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 trypsin and 0.53 mM), fetal bovine serum (FBS), 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2PHO4, and 1.8 mM KH2PO4), and 6 cm culture dishes.</li>
</ol>
2. Preparation and Recovery of Tumor Cells in Suspension
<ol>
	<li>Prepare sterile Dulbecco&#39;s Modified Eagle Media (DMEM) containing 10% or 20% FBS and freshly add 2 mM L-glutamine, 1x PBS, and 0.05% Trypsin-EDTA.</li>
	<li>Culture the cells in DMEM supplemented with 10% FBS at 37 &#176;C and grow them until there is 70-80% confluence in the culture dish.</li>
	<li>Remove the culture media (DMEM) from the dishes, followed by two washes with 2 mL of sterile 1x PBS.</li>
	<li>Add 1 mL of 0.05% Trypsin-EDTA into the dishes to thoroughly cover all the cells. Immediately remove 800 &#956;L of solution and leave only 200 &#956;L in the dishes. Incubate the dishes at 37 &#176;C for 30 s to 1 min (depending on cell type) and wait until the majority of cells are in the suspended state.</li>
	<li>Add 1 mL of fresh DMEM containing 10% FBS directly into the dishes to stop the enzymatic activity of trypsin and vigorously pipette the cell suspension up and down with a 1000 &#181;L pipette to prepare a single cell suspension.</li>
	<li>Transfer the cells from the dish to a 1.5 mL microcentrifuge tube, and spin down the cells at room temperature at 162 x g for 3 min.</li>
	<li>Aspirate the supernatant and resuspend the tumor cells in 1.5 mL DMEM containing 20% FBS and end-over-end rotate the cells in suspension at 37 &#176;C for 2 h. 
	NOTE: If the medium turns yellow during the recovery, exchange the old medium with fresh medium containing 20% FBS.</li>
</ol>
3. Fluorescence Staining and Analysis of Tumor Cells in Suspension With Carboxyfluorescein Succinimidyl Ester (CFSE)
<ol>
	<li>After cell recovery in suspension, count the appropriate viable cell numbers with Trypan Blue in a Neubauer counting chamber for titrating the fluorescence staining doses and for tail vein injection.</li>
	<li>Spin down the cells at 162 x g at room temperature for 3 min and resuspend the pelleted cells in 1 mL of sterilized 1x PBS, followed by room temperature centrifugation at 162 x g for 3 min.</li>
	<li>Resuspend the pelleted cells in 500 &#181;L of 1X PBS containing 10% FBS and 0, 5, 10, 20, or 40 &#181;M CFSE for dose titration, and incubate the cell suspension at 37 oC for 10 min in the dark.</li>
	<li>Spin down the cells at 162 x g at room temperature for 3 min and resuspend the pelleted cells with 4 mL of DMEM containing 1% FBS. Repeat this washing step three more times. Resuspend the pelleted cells with 4 mL of DMEM alone for the final wash.</li>
	<li>Subject the CFSE-labeled cells to fluorescence-activated cell sorting (FACS) analysis to quantify the fluorescence intensity of a single cell, or tail vein injection. 
	NOTE: Keep the cells on ice prior to performing the FACS analysis or tail vein injections.</li>
</ol>
4. Lung Colonization Assay
<ol>
	<li>Prepare the cells labeled with 20 &#181;M CFSE (refer to Steps 3.1 through 3.4). 
	NOTE: Decide the working dose of CFSE for labelling tumor cells, based on the titration results from the FACS analysis (refer to Step 3.3).</li>
	<li>Warm up the tails of 4-6 week-old male C57BL/6 mice (6 mice) with a halide lamp for 5-10 min on a mouse-by-mouse basis to dilate mouse tail veins.</li>
	<li>Thoroughly mix and aspirate CFSE-labeled tumor cells with a 1 mL (26Gx1/2) syringe, avoiding any bubbles in the cell suspension (with a final cell density of 5 x 106 cells/mL).</li>
	<li>Carefully inject 1 x106 CFSE-labeled tumor cells in 200 &#181;L of DMEM into the mouse tail vein, which should be lodged in a mouse restrainer. 
