Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Summary

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

Abstract

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Introduction

Tumor cell motility plays a crucial role in metastasis^1,2. The spread of tumor cells to neighboring and remote healthy tissues makes cancer treatment difficult and contributes to recurrence^3,4. Therefore, it is essential to understand the mechanisms of tumor cell motility and develop relevant therapeutic strategies. Since many tumor cells have altered gene expression profiles, it is crucial to understand which changes in the gene expression profile lead to altered tumor cell motility^5,6.

Protocol

1. Synthesis of mRNA

NOTE: For the mRNA synthesis, all the reagents and equipment must be specially treated to inactivate the RNases before use. See the Table of Materials for details about all the materials, instruments, and reagents used in this protocol.

1. Linearization of DNA
   NOTE: Mouse cDNAs of CrkI and CrkL were cloned into the pFLAG-CMV-5a expression vector to add the FLAG epitope tag at the C-terminus and subcloned into the pcDNA3.1/myc-His vector to incorporate the T7 promoter, as previously described^18,

Results

Crk and CrkL proteins play important roles in the motility of many cell types, including neurons²², T cells²³, fibroblasts^18,19, and a variety of tumor cells¹³. Since Crk and CrkL proteins have been reported to be elevated in glioblastoma^24,25,26, the effects of the overexpression of CrkI, a splice v.......

Discussion

Migration and invasion are important features of tumor cells. Measuring the motility of tumor cells and understanding the underlying mechanism that controls tumor cell motility provide critical insights into therapeutic interventions^2,27. Several methods have been developed to study cell migration⁷. The wound-healing assay using scratches or culture inserts is a simple and frequently used method that provides contrasting images of gap clos.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AlphaImager HP	ProteinSimple	92-13823-00	Agarose gel imaging system
α-Tubulin antibody	Sigma	T9026	Used to detect α-tubulin protein (dilution 1:3,000)
CIM-plate 16	Agilent Technologies, Inc	5665825001	Cell invasion and migration plates
Crk antibody	BD Biosciences	610035	Used to detect CrkI and CrkII proteins (dilution 1:1,500)
CrkL antibody	Santa Cruz	sc-319	Used to detect CrkL protein (dilution 1:1,500)
Dulbecco’s Modified Eagle’s Medium (DMEM)	ATCC	302002	Cell culture medium
Dulbecco's phosphate-buffered saline (DPBS)	Corning	21-031-CV	Buffer used to wash cells
Fetal bovine serum (FBS)	Hyclone	SH30910.03	Culture medium supplement
Heracell VIOS 160i CO2 incubator	Thermo Scientific	51030285	CO2 incubator
IRDye 800CW goat anti-mouse IgG secondary antibody	Li-Cor	926-32210	Secondary antibody for Western blot analysis (dilution 1:10,000)
IRDye 800CW goat anti-rabbit IgG secondary antibody	Li-Cor	926-32211	Secondary antibody for Western blot analysis  (dilution 1:10,000)
Lithium chloride	Invitrogen	AM9480	Used for RNA precipitation
Matrigel matrix	Corning	354234	Extracellular matrix (ECM) gel
MEGAscript T7 transcription kit	Invitrogen	AM1334	Used for RNA synthesis
Millennium RNA markers	Invitrogen	AM7150	Used for formaldehyde agarose gel electrophoresis
Mini centrifuge	ISC BioExpress	C1301P-ISC	Used to spin down cells
Mouse brain QUICK-Clone cDNA	TaKaRa	637301	Source of genes (inserts) for cloning
NanoQuant	Tecan	M200PRO	Nucleic acid quantification system
Neon electroporation system	ThermoFisher Scientific	MPK5000	Electroporation system1
Neon transfection system 10 µL kit	ThermoFisher Scientific	MPK1025	Electroporation kit
Neon transfection system 100 µL kit	ThermoFisher Scientific	MPK10096	Electroporation kit
NorthernMax denaturing gel buffer	Invitrogen	AM8676	Used for formaldehyde agarose gel electrophoresis
NorthernMax formaldehyde load dye	Invitrogen	AM8552	Used for formaldehyde agarose gel electrophoresis
NorthernMax running buffer	Invitrogen	AM8671	Used for formaldehyde agarose gel electrophoresis
Nuclease-free water	Teknova	W3331	Used for various reactions during mRNA synthesis
Odyssey CLx Imager	Li-Cor		Imager for Western blot analysis
pcDNA3.1/myc-His	Invitrogen	V80020	The vector into which inserts (mouse CrkI and CrkL cDNAs) were cloned
pFLAG-CMV-5a	Millipore Sigma	E7523	Source of the FLAG epitope tag
Phenol:chloroform:isoamyl alcohol	Sigma	P2069	Used for DNA extraction
PmeI	New England BioLabs	R0560L	Used to linearize the plasmids for mRNA synthesis
Poly(A) tailing kit	Invitrogen	AM1350	Used for poly(A) tail reaction
Polystyrene tissue culture dish (100 x 20 mm style)	Corning	353003	Used for culturing cells before transfection
Polystyrene tissue culture dish (35 x 10 mm style)	Corning	353001	Used for culturing transfected cells
Proteinase K	Invitrogen	25530049	Used to remove protein in the reaction mixture
Purifier Axiom Class II, Type C1	Labconco Corporation	304410001	Biosafety cabinet for sterile handling of cells
Resuspension Buffer R	ThermoFisher Scientific		A buffer included in the electroporation kits, MPK1025 and MPK10096. The buffer is used to resupend cells before electroporation, and its composition is proprietary information.
RNaseZap	Invitrogen	AM9780	RNA decontamination solution
Scepter	Millipore	C85360	Handheld automated cell counter
ScriptCap 2'-O-methyltransferase kit	Cellscript	C-SCMT0625	Used for capping reaction
ScriptCap m7G capping system	Cellscript	C-SCCE0625	Used for capping reaction
Sodium dodecyl sulfate solution	Invitrogen	15553-035	Detergent used for the proteinase K reaction
Sorvall Legend XT centrifuge	Thermo Scientific	75004532	Benchtop centrifuge to spin down cells
Trypsin-EDTA	Gibco	25300-054	Used for dissociation of cells
U-118MG	ATCC	HTB15	An adherent cell line derived from a human glioblastoma patient
Vinculin antibody	Sigma	V9131	Used to detect vinculin protein (dilution 1:100,000)
xCELLigence RTCA DP	Agilent Technologies, Inc	380601050	Instrument used for real-time cell analysis
1Electroporation parameters and other related information for various cell lines are available on the manufacturer's homepage (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html?).