This work was supported by funding from Department of Defense-Prostate Cancer Research Program (W81XWH-12-1-0124), U54CA143876 from the National Cancer Institute, and the Robert McCooey Genitourinary Oncology Research Fund. We would like to thank Dr. Annarita Lorenzo (Department of Pathology) for providing VE-Cadherin antibodies, and Dr. Marco Seandel (Department of Surgery) for providing HUVECs.

1. Culturing HUVECs on Microslides for Observing CTC-endothelial Interactions
<ol>
	<li>Under the tissue culture hood, first rinse the microslide, channel width of 1 mm with PBS. Gently coat the microslide with 200 &#181;l of 50 &#181;g/ml fibronectin (dissolved in PBS) using a 1-ml Luer-lock syringe.</li>
	<li>Cover the microslide with the lid and keep it inside the tissue culture hood for 30 min. Slow dispensing of the liquid in the microslide prevents bubble formation in the channel.</li>
	<li>Perfuse 200 &#181;l of warm (37 &#176;C) HUVEC growth medium (M199 media, 1M HEPES, 20% FBS, 5 mg/ml heparin, 100 &#181;g/ml endothelial cell growth factor, and L-glutamine) over the microslide and incubate for 20 min at RT. During the perfusion, prepare HUVEC cell suspension.</li>
	<li>Rinse HUVECs with PBS and add 0.05% trypsin-EDTA for 1-2 min at RT. Centrifuge HUVECs in 2 ml growth medium at 180 x g for 5 min.</li>
	<li>Measure the cell concentration using a neubauer hemocytometer and prepare 107 HUVEC cells/100 &#181;l growth medium. Then, carefully remove the medium from the inlet of the microslide using a 200-&#181;l pipette tip.</li>
	<li>Bring the microslide to eye level and using a 1-ml Luer-lock syringe, gently perfuse 200 &#181;l of prepared concentration of HUVECs into the channel. Caution is required at this step to prevent bubble formation. If the bubbles appear, keep perfusing for a slightly longer time until bubbles enter the outlet channel.</li>
	<li>Put an equal volume (~80 &#181;l) of HUVEC media in both the inlet and outlet of the microslide. This prevents the flow of cells in either direction.</li>
	<li>Cover the slide and keep it in the incubator (37 &#176;C) for 1.5 hr.</li>
</ol>
2. Preparation of Flow Chamber Assembly for Overnight HUVEC Culture on a Microslide
<ol>
	<li>Place a sterile 20-ml syringe, female and male Luer connectors, tubing, and a syringe pump in the incubator for 15 min.</li>
	<li>For minimal dead volume, use the tubing with inner diameter of 0.04 inches. Smaller tubing diameter prevents bubble formation.</li>
	<li>Fill the 20-ml syringe with warm (37 &#176;C) HUVEC media (12 ml). Attach the tubing with the connectors onto the syringe. Remove the bubbles. Connect this assembly to the microslide.</li>
	<li>Completely fill the inlet of the microslide with HUVEC media. Bring the filled 20-ml syringe attached to the connector next to the microslide. Gently attach the connector to the microslide.</li>
	<li>Bring the set-up into the incubator and connect it to the syringe pump, set&#160;at 10 &#181;l/min shear rate. Leave the cells O/N in the incubator at 37 &#176;C.</li>
	<li>Next day, disassemble the set-up by removing the connector attached to the inlet of the microslide.</li>
	<li>To upregulate E-selectin expression on ECs, prepare fresh growth medium containing IL-1&#946;&#160;at 10 ng/ml in 4 ml media. Aspirate the media in a 10-ml syringe. Remove the media from the inlet of the microslide and connect the syringe to the slide.</li>
	<li>Set the syringe pump&#160;at 10 &#181;l/min shear rate for 4 hr in the incubator.</li>
</ol>
3. Preparation of Anti-PSMA (J591-488) Labeled Prostate Cancer Cells
<ol>
	<li>During IL-1&#946; incubation of HUVECs, prepare anti-PSMA J591-alexa488 labeled PCa cells. Add 0.05% trypsin-EDTA to MDA cells for 1 min. Do not expose the cells to trypsin for longer times as it can affect the glycopeptides present on the cell surface. Enzyme free cell dissociation reagent can also be used instead of trypsin. Centrifuge at 200 x g for 5 min.</li>
	<li>Resuspend MDA cell pellet in 1 ml H/H buffer (Hanks balanced salt solution/0.1% HSA/10 mM HEPES/1 mM CaCl2). Add anti-PSMA J591-alexa488 antibody&#160;at 20 &#181;g/ml for 30 min at RT in a dark place. Resuspend the cells during the incubation.</li>
	<li>After 30 min, centrifuge the cell solution&#160;at 800 x g for 5 min. Aspirate and resuspend the pellet in 1 ml H/H buffer. Count the labeled MDA cells and bring the final concentration to 1x106 cells/ml. Fill a 5-ml syringe with J591-488 labeled MDA cells and remove the bubbles.</li>
