Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Summary

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line. 

Abstract

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Introduction

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining cancer genotypes and phenotypes is critical to understanding and designing effective therapeutic strategies. Decades of cancer research have led to the development and adaptation of different methods to determine cancer genotypes and phenotypes. One of the latest technical developments is real-time measurement of cell migration and invasion based on cell impedance. Cell adhesion to substrates and cell-cell contacts play an important role in cell-to-cell communication and regulation, development, and maintenance of tissues. Abnormalities in cell adhesion lead to the loss of ce....

Protocol

NOTE: All cell culture materials need to be sterile and the entire experiment must be performed in a biosafety cabinet under sterile conditions.

1. Culture and Electroporation of the U-118MG Glioblastoma Cell Line

1. Culture the U-118MG cell line in 5% fetal bovine serum (FBS) containing Dulbecco’s Modified Eagle Medium (DMEM) (culture medium) and Maintain at 37 °C in a humid atmosphere containing 5% CO₂ incubator (culture conditions).
2. Use 70– 80% confluent healthy cells for electroporation.
3. For harvesting cells, wash the cells growing in culture dishes with 1x PBS an....

Results

It has been suggested that Crk and CrkL are important for cell migration and invasion in different cancer cell lines^13,17. Although Crk and CrkL proteins are structurally and functionally similar to each other and play essential overlapping functions^16,19,20,21, many gene knockdown studies for Crk and CrkL have not clearly addressed whe.......

Discussion

The real-time measurement of cell migration and invasion using the real-time cell analysis system is a simple, quick, and continuous monitoring process with multiple, significant advantages over the traditional methods that provide data at a single time point. As with the traditional methods, experimental conditions must be optimized for each cell line for the real-time cell analysis system, because each cell line may be different in terms of its adhesion to the substrate, growth, cell-to-cell contacts, and migratory and.......

Materials

Name	Company	Catalog Number	Comments
Biosafety cabinet	ThermoFisher Scientific	1300 Series Class II, Type A2
CIM plates	Cell Analysis Division of Agilent Technologies, Inc	5665825001	Cell invasion and migration plates
Crk siRNA	Dharmacon	J-010503-10
CrkL siRNA	Ambion	ID: 3522 and ID: 3524
Dulbecco’s modified eagle’s medium (DMEM)	ATCC	302002	Culture medium used for cell culture
Dulbecco's phosphate-buffered saline (DPBS)	Gibco	21-031-CV	DPBS used to wash the cells
Fetal bovine serum (FBS)	Hyclone	SH30910.03
Heracell VIOS 160i CO2 incubator	ThermoFisher Scientific	51030285	Co2 incubator
Matrigel	BD Bioscience	354234	Extracellular matrix gel
Neon electroporation system	ThermoFisher Scientific	MPK5000	Electroporation system
Neon transfection system 10 µL kit	ThermoFisher Scientific	MPK1025	Electroporation kit
Non-targeting siRNA	Dharmacon	D-001810-01	siRNA for non targated control
Odyssey CLx (Imaging system)	LI-COR Biosciences		Western blot imaging system
RTCA software	Cell Analysis Division of Agilent Technologies, Inc		Instrument used for experiment
Scepter	Millipore	C85360	Handheld automated cell counter
Trypsin-EDTA	Gibco	25300-054
U-118MG	ATCC	ATCC HTB15	Cell lines used for experiments
xCELLigence RTCA DP	Cell Analysis Division of Agilent Technologies, Inc	380601050	Instrument used for experiment