Name: In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells (Video) | JoVE Journal
Uploaded: 2014-05-15T13:00:00.000+00:00
Duration: 11 min 56 s
Description: Learn to visualize prostate circulating tumor cell interactions with E-selectin-expressing endothelial cells using a micro slide flow assembly. This method enables real-time analysis of cancer metastasis mechanisms under physiological flow conditions.

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5 minutes

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/pubmed?term=((Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Targeting+selectins+and+selectin+ligands+in+inflammation+and+cancer%5BTitle%5D)+AND+%22Expert+Opin.+Ther.+Targets%22%5BJournal%5D)">Targeting selectins and selectin ligands in inflammation and cancer.</a> Expert Opin. Ther. Targets. 11, 1473-1491 (2007).</li>
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<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/pubmed?term=((Isolation+of+rare+circulating+tumour+cells+in+cancer+patients+by+microchip+technology%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Heterogeneity+of+prostate-specific+membrane+antigen+%28PSMA%29+expression+in+prostate+carcinoma+with+distant+metastasis%5BTitle%5D)+AND+%22Pathol.+Oncol.+Res%22%5BJournal%5D)">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. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+II+study+of+lutetium-177+labeled+anti-prostate-specific+membrane+antigen+%28PSMA%29+monoclonal+antibody+J591+for+metastatic+castration-resistant+prostate+cancer%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Phase II study of lutetium-177 labeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 for metastatic castration-resistant prostate cancer.</a> Cancer Res. , (2013).</li>
<li>Ganguly, A., Zhang, H., Sharma, R., Parsons, S., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+human+umbilical+vein+endothelial+cells+and+their+use+in+the+study+of+neutrophil+transmigration+under+flow+conditions%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions.</a> J. Vis. Exp. (66), 10-3791 (2012).</li>
<li>Moss, M. A., Zimmer, S., Anderson, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+metastatic+potential+in+the+adhesion+of+human+breast+cancer+cells+to+endothelial+monolayers%5BTitle%5D)+AND+%22Anticancer+Res%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+physiologic+E-selectin-mediated+leukocyte+rolling+on+microvascular+endothelium%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Selectin+binding+is+essential+for+peritoneal+carcinomatosis+in+a+xenograft+model+of+human+pancreatic+adenocarcinoma+in+pfp--%2Frag2--+mice%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">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/pubmed?term=((Rolling+and+adhesion+of+human+tumor+cells+on+vascular+endothelium+under+physiological+flow+conditions%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">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. Adhesion of tumor cells under flow. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRemuzzi%2C+A+%22Adhesion+Protein+Protocols%22">Adhesion Protein Protocols</a>. Dejana, E., Corada, M. 96, Humana Press. 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/pubmed?term=((Monoclonal+antibodies+to+the+extracellular+domain+of+prostate-specific+membrane+antigen+also+react+with+tumor+vascular+endothelium%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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. S., Skoura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow-regulated+endothelial+S1P+receptor-1+signaling+sustains+vascular+development%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">Flow-regulated endothelial S1P receptor-1 signaling sustains vascular development.</a> Dev. Cell. 23 (3), 600-610 (2012).</li>
<li>Yin, X., Rana, K., Ponmudi, V., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Knockdown+of+fucosyltransferase+III+disrupts+the+adhesion+of+circulating+cancer+cells+to+E-selectin+without+affecting+hematopoietic+cell+adhesion%5BTitle%5D)+AND+%22Carbohydr.+Res%22%5BJournal%5D)">Knockdown of fucosyltransferase III disrupts the adhesion of circulating cancer cells to E-selectin without affecting hematopoietic cell adhesion.</a> Carbohydr. Res. 345 (16), 2334-2342 (2010).</li>
<li>Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transmigratory+cup+in+leukocyte+diapedesis+both+through+individual+vascular+endothelial+cells+and+between+them%5BTitle%5D)+AND+%22J+Cell+Bio%22%5BJournal%5D)">A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them.</a> J Cell Bio. 167 (2), 377-388 (2004).</li>
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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><tbody><tr><td>Microslide</td><td>Ibidi</td><td>80331</td><td></td></tr><tr><td>Fibronectin</td><td>Millipore</td><td>FC010</td><td></td></tr><tr><td>Plastic tubing</td><td>Cole Parmer</td><td>EW-96115-08</td><td></td></tr><tr><td>Male Luer adapter</td><td>GlycoTech</td><td>31-001</td><td></td></tr><tr><td>Female Luer adapter</td><td>GlycoTech</td><td>31-001</td><td></td></tr><tr><td>Syringe pump</td><td>Chemyx Inc</td><td>Fusion 100</td><td></td></tr><tr><td>Luer-lock syringe</td><td>BD Biosciences</td><td>309628</td><td></td></tr><tr><td>M199 medium</td><td>Sigma</td><td>M7653</td><td></td></tr><tr><td>Endothelial Mitogen</td><td>Biomedical Technologies</td><td>BT-203</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H9269</td><td></td></tr><tr><td>Anti-PSMA J591-488</td><td>Weill Cornell Medical College-Lab of Urologic Oncology</td><td></td><td></td></tr><tr><td>Interleukin-1 beta</td><td>Peprotech</td><td>200-01B</td><td></td></tr><tr><td>Trypsin</td><td>Millipore</td><td>SM-2002-C</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H-3149</td><td></td></tr><tr><td>HUVECs</td><td>Weill Cornell Medical College-Department of Surgery</td><td></td><td>provided by Marco Seandel</td></tr><tr><td>VE-Cadherin</td><td>Santa Cruz</td><td>sc-5648</td><td></td></tr><tr><td>10x objective</td><td>Zeiss Plan Neofluar</td><td></td><td></td></tr><tr><td>Enzyme free cell dissociation reagent</td><td>Millipore</td><td>S-004-C</td><td></td></tr><tr><td>RPMI-1640</td><td>Lonza</td><td>12-702-F</td><td></td></tr><tr><td>Ficoll-paque plus</td><td>GE healthcare</td><td>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