</ol>
4. Preparation of Anti-PSMA (J591-488) Labeled CTCs Enriched from PCa Patients
<ol>
	<li>Collect 7.5 ml blood from prostate cancer patients in a blue cap tube (containing sodium citrate). Gently dilute blood 1:1 in 0.1% BSA/1 mM EDTA/PBS.</li>
	<li>Add 5.3 ml Ficoll-paque plus in a 50-ml conical tube. Layer diluted blood on top of the ficoll-paque. Centrifuge at 400 x g for 30 min.</li>
	<li>Pre-coat all the tubes or tips coming in contact with CTCs with 2% FBS/RPMI-1640/ 1 mM CaCl2/ 4 mM MgCl2 (R/S buffer) to prevent non-specific binding of CTCs to the surfaces. Maintain 4 &#176;C temperature of media and buffers coming in contact with CTCs.</li>
	<li>Collect the peripheral blood mononuclear cell (PBMCs) fraction containing CTCs from the interface in another 50-ml conical tube containing 30 ml R/S buffer. Centrifuge at 400 x g for 8 min.</li>
	<li>Resuspend the pellet and wash again in R/S buffer at 400 x g for 8 min. After centrifugation, resuspend the pellet in H/H buffer. Add anti-PSMA J591-488 antibody&#160;at&#160;20 &#181;g/ml for 30 min at RT in a dark place.</li>
	<li>After 30 min, centrifuge the PBMCs containing J591-488 labeled CTCs at 800 x g for 5 min. Resuspend in H/H buffer. Fill a 1-ml syringe (pre-coated with R/S buffer) with the sample.</li>
</ol>
5. Preparation of Microscope, Syringe Pump and Flow Chamber Assembly
<ol>
	<li>Turn on the inverted microscope and set the Kohler illumination at 10X objective. Bring the syringe pump at the same level as the sample stage of the microscope and set it at 1 dyn/cm2 shear stress (~ 10 &#181;l/min).</li>
	<li>Open the Zeiss Axiovision software. Select the objective on the computer screen. Create a new folder under the tools option in the software.</li>
	<li>Open the smart experiments option in Axiovision and adjust the settings to record short 30 sec videos for 30 min.</li>
	<li>For live fluorescent video, set the image options- 12 msec Exposure, 2 x 2 bin, 5 Gain. These parameters help in attaining videos close to the video frame rate (~ 23 frames per sec) with the Zeiss mRm camera.</li>
	<li>To visualize full width of the flow channel at 10 X objective on the computer screen, use a C-mount adapter (0.63 x f/60 mm Interface).</li>
	<li>Switch on the mercury lamp.</li>
	<li>Place the syringe and the connector containing the cells onto the syringe pump.</li>
	<li>Bring IL-1&#946; stimulated HUVECs from the incubator. Attach the connector to the filled inlet channel of the microslide containing HUVECs.</li>
	<li>Connect the outlet channel with the connector attached to the tubing. Put the tubing into a dish or 15 ml conical tube to collect the flow through.</li>
	<li>Start the infusion through the microslide&#160;at 10 &#181;l/min. Observe the interactions between endothelial cells and labeled MDA cells or labeled CTCs derived from patients under 488-nm filter on the epifluorescence microscope.</li>
	<li>Start recording the experiment as 30 sec short videos. During playback analysis, measure the rolling velocity. Rolling velocity is measured by dividing the distance traveled by the cells over time.</li>
</ol>
6. Immunostaining of the Microslide
<ol>
	<li>Remove the media from the inlet and outlet of the microslide after perfusing MDA cells on IL-1&#946;-stimulated HUVECs for 10 min.</li>
	<li>Using a 200-&#181;l tip, put warm (37 &#176;C) PBS containing calcium and magnesium into the inlet of the microslide for 5 min. Tilt the microslide using its lid as a prop on the bench. Keep the microslide in a tilted position for all the incubations during immunostaining.</li>
	<li>Fix HUVECs by perfusing warm 2% formaldehyde into the inlet</li>
	<li>Incubate for 20 min at RT. Rinse twice with PBS for 5 min each.</li>
	<li>Add triton-X 100 (0.1%) in 5% BSA in PBS for 10 min at RT.</li>
	<li>Rinse twice with PBS for 5 min each. Block with 5% BSA in PBS for 30 min.</li>
	<li>Incubate with primary antibody goat anti-VE-Cadherin (1:100) in 2.5% BSA O/N at 4 &#176;C.</li>
	<li>Next day, wash twice with PBS for 5 min each. Add secondary donkey anti-goat 647 antibody for 45 min. Wash twice with PBS for 5 min each.</li>
	<li>Incubate at RT with humanized J591-alexa488 conjugated antibody for 1 hr.</li>
	<li>Wash twice with PBS for 5 min each. Add DAPI for 5 min. Wash with distilled water for 5 min.</li>
	<li>Add PBS (or mowiol for long-term storage). Visualize the microslide under the confocal microscope.</li>
</ol>

