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

Summary

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

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

Metastasis is the major cause of cancer death^1,2. 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 metastasis^3,4,5,6. Therefore, vigorous efforts have currently been made in characterizing circulating tumor cell....

Protocol

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 KH₂PO_....

Results

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 molecules^26,27 in suspended tumo.......

Discussion

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 processes^18,19,20,21. Although labeling cells with long term cell tracke.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
Bovine Serum Albumin (BSA)	Cyrusbioscience (Taipei, Taiwan)	101-9048-46-8
Bouin's Fluid	MCC(medical chemical corporation)/POISON	456-A-1GL
CFSE Proliferation Dye	ebiosciences	65-0850-85	Full name: Carboxyfluorescein succinimidyl ester
Dulbecco's Modified Eagle Media (DMEM)	(Gibco)ThermoFisher Scientific	12100-061
Ethylenediaminetetraacetic acid (EDTA)	Cyrusbioscience (Taipei, Taiwan)	101-6381-92-6	For prepared trypsin-EDTA solution( Final concentration: 0.53mM )
Fetal bovine serum (FBS)	(Gibco)ThermoFisher Scientific	10437-028
Lewis lung carcinoma (LLC)	ATCC, Manassas, VA, USA	CRL-1642
L-Glutamine, USP	(Gibco)ThermoFisher Scientific	21051-024
Potassium chloride (KCl)	Cyrusbioscience (Taipei, Taiwan)	101-7447-40-7	For prepared 1X PBS ( Final concentration: 2.7mM )
Potassium phosphate monobasic (KH2PO4)	Cyrusbioscience (Taipei, Taiwan)	101-7778-77-0	For prepared 1X PBS ( Final concentration: 1.8mM )
Sodium chloride (NaCl)	Cyrusbioscience (Taipei, Taiwan)	101-7647-14-5	For prepared 1X PBS ( Final concentration: 137mM )
Sodium phosphate dibasic (Na2HPO4)	Cyrusbioscience (Taipei, Taiwan)	101-10039-32-4	For prepared 1X PBS ( Final concentration: 10mM )
Trypan Blue	Sigma Aldrich	T6146	0.5 g mix with 100 mL 1X PBS
Trypsin	Sigma Aldrich	T4799	For prepared trypsin-EDTA solution ( Final concentration: 5g/L )
Zoletil 50	Virbac		To dilute with 1X PBS
Name	Company	Catalog Number	Comments
Equipment
Compact Tabletop Centrifuge 2420	KUBOTA 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 mixer	C.T.I YOUNG CHENN	TS-20	For suspended cells recovery
FACSCalibur (FACS)	BD biosciences
Forceps	Dimeda	10.102.14
Forma Direct Heat CO2 incubator	Thermo Fisher Scientific Inc.	HEPA CLASS 100
Mouse restrainer (Cylindrical Restrainer 15-30 gm)	Stoelting	51338
Multiphoton Confocal Microscope BX61WI	Olympus	FV1000MPE
Neubauer counting chamber	Marienfeld-Superior	640010
Surgical scissor	Dimeda	08.370.11
Surgical sutures	UNIK SURGICAL SUTURES MFG. CO.	NO. 0034	Black Braided silk; non-absorbable (25YD; U.S.P. 4/0)
1.5 mL microcentrifuge tube	Wuxi NEST Biotechnology Co.	615001
15 mL Greiner tube	Greiner bio-one	188271