	NOTE: During the injection, if the needle is directly placed in the lumen of the tail vein, tumor cells should be easily delivered into the circulation without needing to push hard on the syringe. If it is hard to push the tumor cells through, they have most likely been injected outside of the tail vein, into the subcutaneous space.</li>
	<li>Divide the mice that intravenously received the CFSE-labeled tumor cells into two groups: one group will undergo lung perfusion followed by confocal microscopy and ImageJ quantification in the lung colonization assay and the other one will be left alone for 4-5 weeks in the long term lung colonization assay.</li>
	<li>At 20, 38, or 45 h and with 1x 106 tumor cells intravenously injected during a time course- and dose-dependent titration, anesthetize tumor-bearing mice with 50-75 mg/kg zoletil 50, which is a well-accepted and proper anesthetizer23. 
	NOTE: Use vet ointment on the mice&#39;s eyes to prevent dryness while under anesthesia.</li>
	<li>Longitudinally cut the skin and subcutaneous tissues from the abdomen to the chest region with surgical scissors. Open the pleural cavity with surgical scissors and forceps to fully expose the heart and lungs. 
	NOTE: Be careful to avoid cutting off blood vessels.</li>
	<li>Tie the superior vena cava (SVC) and inferior vena cava (IVC) with sterile surgical sutures to prevent the backflow of perfusion solution during lung perfusion.</li>
	<li>Cut the left ventricle wall with surgical scissors to open a fissure (about 2-4 mm) to drain the perfusion solution from the lungs.</li>
	<li>Inject 1x PBS into the right ventricle with a 3 mL syringe and remove the drained solution with constant suction during lung perfusion until the lungs turn from a reddish color to completely pale.</li>
</ol>
5. Confocal Microscopic Imaging and Analysis of Lung-Colonized Tumor Cells
<ol>
	<li>Remove the now-pale lungs from the pleural cavity and separate the five lobes by cutting away the trachea and connective tissue ligaments with surgical scissor and forceps24.</li>
	<li>Prepare a stretcher-like lung holder as shown in Figure 3B by winding a nylon suture around two metal rods to produce a reticulate texture with a space between the two rods for the placement of lung lobes. Set the lung holder in a 6 cm dish. 
	NOTE: The lung holder will be used to stabilize the lung lobes underneath the objective lens of a confocal microscope.</li>
	<li>Lodge and fix the lung lobes on the reticulate texture of the lung holder. Cover and moisten the lung lobes with 1x PBS.</li>
	<li>Subject the 6-cm culture dish harboring the lung lobes on the lung holder to confocal microscopy to capture fluorescence images recording the lung-colonizing tumor cells.</li>
	<li>For imaging, use objective lenses (5X, MPLN, NA: 0.1, WD: 20, Air).</li>
	<li>Rotate the filter wheel to NIBA (excitation/emission; 470-495 nm/510-550 nm) and use a 488 nm laser to excite CFSE, to clearly visualize images of fluorescence dots in the lung lobes.</li>
	<li>Rotate the filter wheel to R690 (for MaiTai HP DeepSee laser) to scan with 2.0 &#181;s/pixel scanning speed and optimize the HV, Gain, and offset levels.</li>
	<li>Capture fluorescence images (512 x 512 pixels) by using the microscope software with a 10 &#181;s/pixel scanning speed.</li>
	<li>Quantify and statistically analyze the microscopic images using ImageJ and GraphPad software as previously described21.</li>
</ol>
6. Long term lung colonization assays
NOTE: Repeat steps 4.1 through 4.5.
<ol>
	<li>Sacrifice the mice after tumor cell inoculation for 4-5 weeks and fix their lungs with Bouin&#39;s Fluid25 for 1 to 2 days.</li>
	<li>Quantify and statistically analyze the tumor nodules from the mice lungs with software21.</li>
</ol>

