Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Summary

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Abstract

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

Introduction

Cancer remains the second leading cause of death worldwide¹ and metastasis is responsible for the majority of these deaths^2,3. However, a limited understanding of the molecular mechanisms that govern metastatic colonization and subsequent growth has hindered the development of effective treatments for metastatic disease. The identification of novel therapeutic targets requires an assay to test how perturbed expression or function of a candidate gene influences metastasis formation and growth. While autochthonous mouse models have their advantages, they are time-consuming and expensive t....

Protocol

This protocol involves the use of mice and biohazardous materials and requires approval from the appropriate institutional safety committees. All of the described in vivo work here is approved by the Albany Medical College institutional animal care and use committee (IACUC).

NOTE: For a protocol overview, see schematic in Figure 1.

1. Packaging all required retroviruses and lentiviruses

NOTE: The described protocol uses lentiviral or retroviral vectors to stably express a luciferase enzyme and fluorescent protein as well as to manipulate the expression of a ....

Results

To demonstrate the above approach, we performed a proof of concept experiment in which a critical replication factor, RPA3 was knocked down in a metastatic mouse mammary carcinoma cell line (4T1²²). While the protocol describes labeling the cells with both luciferase and fluorescent proteins prior to genetic manipulation, we used a modified approach because THE RNAi vectors also deliver ZsGreen (Figure 2A). First, 4T1 cells were stably transduced with.......

Discussion

Critical steps of the method
It is critical to optimize the number of cells injected (step 3) for a given cell line and mouse strain as this can greatly influence the number of metastases that form and the length of the experiment. If too many cells are injected or the metastases grow for too long, the metastases may be difficult to count making the effects of the genetic manipulation difficult to assess. However, if too few cells are injected, few or no metastases may form. Thus, a preliminary exp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% SDS-PAGE Gel			For western blot
2.5% Trypsin	Gibco	15090-046	Trypsin for tissue culture
96 well flat bottom white assay plate	Corning	3922	For measuring luciferase and renilla signal in cultured cells
Alcohol wipes			For sterolizing the injection site before tail vein injecitons
BALB/C mice (female, 6 weeks)	Taconic	BALB-F	For tail vein metastatic colonization and burden assays
BSA regular	VWR Ameresco	97061-416	For western blot
Cell lysis buffer	Cell Signaling		For collecting protien samples
Celltreat Syringe Filters, PES 30mm, 0.45 μm	Celltreat	40-229749-CS	For filtering viral supernatant
CO2 and euthanasia chamber			For euthanasing the mice
Dual-luciferase reporter assay kit	Promega	E1960	For measuring luciferase and renilla signal in cultured cells
Dulbecco&39;s phosphate buffered saline	Himedia	TS1006	For PBS
EDTA	VWR	97061-406	Used to dilute trypsin for tissue culture
FBS 100% US origin	VWR	97068-085	Component of complete growth media
Fujifilm LAS-3000 gel imager	Fujifilm		For western blot
GAPDH(14C10) Rabibit mAb	Cell Signaling	2118	For western blot
Goat anti-rabbit IgG (H+L) Secondary Antibody, HRP conjugate	Thermo Scientific	31460	For western blot
Human embryonic kidney cells, HEK-293FT	Invitrogen	R70007	Cell line used for packging virus
HyClone DMEM/High clucose	GE Healthcare life sciences	SH30243.01	Component of complete growth media
Hygromycin B, Ultra Pure Grade	VWR Ameresco	97064-810	For antibiotic selection of infected cells
I3-P/i3 Multi-Mode Microplate/EA	Molecular devices		For measuring luciferase and renilla signal in cultured cells
Imagej			Used for image analysis of lung metastases: threshold set to 25 & 100
Immuno-Blot PVDF Membrane	Biorad	1620177	For western blot
Isoflurane			For mouse anesthesia
IVIS Lumina XRMS In Vivo Imaging System (in vivo live animal imaging device)	PerkinElmer	CLS136340	For in vivo imaging of metastatic burden
Leica M205 FA & Lecia DCF3000 G (GFP and bright field filters)	Leica Microsystems		Microscope and camera for visualing, counting and taking pcitures of metastases in the lungs; 10X magnifacation, 3.5 sec exposure, 1.4 gain
L-Glutamine	Gibco	25030-081	Component of complete growth media
Lipofectamine 3000	Life technologies	L3000008	For YAP/TAZ-TEAD reporter transfection
Living Image 3.2 (image software program)	PerkinElmer		Software for IVIS
Mouse breast cancer cells, 4T1	Karmanos Cancer Institute	Aslakson, CJ et al.,1992	Mouse metastatic breast cance cell line
Multi-Gauge version 3.0	Fujifilm		Software for quantifying western blot band intensity
Opti-MEM (transfection buffer)	Gibco	31985-062	For packaging virus and transfection
Penicillin Streptomycin	Gibco	15140-122	Component of complete growth media
Pierce BCA protein assay kit	Thermo Scientific	23225	For quantifying protein concentration
Pierce Phosphatase Inhibitor Mini Tablets	Thermo Scientific	A32957	Added to cell lysis buffer
Pierce Protease Inhibitor Mini Tablets	Thermo Scientific	A32953	Added to cell lysis buffer
Polybrene (hexadimethrine bromide)	Sigma-Aldrich	45-H9268	For infection
Puromycin	Sigma-Aldrich	45-P7255	For antibiotic selection of infected cells
Rodent restrainer			For restraining mice during tail vein injeciton
SDS-PAGE running buffer			For western blot
TAZ (V3886) Antibody	Cell Signaling	4883	For western blot
TBST buffer			For western blot
TC20 automated cell counter	Bio-Rad		For counting cells
Vectors			See Table 1 for complete list of vectors
VWR Inverted Fluorescence Microscope	VWR	89404-464	For visualizing fluorescence in ZSGreen labeled cells
Western transfer buffer			For western blot
XenoLight D-Luciferin K+ Salt	PerkinElmer	122799	Substrate injected into mice for in vivo bioluminescent IVIS images
X-tremeGENE 9 DNA transfection reagent (lipid solution for transfection)	Roche	6365787001	For packaging virus
YAP (D8H1X) XP Rabbit mAb	Cell Signaling	14074	For western blot