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...

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

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

The overall goal of this procedure is to examine the interactions between rare prostate cancer cells and e selectin expressing endothelial cells using a micro slide flow assembly. This is accomplished by first coating a micro slide with fibronectin. The second step is to culture human umbilical vein endothelial cells or VE in a dynamic flow system on a micro slide with a small channel width.Next, the prostate cancer cells are labeled with an anti prostate specific membrane antigen or PSMA humanized monoclonal antibody, which gets internalized. The final step is to perfuse the anti PSMA labeled prostate cancer cells over the e selectin expressing ve. Ultimately, video microscopy is used to show the interactions between prostate cancer cells and the endothelial cells.The main advantage of this technique over existing methods, such as parallel plate flow chamber and other dynamic assemblies, is that with this technique you can examine the interactions between rare prostate cancer cells, such as circulating tumor cells and cultured endothelial cells in a dynamic flow system. This method can help answer key questions in the field of metastasis, such as how do prostate tumor cells they metastasize through the blood vascular system? Do they use the same mechanism as being used by leukocytes during their ization?Generally, individuals who are new to this technique will struggle because it involves culturing a mono layer of endothelial cells in a narrow channel under perfusion Under the tissue culture hood. First, rinse the micro slide with phosphate buffered saline or PBS. Then gently coat the micro slide with 200 microliters of 50 micrograms per milliliter fibronectin using a one milliliter lure lock syringe.Cover the micro slide with the lid and keep it inside the tissue culture hood for 30 minutes. Next, perfuse 200 microliters of warm qve growth medium over the micro slide and incubate for 20 minutes at room temperature. Slow dispensing of the liquid in the micro slide prevents bubble formation in the channel.During the perfusion, prepare the vex suspension by rinsing vex with PBS and adding 0.1%collagenase plus 0.05%EDTA in PBS for one to two minutes at room temperature. Centrifuge the hve in two milliliters of growth medium at 180 times G for five minutes. Then measure the cell concentration using a hemo cytometer and prepare 10 million cells per 100 microliters of growth medium.Next, carefully remove the medium from the inlet of the micro slide. Using a 200 microliter pipette tip, bring the micro slide to eye level and using a one milliliter lure lock syringe gently perfu 200 microliters of the prepared concentration of vex 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.Next pipet an equal volume of approximately 80 microliters of VE medium into both the inlet and outlet of the micro slide. This prevents the flow of cells in either direction. Cover the slide and keep it in a 37 degrees Celsius incubator for 1.5 hours.To begin flow chamber assembly, place a sterile 20 milliliter syringe, female and male lure connectors, a syringe pump and tubing in the incubator for 15 minutes for minimal dead volume. Used tubing with an inner diameter of 0.04 inches. Smaller tubing diameter prevents bubble formation.Next, fill the 20 milliliter syringe with 12 milliliters of warm VE medium. Remove the bubble from the syringe completely. Fill the inlet of the micro slide with VE medium.Connect this assembly to the micro slide. Gently attach the outlet connector to the micro slide. Bring the setup into the incubator and connect it to the syringe pump.Then set the pump to a 10 microliter per minute shear rate before leaving the cells overnight in the incubator at 37 degrees Celsius. The next day disassemble the setup by removing the connector attached to the inlet of the micro slide. Then remove the outlet connector two to upregulate e selectin expression on endothelial cells.Prepare fresh growth medium containing interleukin one beta. Aspirate the medium in a 10 milliliter syringe. Remove the leftover medium from the inlet of the slide, then add fresh, medium completely filling the inlet.Connect the syringe to the slide. Set the syringe pump at a 10 microliter per minute shear rate for four hours in the incubator during interleukin one beta incubation of vex, prepare anti PSMA Alexa 4 88 antibody labeled prostate cancer cells. First, add 0.05%trypsin EDTA to the MDA prostate cancer cells for one minute.Do not expose the cells to trypsin for longer times as it can affect the glycopeptides present on the cell surface. Centrifuge the cells at 200 times G for five minutes. Next, resuspend the MDA cell palate in one milliliter of Hank's Buffer and add the anti PSMA antibody incubate for 30 minutes at room temperature in