<ol>
	<li>Dimitroff, C. J., Lechpammer, M., Long-Woodward, D., Kutok, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin.">Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin.</a> Cancer Res. 64, 5261-5269 (2004).</li><li>Barthel, S. R., Gavino, J. D., Descheny, L., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+selectins+and+selectin+ligands+in+inflammation+and+cancer.">Targeting selectins and selectin ligands in inflammation and cancer.</a> Expert Opin. Ther. Targets. 11, 1473-1491 (2007).</li><li>Konstantopoulos, K., Thomas, S. N. <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+in+transit:+the+vascular+interactions+of+tumor+cells.">Cancer cells in transit: the vascular interactions of tumor cells.</a> Annu. Rev. Biomed. Eng. 11, 177-202 (2009).</li><li>Nagrath, S., Sequist, L. V., Maheswaran, S., Bell, D. W., Irimia, D., Ulkus, 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=Isolation+of+rare+circulating+tumour+cells+in+cancer+patients+by+microchip+technology.">Isolation of rare circulating tumour cells in cancer patients by microchip technology.</a> Nature. 450 (7173), 1235-1239 (2007).</li><li>Mannweiler, S., Amersdorfer, P., Trajanoski, S., Terrett, J. A., King, D., Mehes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+prostate-specific+membrane+antigen+(PSMA)+expression+in+prostate+carcinoma+with+distant+metastasis.">Heterogeneity of prostate-specific membrane antigen (PSMA) expression in prostate carcinoma with distant metastasis.</a> Pathol. Oncol. Res. 15 (2), 167-172 (2009).</li><li>Tagawa, S. T., Milowsky, M. I., Morris, M. J., Vallabhajosula, S., Christos, P. J., Akhtar, N. 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(66), 10-3791 (2012).</li><li>Moss, M. A., Zimmer, S., Anderson, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+metastatic+potential+in+the+adhesion+of+human+breast+cancer+cells+to+endothelial+monolayers.">Role of metastatic potential in the adhesion of human breast cancer cells to endothelial monolayers.</a> Anticancer Res. 20, 1425-1433 (2000).</li><li>Wiese, G., Barthel, S. R., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+physiologic+E-selectin-mediated+leukocyte+rolling+on+microvascular+endothelium.">Analysis of physiologic E-selectin-mediated leukocyte rolling on microvascular endothelium.</a> J Vis Exp. 24, (2009).</li><li>Gebauer, F., Wicklein, D., Stubke, K., Nehmann, N., Schmidt, A., Salamon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectin+binding+is+essential+for+peritoneal+carcinomatosis+in+a+xenograft+model+of+human+pancreatic+adenocarcinoma+in+pfp--/rag2--+mice.">Selectin binding is essential for peritoneal carcinomatosis in a xenograft model of human pancreatic adenocarcinoma in pfp--/rag2-- mice.</a> Gut. 62 (5), 741-750 (2013).</li><li>Giavazzi, R., Foppolo, M., Dossi, R., Remuzzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+and+adhesion+of+human+tumor+cells+on+vascular+endothelium+under+physiological+flow+conditions.">Rolling and adhesion of human tumor cells on vascular endothelium under physiological flow conditions.</a> J. Clin. Invest. 92 (6), 3038-3044 (1993).</li><li>Remuzzi, A., Giavazzi, R., Dejana, E., Corada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+tumor+cells+under+flow.">Adhesion of tumor cells under flow.</a> Adhesion Protein Protocols. 96, 153-157 (1999).</li><li>Liu, H., Moy, P., Kim, S., Xia, Y., Rajasekaran, A., Navarro, V., 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=Monoclonal+antibodies+to+the+extracellular+domain+of+prostate-specific+membrane+antigen+also+react+with+tumor+vascular+endothelium.">Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium.</a> Cancer Res. 57 (17), 3629-3634 (1997).</li><li>Jung, B., Obinata, H., Galvani, S., Mendelson, K., Ding, B. 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Dr. Bander is the inventor on patents that are assigned to Cornell Research Foundation (&#34;CRF&#34;) for the J591 antibody used in this article.&#160;Dr. Bander is a consultant to and owns stock in BZL Biologics, the company to which the patents have been licensed by CRF for further research and development.