The authors have nothing to disclose.

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 tracker CFSE allowed us to trace the intravenously injected CTCs for up to three days prior to the lung perfusion and retain sufficient green fluorescence for quantitative purposes, it was impossible to directly determine whether the tumor cells were located within the vessel lumen or had already extravasated from the blood vessels. To solve this problem, red fluorescent rhodamine-conjugated dextran which is able to non-specifically bind to lectins expressed on endothelia may be used to label the lung vasculature during lung perfusion28,29,30. Despite the fact that CFSE is a long term cell tracker, its fluorescence intensity is halved every cell division27, limiting the potential of using it in labeling techniques. To circumvent this problem, it should be ascertained that the labeling dosage of CFSE is sufficient for cells to remain detectable for the entire duration of in vivo experiments and that any treatment applied to the cells has no effect on cell proliferation. Because the comparison and quantification of the lung-colonizing shScr and shFN CTCs were done in separate mice, it might be argued that the differences between the two groups were due to individual variation. To exclude such variation, a mixture of two types of tumor cells labeled with distinct fluoresce dyes (e.g., CFSE/CM-diI) or stably transfected with GFP/dsRed may be concomitantly intravenously injected into the same animal in the lung colonization assay31,32,33. It is worth noting that cloning effects resulting from the cell cloning technology that is attempted to sort out stably fluorescent protein-transfected cell clones with sufficiently strong fluorescence34,35,36,37 may make the cloned cells unrepresentative to represent the entire heterogeneous populations38,39,40. If a stable transfection of any fluorescent protein into tumor cells is desired for the lung colonization assays, elevating the transfection efficiency of the tumor cells as a whole should better preserve the original characteristics than cloning the cell populations with sufficient fluorescence41,42.
The lung perfusion steps in the lung colonization assays are necessary in that some intravenously injected tumor cells, instead of specifically arresting to the lung capillary system, tend to be mechanically trapped and jammed within the capillary networks of lung tissues due to the averagely larger diameters of CTCs than the width of lung capillary lumens43,44. Quantification of the lung-colonizing tumor cells without perfusing the lungs may result in an overestimation by mistakenly counting the mechanically jammed cells45,46,47. Even if the lungs have been perfused, additional problems remain. For instance, it is still difficult to differentiate whether the lung-colonizing tumor cells are located on the lumenal endothelia or have already extravasated from the blood vessels. To resolve this issue, staining the blood vessels with Rhodamine-conjugated dextran as aforementioned may be useful30,48,49. Alternatively, if quantification of tumor extravasation is desired, calcium-chelator EDTA/EGTA or trypsin, a non-specific protease, can be used in the perfusion buffer to impede calcium-dependent and -independent tumor cell adhesion events50,51,52. Thus, the tumor cells remaining in the lung tissues may be considered as extravasated.
For quantification of the lung-colonizing tumor cells, the perfused lungs are often subjected to traditional confocal microscopy. However, the measurability of tissue depth is limited due in part to tissue autofluorescence and fluorescence scattering effects, rendering deeper tissues undetectable53,54. A two photon microscope with higher sensitivity than a traditional confocal microscope is suitable to resolve such problems in that the near-infrared radiation used in two-photon excitation with significantly less absorption by biological specimens than UV or blue-green light makes the technique more appropriate for imaging thick specimens55,56,57. Lung-colonizing tumor cells are counted by averaging tumor cell numbers in several representative confocal images, which do not include the entire number of lung-colonizing tumor cells in the whole lungs of an individual animal. To quantify the whole lungs in the lung colonization assays, tumor cells stably transfected with luciferase could be employed and the perfused lungs could then be subjected to IVIS imaging after intravenous inoculation of suspended tumor cells in the presence of luciferin46,58,59. Alternatively, the perfused lungs could be minced into pieces and subjected to enzymatic digestion with proteases, e.g., collagenase, releasing single cells into suspension60,61. The tumor cells with fluorescence dyes or stably transfected with fluorescence proteins could then be quantified with fluorescence-activated cell sorting (FACS) analysis56,62.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 tumor cells and remains in the cells for quite a long period of time. We found that tumor cells were effectively labeled with CFSE in a dose-dependent manner, as illustrated in Figure 1B. The CFSE-labeling in 6 x105 LLC cells/mL almost reached a plateau at 20 &#181;M as illustrated in Figure 1C.
Owing to the abundant narrowed capillary system which might physically impede the flow of CTCs within the lung vasculature, we performed lung perfusion in mice bearing intravenously injected CFSE-labeled LLC cells to clear non-specifically trapped CTCs, as illustrated in Figure 1A prior to lung removal at each time point as depicted in Figure 4. Before perfusion, the lungs were clearly reddish due to the presence of blood, as illustrated in the left panel of Figure 2. The lungs became pale after perfusion with 10-15 mL of 1x PBS, as illustrated in the right panel of Figure 2.
Immediately after the perfused mouse lungs were removed from the pleural space, the lung lobes were separated and mounted on the lung holder placed in dishes as illustrated in Figure 3A with 1x PBS thoroughly covering the lung lobes. The lung lobes mounted on the lung holder as illustrated in Figure 3B were subjected to confocal fluorescence microscopy as illustrated in Figure 3A. The CFSE-labeled tumor cells colonizing the lungs were imaged as illustrated in the upper panel of Figure 3C and turned into black-and-white images as illustrated in the lower panel of Figure 3C to facilitate the quantification of lung colonization with Image J software.