a dark place, resus suspending the cells during the incubation.After 30 minutes, centrifuge the cell solution at 800 times G for five minutes. Aspirate and resuspend the pellet in one milliliter of Hank's buffer. Count the labeled MDA cells and bring the final concentration to 1 million cells per milliliter.Fill a five milliliter syringe with the labeled MDA cells and remove the bubbles. Turn on the inverted microscope and set the curler illumination at 10 x objective. Bring the syringe pump at the same level as the sample stage of the microscope and set it at one dime per centimeter square shear stress.Next, open the Zeiss Axio vision software. Select the objective on the computer screen. Create a new folder under the tools option in the software.Open the smart experiments option in Axio vision and adjust the settings to record short 32nd videos for 30 minutes For live fluorescent video, set the image options. These parameters help in attaining videos close to the video frame rate with the Zeiss MRM camera to visualize full width of the flow channel at 10 x objective on the computer screen, use a CM mount adapter switch on the mercury lamp before placing the syringe and the connector containing the cells onto the syringe pump. Next, attach the connector to the filled inlet channel of the micro slide containing interleukin one beta stimulated ax.Connect the outlet channel with the connector attached to the tubing. Put the tubing into a dish or a 15 milliliter conical tube to collect the flow through. Start the infusion through the micro slide at 10 microliters per minute.Observe the interactions between endothelial cells and labeled MDA cells or labeled CTCs derived from patients under 488 nanometer. Filter on the epi fluorescence microscope. Start recording the experiment as 32nd short videos.During playback analysis, measure the rolling velocity. Rolling velocity is measured by dividing the distance traveled by the cells over time. Shown here is an overnight culture of a monolayer of endothelial cells.On the micro slide. The scalings show that 100%of the micro slide is visible using a five x objective while 70%is visible using a 10 x objective for selected mediated interactions, cells rolling at the edges are not considered, which makes more than 70%of the micro slide available for video recording and playback analysis. The box plot shows the distribution of the rolling velocities of both unlabeled and anti PSMA, labeled MDA cells on interleukin one beta stimulated endothelial cells at different shear stress conditions.No significant difference was observed in the rolling velocities at one and five dines centimeter square at higher sheer stress of 10 dimes per centimeter square. A statistically significant difference was observed between the rolling velocities of unlabeled and anti PSMA, labeled MDA cells, A time stitched video taken using the blood from a prostate cancer patient shows three types of interactions between labeled prostate CTCs and lectin expressing endothelial cells tethering where CTCs attach and reattach stable adhesion where CTCs did not detach even after 30 seconds and no interactions as non interacting CTCs enter the field of view. Micro slides can be easily immuno stain and fluorescently imaged the firm adhesion of anti PSMA, labeled MDA cells.After profusion over interleukin one beta stimulated endothelial cells can be seen here. After watching this video, you should have a good understanding of how to examine the interactions between rare cells such as circulating prostate tumor cells and S selectin expressing endothelial cells following this procedure. Other methods such as immunofluorescence can also be performed in order to answer additional questions such as the presence of s selectin.Ligands.Techniques like these can help the researchers to understand different steps involved in metastasis.

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This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress.  This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

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Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

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

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

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

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In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

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Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Learn to visualize prostate circulating tumor cell interactions with E-selectin-expressing endothelial cells using a micro slide flow assembly. This method enables real-time analysis of cancer metastasis mechanisms 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 (Video) | JoVE Journal

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

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