin layer of gelatin on the PAAs in the microwells at a steep angle (60&#176; or greater) to optimize drying for 60 min.</li>
	<li>Treat the microwells with 2 ml of a chilled 0.5% glutaraldehyde solution on ice for 15 min followed by room temperature for 30 min.</li>
	<li>Wash the microwells with 2 ml of a 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Treat the microwells with 2 ml of a sodium borohydride solution for 1 min. Gently tap dishes on bench to remove bubbles that form on the surface of the gelatin.</li>
	<li>Aspirate and wash the microwells with 2 ml of a 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Dilute a FITC-labeled human plasma fibronectin solution to 50 &#181;g/ml with a 1x PBS and centrifuge at 175,000 x g at 4 &#176;C for 15 min. Either purchase commercially available labeled fibronectin or label it as follows:
	<ol>
		<li>Dissolve 5 mg of fibronectin in 10 ml of borate buffer (170 mM sodium metaborate tetrahydrate and 40 mM NaCl) and place in dialysis tubing.</li>
		<li>Label the fibronectin by placing the tubing in 200 ml of borate buffer containing 6 mg of dissolved FITC at room temperature on a magnetic stirrer set at the lowest setting for 1.5 hr in the dark.</li>
		<li>Dialyze with 500 ml of a 1x PBS on a magnetic stirrer set at the lowest setting for 3 days in a dark cold room (4 &#176;C) with two volume changes a day.</li>
		<li>Calculate FITC-labeled fibronectin concentration based on the optical density of diluted aliquots (1:200) at 280 nm and 493 nm as [OD280 &#8211; (0.36 x OD493)] / 1.4 times the dilution factor of 200 times 2 (which accounts for the glycerol concentrating the fibronectin in the next step) resulting in mg/ml units.</li>
		<li>Dialyze overnight in a 50% glycerol solution on a magnetic stirrer set at the lowest setting overnight in a dark cold room at 4 &#176;C.)</li>
	</ol>
	</li>
	<li>Treat the microwells with 150 &#181;l of the FITC-labeled fibronectin solution at room temperature for 60 min in the dark.</li>
	<li>Carefully aspirate the FITC-labeled fibronectin solution from the bottom of the microwells when holding the glass bottom dishes at a 45&#176; angle and then fill the glass bottom dishes with 70% ethanol solution at room temperature for 10 min in a dark cell culture hood for sterilization. Also fill glass bottom dish lids or wipe clean with low lint wipes soaked in 70% ethanol.</li>
	<li>Wash glass bottom dishes by filling them with 1x PBS for 5 min. Repeat two more times with 2 ml of 1x PBS for a total of 15 min.</li>
</ol>
5. Preparation and Imaging of Invadopodia Assays
<ol>
	<li>Add 25,000 cancer cells in 2 ml of invadopodia medium (1:1 DMEM and RPMI 1640 with 5% low-protein serum, 10% fetal bovine serum, and 20 ng/ml of freshly added epidermal growth factor) to the glass bottom dishes and incubate overnight.</li>
	<li>Aspirate and treat the microwells with 2 ml of a 3.7% paraformaldehyde solution at room temperature in the dark for 20 min to fix the cells.</li>
	<li>After two quick washes with 2 ml of 1x PBS, treat the microwells with 2 ml of a 0.1% Triton X-100 solution at room temperature in the dark for 5 min to permeabilize the cells.</li>
	<li>After one quick wash with 2 ml of 1x PBS, treat the microwells with 3% bovine serum albumin blocking solution at room temperature in the dark for 60 min to block the cells.</li>
	<li>Incubate the microwells with 150 &#181;l of mouse anti-cortactin antibody (1:750) in blocking solution at room temperature in the dark for 60 min.</li>
	<li>Wash microwells with 2 ml of 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Incubate the microwells with 150 &#181;l of goat anti-mouse 633 antibody (1:500) and 546 phalloidin (1:750) in blocking solution at room temperature in the dark for 60 min.</li>
	<li>Wash microwells with 2 ml of 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Add six drops of mounting medium to fill each microwell and cover with 22 x 22 coverslips.</li>
	<li>Image cortactin and actin to identify invadopodia using excitation and emission filters of 632/647 nm and 556/570 nm, respectively, using a high NA, 40X oil immersion lens on a wide-field inverted microscope. Similarly, image fibronectin to identify ECM degradation using an excitation and emission filter of 492/518 nm.</li>
</ol>
6. Preparation and Imaging of Traction Force Assays
<ol>
	<li>Add 15,000 cancer cells in 2 ml of invadopodia medium to the glass bottom dishes and incubate overnight.</li>
	<li>Aspirate medium in the glass bottom dishes and replace with 2 ml of L-15 medium, with the same supplements (5% low-protein serum, 10% fetal bovine serum, and 20 ng/ml of freshly added epidermal growth factor), pre-warmed to 37 &#176;C.</li>
	<li>Incubate the glass bottom dishes in an environmental chamber on an inverted microscope pre-equilibrated to 37 &#176;C with high humidity for 1 hr.</li>
	<li>Take four sets of images using a high NA, 40X lens on a wide-field inverted microscope with an automated stage to mark cellular positions:
	<ol>
		<li>Image cells on top of the PAAs using phase contrast (&#8220;phase&#8221; image).</li>
		<li>Image fluorescent microspheres using excitation and emission filters of 560/645 nm by focusing slightly under the cells until the first layer of microspheres at the top of the PAAs comes into focus (&#8220;stressed&#8221; image).</li>
		<li>In addition, focus to the bottom of the PAAs (using the microspheres as markers) and take an image (&#8220;bottom&#8221; image). Note: The purpose of this image is to record the z-position to get the actual PAA thickness at each position for traction force calculations as described in Representative Results.</li>
		<li>After these images have been acquired at multiple positions, gently mix in 220 &#181;l of 10% Triton-X solution to remove cells. Image microspheres at the top of the PAAs at each position as previously described (&#8220;null&#8221; image).</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

This work was supported by the National Cancer Institute of the National Institutes of Health under Award Number K25CA143412 (Parekh). We would like to acknowledge that additional support was provided by the Department of Otolaryngology. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

We present a method for fabricating PAAs that can be used as the basis for invadopodia and traction force assays to correlate invasive and contractile cellular behaviors. While PAAs have long been used to look at rigidity effects on cells and calculate traction forces18,24,27, this protocol is the first to develop parallel assays based on PAAs with the same rigidities to correlate invasive and contractile cellular behaviors in response to matrix mechanical properties. Properly activating the coverslips of the ...

