Analysis of Side Population in Solid Tumor Cell Lines

Summary

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Abstract

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

Introduction

Cancer stem cells (CSCs) are subsets of cells with self-renewal ability and multiple differentiation potential, which play a vital role in tumor growth, metastasis, and recurrence^1,2. Currently, CSCs have been identified to exist in a variety of malignant tumors, including lung, brain, pancreas, prostate, breast, and liver cancers^{3,4,5,6,7,8,9}. Identification of CSCs in these tumo....

Protocol

1. Cell preparation

1. Cell digestion and neutralization
   1. Seed tumor cells (such as MDA-MB-231 cells) in a 6 well plate, and culture them in a 37 °C incubator supplied with 5% CO₂.
   2. Harvest cells when their density reaches about 90%. Aspirate the culture medium thoroughly and wash the cells 2x with 3 mL of phosphate buffered saline (PBS).
      NOTE: To examine the effects of signaling pathway inhibitors (e.g., FRA1 inhibitor), or activators (e.g., STAT3 activator) on stemness features of tumor cells, tumor cells are seeded in a 6 well plate and pretreated with inhibitors or activators for a certain amount of time before....

Results

Four experimental SP analyses were performed according to this method. In the first one, we detected the proportion of SP cells in MDA-MB-231, which is a triple negative human breast cancer cell line, under normal conditions. After cell counting, Hoechst 33342 was added into one tube containing 1 x 10⁶ cells to a final concentration of 3 µg/mL. Reserpine and Hoechst 33342 were added to another tube to final concentrations of 40 µM and 3 µg/mL, respectively. PI was added to both tubes. The dot plot of FSC-A (X-.......

Discussion

There are several key points to keep in mind for the SP assay. The first is the selection of a proper blocker, such as Verapamil or Reserpine, for each cell line, because the "gate" location of the SP cells is determined according to the position at which a large number of SP cells disappear after the addition of the blocker. For the MDA-MB-231 cell line, Reserpine works well. However, for other cell lines, different blockers might work better.

The second is the concentration of Hoechs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 well cell culture plate	CORNING	3516	9.5 cm2 (approx.)
Colivelin	MCE	HY-P1061A	Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-Pro-Ala-Gly-Ala-Ser-Arg-Leu-Leu-Leu-Leu-Thr-Gly-Glu-Ile-Asp-Leu-Pro
Fetal bovine serum (FBS)	Biological Industries (BIOIND)	04-001-1ACS
Flow cytometer	BD Biosciences		BD LSRFortessa
Flow cytometer software	BD Biosciences		FACSDiva
Flow cytometry analysis software	BD Biosciences		FlowJo
Hoechst 33342	Sigma-Aldrich	B2261	bisBenzimide H 33342 trihydrochloride
Polystyrene round bottom test tube	CORNING	352054	12 x 75 mm, 5 mL
Propidium iodide (PI)	Sigma-Aldrich	P4170	3,8-Diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide
Reserpine	Sigma-Aldrich	83580	(3β, 16β, 17α, 18β, 20α)-11,17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester
SKLB816	Provided by Dr. Shengyong Yang, Sichuan University
Trypsin-EDTA (0.25%), phenol red	Gibco	25200072
Verapamil hydrochloride	Sigma-Aldrich	V4629	5-[N-(3,4-Dimethoxyphenylethyl)methylamino] -2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile hydrochloride