Name: the establishment of a lung colonization assay for circulating tumor cell visualization in lung tissues
Uploaded: 2018-06-16T12:30:00
Description: Watch this Scientific Journal Video about The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues at JoVE.com

The authors wish to thank Dr. Ming-Min Chang and Ms. Ya-Hsin Cheng for their technical support. This work was supported by Taiwan&#39;s Ministry of Science and Technology (MOST- 103-2325-B-006 -009, MOST- 104-2325-B-006-001, MOST- 105-2325-B-006-001, and MOST-106-2320-B-006-068-MY3) and the Ministry of Health and Welfare (MOHW106-TDU-B-211-144004). We are also grateful for the support from the Core Research Laboratory of the College of Medicine, National Cheng Kung University, for their multi-photon confocal microscope.

All experiments on mice were performed according to the guidelines of our institute (the Guide for Care and Use of Laboratory Animals, NCKU Medical College).
1. Preparation of Instruments, Culture Media, and Dishes
<ol>
	<li>Before starting the experimental protocols, obtain sterile surgical sutures, 1 mL syringes with 26G/0.5 cm needle (for tail vein injection), 3 mL syringes with 24G/3 cm needle (for lung perfusion), Dulbecco&#39;s Modified Eagle Media (DMEM), trypsin-EDTA solution (5 g/L ...

Together with long term lung colonization assays, the short term methodology we employed here to evaluate in vivo lung colonization by CTCs in distant organs clearly unveiled and differentiated the specific role of polyFN assembled on CTCs in colonizing the lungs, which then led to the extravasation and metastatic processes18,19,20,21. Although labeling cells with long term cell tracke...

Metastasis is the major cause of cancer death1,2. Tumor cells derived from primary tissues enter the circulation in suspension and survive various hematogenous challenges, e.g., anoikis, immune assaults, and damages due to shear stress from blood pressure or geometric constraints, before they are able to colonize distant organs, a key step dictating the success of metastasis3,4,5,6. Therefore, vigorous efforts have currently been made in characterizing circulating tumor cells (CTCs) and correlating these characteristics with tumor malignancy, metastasis, and survival rates of cancer patients7,8,9. Since the process of cancer metastasis specifically depicts an in vivo event, animal models are the only approach that captures the full systemic process of metastasis10,11,12.
CTCs become metastatic tumor tissues through multiple cellular events including the colonization of distant organs1,2. However, the most commonly used metastasis assays13,14,15 do not provide a way to observe CTC colonization of distant organs. Therefore, an in vivo assay design for CTC colonization visualization is urgently needed. Although several in vivo and ex vivo short term lung colonization assays have been designed, problems and disadvantages remain. For instance, while green fluorescence protein (GFP)-overexpressing tumor cells have been used in these assays22,23, it takes time to stably transfect and clone tumor cells with sufficient GFP fluorescent intensity under the microscope. Similarly, although transient staining of tumor cells with the long term cell tracer CFSE has been employed to replace the GFP-expressing tumor cells, it remains difficult to judge whether the CFSE-labeled tumor cells are attached or merely present within the vasculature of the excised distant organs16,17.
Polymeric fibronectin (polyFN) assembled on surfaces of CTCs has been found to critically contribute to the final establishment of metastatic tumor tissues18,19,20,21,22. Here, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with CFSE were intravenously inoculated into C57BL/6 mice. After 2-3 days, mouse lungs were first perfused with phosphate buffered saline (PBS) to completely remove unattached CTCs within the vasculature before being subjected to confocal microscopy and quantification of lung-colonizing LLCs. We clearly showed that the numbers of lung-colonizing shFN LLCs and tumor nodules were significantly reduced in comparison with shScr LLCs,substantially corroborating the role of polyFN assembly on CTCs in facilitating the colonization and growth of CTCs in the lungs. Our study warrants further investigation for the role of polyFN in cancer metastasis.

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	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;(BSA)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#39;s Fluid</td>
			<td>MCC(medical chemical corporation)/POISON</td>
			<td>456-A-1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE Proliferation Dye</td>
			<td>ebiosciences</td>
			<td>65-0850-85</td>
			<td>Full name: Carboxyfluorescein succinimidyl ester</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media (DMEM)&#160;</td>
			<td>(Gibco)ThermoFisher Scientific</td>
			<td>12100-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-6381-92-6</td>
			<td>For prepared trypsin-EDTA solution( Final concentration: 0.53mM )&#160;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>(Gibco)ThermoFisher Scientific</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Lewis lung carcinoma (LLC)</td>
			<td>ATCC, Manassas, VA, USA</td>
			<td>CRL-1642</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, USP&#160;</td>
			<td>(Gibco)ThermoFisher Scientific</td>
			<td>21051-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-7447-40-7</td>
			<td>For prepared 1X PBS ( Final concentration: 2.7mM )</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-7778-77-0</td>
			<td>For prepared 1X PBS ( Final concentration: 1.8mM )</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-7647-14-5</td>
			<td>For prepared 1X PBS ( Final concentration: 137mM )</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-10039-32-4</td>
			<td>For prepared 1X PBS ( Final concentration: 10mM )</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma Aldrich</td>
			<td>T6146</td>
			<td>0.5 g mix with 100 mL 1X PBS</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma Aldrich</td>
			<td>T4799</td>
			<td>For prepared trypsin-EDTA solution ( Final concentration: 5g/L )</td>
		</tr>
		<tr>
			<td>Zoletil 50</td>
			<td>Virbac</td>
			<td></td>
			<td>To dilute with 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Tabletop Centrifuge 2420</td>
			<td>KUBOTA Co.</td>
			<td>2420</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish (6cm)</td>
			<td>Wuxi NEST Biotechnology Co.</td>
			<td>705001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringe (with needle)</td>
			<td>Perfect Medical Industry Co.</td>
			<td></td>
			<td>24G/3 cm;3 ml &#38; 26G/0.5 cm;1 ml</td>
		</tr>
		<tr>
			<td>End over end mixer</td>
			<td>C.T.I YOUNG CHENN</td>
			<td>TS-20</td>
			<td>For suspended cells recovery&#160;</td>
		</tr>
		<tr>
			<td>FACSCalibur (FACS)</td>
			<td>BD biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dimeda</td>
			<td>10.102.14</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Direct Heat CO2 incubator</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>HEPA CLASS 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse restrainer (Cylindrical Restrainer 15-30 gm)</td>
			<td>Stoelting</td>
			<td>51338</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiphoton Confocal Microscope BX61WI</td>
			<td>Olympus</td>
			<td>FV1000MPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld-Superior</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Dimeda</td>
			<td>08.370.11</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures&#160;</td>
			<td>UNIK SURGICAL SUTURES MFG. CO.</td>
			<td>NO. 0034</td>
			<td>Black Braided silk; non-absorbable (25YD; U.S.P. 4/0)</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Wuxi NEST Biotechnology Co.</td>
			<td>615001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Greiner tube</td>
			<td>Greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
	</tbody>
</table>

the establishment of a lung colonization assay for circulating tumor cell visualization in lung tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

Prior to performing the in vivo lung colonization assay as illustrated in Figure 1A to test whether polyFN on suspended tumor cells mediates lung colonization and/or extravasation in facilitating metastasis, we first titrated different concentrations of CFSE, a fluorescent compound that is cell permeable and covalently conjugates intracellular amine-containing molecules26,27 in suspended tumo...

Watch this Scientific Journal Video about The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues at JoVE.com

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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