Method Article

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

DOI:

10.3791/56761

⸱

June 16th, 2018

 ,  ,  ,  ,  ,  , 
1The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, 2Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 3Trauma Office, Children's National Health System

In This Article

Summary

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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.

Abstract

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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 μM 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.

Introduction

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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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Protocol

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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

  1. 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'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.

2. Preparation and Recovery of Tumor Cells in Suspension

  1. Prepare sterile Dulbecco's Modified Eagle Media (DMEM) containing 10% or 20% FBS and freshly add 2 mM L-glutamine, 1x PBS, and 0.05% Trypsin-EDTA.
  2. Culture the cells in DMEM supplemented with 10% FBS at 37 °C and grow them until there is 70-80% confluence in the culture dish.
  3. Remove the culture media (DMEM) from the dishes, followed by two washes with 2 mL of sterile 1x PBS.
  4. Add 1 mL of 0.05% Trypsin-EDTA into the dishes to thoroughly cover all the cells. Immediately remove 800 μL of solution and leave only 200 μL in the dishes. Incubate the dishes at 37 °C for 30 s to 1 min (depending on cell type) and wait until the majority of cells are in the suspended state.
  5. 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 µL pipette to prepare a single cell suspension.
  6. 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.
  7. 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 °C for 2 h.
    NOTE: If the medium turns yellow during the recovery, exchange the old medium with fresh medium containing 20% FBS.

3. Fluorescence Staining and Analysis of Tumor Cells in Suspension With Carboxyfluorescein Succinimidyl Ester (CFSE)

  1. 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.
  2. 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.
  3. Resuspend the pelleted cells in 500 µL of 1X PBS containing 10% FBS and 0, 5, 10, 20, or 40 µM CFSE for dose titration, and incubate the cell suspension at 37 oC for 10 min in the dark.
  4. 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.
  5. 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.

4. Lung Colonization Assay

  1. Prepare the cells labeled with 20 µM 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).
  2. 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.
  3. 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).
  4. Carefully inject 1 x106 CFSE-labeled tumor cells in 200 µL 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.
  5. 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.
  6. 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's eyes to prevent dryness while under anesthesia.
  7. 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.
  8. 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.
  9. Cut the left ventricle wall with surgical scissors to open a fissure (about 2-4 mm) to drain the perfusion solution from the lungs.
  10. 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.

5. Confocal Microscopic Imaging and Analysis of Lung-Colonized Tumor Cells

  1. 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.
  2. 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.
  3. Lodge and fix the lung lobes on the reticulate texture of the lung holder. Cover and moisten the lung lobes with 1x PBS.
  4. 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.
  5. For imaging, use objective lenses (5X, MPLN, NA: 0.1, WD: 20, Air).
  6. 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.
  7. Rotate the filter wheel to R690 (for MaiTai HP DeepSee laser) to scan with 2.0 µs/pixel scanning speed and optimize the HV, Gain, and offset levels.
  8. Capture fluorescence images (512 x 512 pixels) by using the microscope software with a 10 µs/pixel scanning speed.
  9. Quantify and statistically analyze the microscopic images using ImageJ and GraphPad software as previously described21.

6. Long term lung colonization assays

NOTE: Repeat steps 4.1 through 4.5.

  1. Sacrifice the mice after tumor cell inoculation for 4-5 weeks and fix their lungs with Bouin's Fluid25 for 1 to 2 days.
  2. Quantify and statistically analyze the tumor nodules from the mice lungs with software21.

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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The authors wish to thank Dr. Ming-Min Chang and Ms. Ya-Hsin Cheng for their technical support. This work was supported by Taiwan'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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Material
Bovine Serum Albumin (BSA)Cyrusbioscience (Taipei, Taiwan)101-9048-46-8
Bouin's FluidMCC(medical chemical corporation)/POISON456-A-1GL
CFSE Proliferation Dyeebiosciences65-0850-85Full name: Carboxyfluorescein succinimidyl ester
Dulbecco's Modified Eagle Media (DMEM) (Gibco)ThermoFisher Scientific12100-061
Ethylenediaminetetraacetic acid (EDTA)Cyrusbioscience (Taipei, Taiwan)101-6381-92-6For prepared trypsin-EDTA solution( Final concentration: 0.53mM ) 
Fetal bovine serum (FBS)(Gibco)ThermoFisher Scientific10437-028
Lewis lung carcinoma (LLC)ATCC, Manassas, VA, USACRL-1642
L-Glutamine, USP (Gibco)ThermoFisher Scientific21051-024
Potassium chloride (KCl)Cyrusbioscience (Taipei, Taiwan)101-7447-40-7For prepared 1X PBS ( Final concentration: 2.7mM )
Potassium phosphate monobasic (KH2PO4)Cyrusbioscience (Taipei, Taiwan)101-7778-77-0For prepared 1X PBS ( Final concentration: 1.8mM )
Sodium chloride (NaCl)Cyrusbioscience (Taipei, Taiwan)101-7647-14-5For prepared 1X PBS ( Final concentration: 137mM )
Sodium phosphate dibasic (Na2HPO4)Cyrusbioscience (Taipei, Taiwan)101-10039-32-4For prepared 1X PBS ( Final concentration: 10mM )
Trypan BlueSigma AldrichT61460.5 g mix with 100 mL 1X PBS
TrypsinSigma AldrichT4799For prepared trypsin-EDTA solution ( Final concentration: 5g/L )
Zoletil 50VirbacTo dilute with 1X PBS 
NameCompanyCatalog NumberComments
Equipment
Compact Tabletop Centrifuge 2420KUBOTA Co.2420
Culture dish (6cm)Wuxi NEST Biotechnology Co.705001
Disposable syringe (with needle)Perfect Medical Industry Co.24G/3 cm;3 ml & 26G/0.5 cm;1 ml
End over end mixerC.T.I YOUNG CHENNTS-20For suspended cells recovery 
FACSCalibur (FACS)BD biosciences
ForcepsDimeda10.102.14
Forma Direct Heat CO2 incubatorThermo Fisher Scientific Inc.HEPA CLASS 100
Mouse restrainer (Cylindrical Restrainer 15-30 gm)Stoelting51338
Multiphoton Confocal Microscope BX61WIOlympusFV1000MPE
Neubauer counting chamberMarienfeld-Superior640010
Surgical scissorDimeda08.370.11
Surgical sutures UNIK SURGICAL SUTURES MFG. CO.NO. 0034Black Braided silk; non-absorbable (25YD; U.S.P. 4/0)
1.5 mL microcentrifuge tubeWuxi NEST Biotechnology Co.615001
15 mL Greiner tubeGreiner bio-one188271

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Circulating Tumor CellsCFSE LabelingLung PerfusionConfocal MicroscopyFibronectin AssemblyLewis Lung CarcinomaIntravenous InoculationC57BL 6 MiceFluorescence Imaging

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