Due to the low number of CTCs among blood cells, it is difficult to isolate CTCs as a pure population of cells.&#160;In order to study CTC/EC interactions, the rare and impure population of CTCs poses two major challenges: a) Identification of CTCs among blood cells; b) Observation of CTC/EC interactions.

To overcome the first limitation of identifying prostate CTCs among blood cells, we took advantage of the fact that virtually all prostate tumor cells express PSM...

Metastasis is a complex multi-step process that remains poorly understood.&#160;The E-selectin/selectin ligand axis has been shown to play an important role in tumor metastasis by promoting primary adhesive interactions between the vascular endothelium and cancer cells1,2.&#160;Endothelial (E)-selectin is a transmembrane protein expressed by activated endothelial cells, while different E-selectin ligand(s) are expressed by tumor cells3.&#160;Numerous in vitro approaches have been successfully employed to model E-selectin/selectin ligand interactions between tumor cells and endothelial cells (ECs)1.&#160;To study these interactions, different flow chamber systems are being employed to simulate blood vascular system.&#160;Among flow chamber assemblies, parallel-plate flow chamber (PPFC) in conjunction with ECs is routinely used as an in vitro model simulating in vivo shear stress conditions.&#160;In this method, ECs are grown on a 35-mm dish and after achieving a monolayer, ECs are attached to the PPFC and shear stress based experiments are performed.&#160;