We intravenously injected either shScr or shFN LLC cells that were labeled with 20 &#181;M CFSE and waited for a time course from 24-72 h before performing the lung perfusions and confocal microscopic imaging. Quantification of the lung-colonizing tumor cells in 6 mice as illustrated in Figure 4A with ImageJ software revealed that significantly fewer shFN LLC cells than shScr LLC cells were counted at the 38 h and 45 h time points, as illustrated in Figure 4B. The reason why the numbers of both lung-colonized shScr and shFN LLC cells gradually diminished was very likely due to the continuous cytotoxicity exerted by the circulation&#39;s immunity even against the already colonized LLC cells. Altogether, these results suggest that polyFN assembled on CTCs is indeed required in mediating lung colonization by LLC cells, leading to complete metastasis in the lungs as revealed by the results in which some mice intravenously receiving aliquots of the CFSE-labeled shScr and shFN LLC cells for the lung colonization assays as illustrated in Figure 4 were left aloneuntil lung tumor nodules were developed in the experimental tumor metastasis assay as illustrated in Figure 5A and 5B.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56761/56761fig1.jpg" /> 
Figure 1: Titration of the CFSE concentrations for labeling tumor cells in the lung colonization assay. (A) Schematic illustration of procedures for the lung colonization assay and the experimental metastasis assay as detailed in the Protocols section. (B) FACS analysis for the CFSE fluorescence intensities of LLC cells that were stained with various concentrations of CFSE. (C) Fluorescence intensities were plotted as functions of various CFSE concentrations. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56761/56761fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56761/56761fig2.jpg" /> 
Figure 2: Lungs before and after perfusion. Representative images of lungs before (unperfused; Unperf.) and after (perfused; Perf.) performing lung perfusion. Gold star: heart. White arrow: the tie that closed the IVC/SVC. Red arrows: the lungs of the same mouse before and after perfusion. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56761/56761fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56761/56761fig3.jpg" /> 
Figure 3: Mounting of the perfused lung lobes on the lung holder for fluorescence confocal microscopy. (A) Scheme of the lung-mounting on the lung holder for the confocal microscopy. (B) A representative image of 3 perfused lung lobes on the lung holder. (C) Representative images of lung-colonizing LLC cells with CFSE fluorescence for quantification by Image J software. Upper panel: regular imaging. Lower panel: reversed black-and-white imaging. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56761/56761fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56761/56761fig4.jpg" /> 
Figure 4: Time course imaging, quantification, and statistical analysis for the lung colonized shScr and shFN LLC cells after lung perfusion in the lung colonization assay. (A) Representative black-and-white images (converted from fluorescence images) of shScr and shFN LLC cells that were colonized in the lung vasculature at various time points as depicted after extensive lung perfusion. Dotted lines denote the edge of the in-focus confocal lung tissue areas. (B) Quantification and statistical analysis for the lung-colonizing LLC cells as visualized in (A) by averaging 5 absolute representative images/lung lobe/mouse. Note: Each bar indicates the averaged fluorescence intensity of each in-focus confocal area. Data were statistically analyzed from 6 mice and reported as mean &#177; SD; n.s. means nonsignificant; * means p&#60;0.05; and ** means p&#60;0.01; Two-way ANOVA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56761/56761fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56761/56761fig5.jpg" /> 
Figure 5: Silencing FN expression in CFSE-labeled LLC cells diminishes tumor nodules in the lungs in the long term in vivo CTC colonization assay. (A) Bouin&#39;s Fluid-fixed mouse lungs bearing tumor nodules derived from CFSE-labeled shScr or shFN LLC cells. (B) Quantification and statistical analysis for the tumor nodules in the lungs. Data are reported as mean &#177; SD; ***: p&#60;0.001; n=6 per group; Unpaired t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56761/56761fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

the-establishment-lung-colonization-assay-for-circulating-tumor-cell

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.2K Views.  National Cheng Kung University. 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.

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

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image analysis. Digital scanning is a fast way to scan a stack of slides and produce digital images with high quality. It can produce concordant results with conventional light microscopy (CLM) by pathologists. Furthermore, the availability of digital images makes it possible that the same slide can be concurrently observed by multiple people. Moreover, digital images of slides can be stored in a database, which means the long-term deterioration of glass slides is avoided. The limitations of this technique are as follows. First, it needs high-quality prepared tissue and the original immunohistochemistry (IHC) slides without any damage or excess sealant residue. Second, tumor or nontumor areas should be specified by experienced pathologists before the analysis using software, in order to avoid any confusion about the tumor or nontumor areas during scoring. Third, the operator needs to control the color reproduction throughout the digitization process in whole-slide imaging.

ZW10 interacting protein (ZWINT) participates in the mitotic spindle checkpoint and the pathogenesis of carcinoma. Here, we introduce a methodology of the immunostaining of ZWINT in human lung cancer tissues, followed by the digital scanning of whole slides and image analysis. This methodology can provide high-quality digital images and reliable results.

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...