Name: in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells
Uploaded: 2014-05-15T13:00:00
Description: 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

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...

<ol>
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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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