However, PPFC and other current systems present many limitations to studying adhesive interactions between circulating tumor cells (CTCs) derived from patients and ECs, primarily, because CTCs are a rare population of cells, shed from the primary tumor, circulating among millions of blood cells (1 CTC per 109 blood cells)4. Hence, unlike unlimited supply of cultured cell lines, low CTC counts lead to very few and rare CTC/EC interactions, requiring proper flow channel width to record the interactions for playback analysis.&#160;Additionally, since patient derived CTCs are an impure population, therefore an identification marker is required to track CTCs in specific.&#160;To solve this problem, we developed a new method to identify prostate cancer (PCa) CTCs by taking advantage of the fact that virtually all of these CTCs express prostate specific membrane antigen (PSMA) on their cell surface5,6.&#160;In this report, we used the prostate cancer cell line, MDA PCa2b (MDA), to demonstrate the potential utility of our new system to study prostate CTC interactions with ECs, eventually to understand the mechanism of metastasis.

Our methodology can be applied for various shear based experiments simulating in vivo vascular system7-9.&#160;Besides examining PCa CTC/EC interactions, the current flow chamber system could be easily adapted for analyzing peripheral blood mononuclear cells or tumor cells&#8217; interactions with ECs.&#160;The ease of disassembling and reassembling of the flow chamber, a microslide III (0.1) (hereafter referred as microslide), allows culturing ECs under perfusion and stimulating ECs with different cytokines to induce protein expression.&#160;Besides, cultured ECs, recombinant proteins such as E- and P-selectin can be coated onto the microslide and interactions with tumor cells can be observed under laminar flow conditions10.

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Microslide</td>
			<td style="width:192px;">Ibidi</td>
			<td style="width:119px;">80331</td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fibronectin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Plastic tubing</td>
			<td style="width:192px;">Tygon</td>
			<td style="width:119px;">AAQ04103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Male luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Female luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe pump</td>
			<td style="width:192px;">Chemyx Inc</td>
			<td style="width:119px;">Fusion 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Luer-lock syringe</td>
			<td style="width:192px;">BD Biosciences</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">M199 medium</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">M7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Endothelial Mitogen</td>
			<td style="width:192px;">Biomedical Technologies</td>
			<td style="width:119px;">BT-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">HBSS</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H9269</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Anti-PSMA J591-488</td>
			<td style="width:192px;">Weill Cornell Medical College-Lab of Urologic Oncology</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Interleukin-1 beta</td>
			<td style="width:192px;">Peprotech</td>
			<td style="width:119px;">200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Trypsin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">SM-2002-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heparin</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H-3149</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">HUVECs</td>
			<td style="width:192px;">Weill Cornell Medical College-Department of Surgery</td>
			<td style="width:119px;"></td>
			<td>provided by Marco Seandel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">VE-Cadherin</td>
			<td style="width:192px;">Santa Cruz</td>
			<td style="width:119px;">sc-5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10x objective</td>
			<td style="width:192px;">Zeiss Plan Neofluar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">Enzyme free cell dissociation reagent</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">S-004-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI-1640</td>
			<td style="width:192px;">Lonza</td>
			<td style="width:119px;">12-702-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ficoll-paque plus</td>
			<td style="width:192px;">GE healthcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

Figure 1 shows an O/N culture of a monolayer of ECs on the microslide. The scalings on Figure 1A shows that 100% of the microslide is visible using 5X objective while 70% is visible using a 10X objective (Figure 1B). For E-selectin mediated interactions, cells rolling at the edges are not considered which makes more than 70% of the microslide available for video-recording and playback analysis. In our experience, initially this set-up assembly needs some practice to cult...

Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

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Research

JoVE Journal

Medicine

9.0K Görüntüleme.  Weill Cornell Medical College.  Bizim rapor fizyolojik akış koşulları altında prostat kanserinde CTC / EC etkileşimleri görselleştirmek ve analiz etmek için eşsiz bir yöntem açıklanır. 

Video: In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

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 ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

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 ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...