The rigidity of the tumor-associated ECM has been identified as a significant factor in driving malignant behavior by increasing actomyosin contractility1-3. While this effect has primarily been demonstrated with breast cancer cells, matrix rigidity has been found to alter invasive properties of cells derived from a variety of cancers4-8 suggesting that tumor rigidity may play a role in other type of cancers. To penetrate cross-linked tissues during invasive migration, cancer cells utilize actin-rich adhesive protrusions known as invadopodia that localize proteinases to focally degrade the ECM9. Invadopodia are considered a hallmark of invasive cells and have been implicated in tumor cell invasion and metastasis10,11. Previous work has shown that matrix rigidity can regulate invadopodia numbers and associated ECM degradation4,12 through myosin II activity and mechanosensitive proteins12. Given the correlation between tumor density and cancer aggressiveness13,14, these results suggest a mechanism by which cancer cells may respond to rigid tumor tissues to drive invasion and metastasis through actomyosin contractility.
In vitro ECM rigidity and in vivo tissue density have been shown to regulate invasive behavior of cancer cells1,15-17. While actomyosin contractility appears to be important in this process, current studies conflict as to whether metastatic capacity is correlated to increased or decreased contractile forces6,18-20. Furthermore, it remains unknown whether these forces directly mediate invadopodia activity21. We recently found that cancer cell contractile forces were dependent on matrix rigidity and were predictive of ECM degradation by invadopodia5. These results suggest that cellular forces may play an important role in cancer progression by mediating invadopodia activity in response to the mechanical properties of the tumor microenvironment.
In order to correlate invasive and contractile properties of cancer cells5, we modified a protocol for creating PAAs with different rigidities that was previously used to investigate rigidity-dependent invadopodia activity4,12,22. By chemically crosslinking human plasma fibronectin throughout the PAAs, these modified hydrogels can be used as the basis for both invadopodia and traction force assays to ensure that cells experienced the same rigidities in both experiments5. In the invadopodia assays, the fibronectin provides a natural binding domain for gelatin to link the overlaid ECM to the PAAs to detect matrix degradation. In the traction force assays, the fibronectin provides a ligand for direct cellular adhesion to detect microsphere displacements used to calculate cellular traction forces. This method results in what we have called soft, hard, and rigid PAAs that are bound to glass bottom dishes and have elastic moduli, E, of 1,023, 7,307, and 22,692 Pa5 which span the range of mechanical properties reported for normal and cancerous tissues23.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-Aminopropyltrimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic acid NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>A8060</td>
			<td>prepare fresh in fume hood 10 mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Life Technologies</td>
			<td>A22283</td>
			<td>can also use rhodamine</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>prepare fresh 10% solution in 1X PBS</td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>18606</td>
			<td>use six drops to fill microwells</td>
		</tr>
		<tr>
			<td>BIS (2%)</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td>make 3% for blocking solution in 1X PBS and store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9777</td>
			<td>pre-equilibrate in borate buffer for 15-30 min</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>use to make acrylic acid NHS ester solution</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PHARMCO-AAPER</td>
			<td>E200</td>
			<td>dilute with ultrapure water to 70%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30070.03</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Polysciences</td>
			<td>00639</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (35 mm coverslips)</td>
			<td>MatTek</td>
			<td>P35G-0-14-C</td>
			<td>coverslips are uncoated</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%)</td>
			<td>Polysciences</td>
			<td>01909</td>
			<td>dilute with 1X PBS to 0.5%</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa Fluor 633 antibody&#160;</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Life Technologies</td>
			<td>33016-015</td>
			<td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>PX-1565-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>mouse anti-cortactin 4F11 antibody&#160;</td>
			<td>EMD Millipore</td>
			<td>05-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>SX-0720-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>use to make 10X PBS stock and borate buffer</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>dilute with ultrapure water to 0.1 N</td>
		</tr>
		<tr>
			<td>Nu-Serum (low-protein serum)</td>
			<td>BD Biosciences</td>
			<td>355500</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros</td>
			<td>416785000</td>
			<td>typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td>
		</tr>
		<tr>
			<td>PBS (sterile)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>use for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Cellgro</td>
			<td>10-040-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>prepare fresh in fume hood 1 mg/ml in 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>sodium metaborate tetrahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0251</td>
			<td>use to make borate buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td>make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining</td>
		</tr>
	</tbody>
</table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

In the invadopodia assay, invadopodia are typically identified by colocalization of markers like actin and cortactin at punctate structures within the cell body (Figure 1). Both actively degrading and non-degrading invadopodia can be counted and are differentiated by whether these structures are colocalized with black areas lacking fluorescent signal in the FITC-labeled fibronectin (Figure 1). Invadopodia are manually counted, and ECM degradation per cell is determined by manually thresh...

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

Research

JoVE Journal

Medicine

12.0K Views.  Vanderbilt University Medical Center. Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

Video: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...