Sorting and identification of side population cells in the human cervical cancer cell line HeLa

Wenjuan Qi,Chao Zhao,Ning Liu,Xiaoping Li,Lihui Wei,Weidong Yu,Lijun Zhao

doi:10.1186/1475-2867-14-3

Abstract

BackgroundSeveral reports have revealed that cancer stem cells (CSCs) exist in many types of solid tumors. Some studies have demonstrated that side population (SP) cells isolated from diverse cancer lines harbor cancer stem-like properties, but there are few reports examining the characteristic of SP cells in human cervical cancer. The aim of this study is 1) to find out a feasible way to detect the tumor stem-like cells in cervical cancer, and 2) to analyze the properties of the SP cells being sorted.MethodsIsolated SP and non-SP cells from human cervical cancer cell line Hela by Hoechst 33342 dying method and flow cytometry analysis. Observing morphology of SP and non-SP cells. The expression of various biomarkers putatively related to cancer stem cells were investigated by immucytochemistry of SP and non-SP cells. We also analyzed cell cycle and cell apoptosis for sorted cells. The oncogenicity of the SP and non-SP cells were analyzed by tumor formation in nonobesediabeti- c/severe combined immune- deficient (NOD/SCID) mice. The drug-resistant and radiation-resistant index between SP, non-SP and Hela cells was estimated by MTS assay.ResultsThe fraction of SP cells in Hela was approximately 1.07 ± 0.32%. SP cells were smaller and rounder in shape than non-SP cells, and mostly showed colony-like growth. Immunocytochemistry showed that stem cell makers (Oct3/4, CD133, BCRP) were highly expressed in SP cells. Moreover, the number of apoptotic cells among non-SP cells (17.6 ± 3.7%) was significantly higher compared with that among SP cells (4.4 ± 1.2%). The HE staining of in vivo grown tumors result from SP cells showed more poor differentiation, though no significant differences were shown between SP and non-SP cells in NOD/SCID mice tumorigenicity. Furthermore, SP cells demonstrated a higher degree of drug resistance against trichostatin A (TSA) compared with that of non-SP and Hela cells. SP cells were also found to be more resistant against radiotherapy.ConclusionsSP cells possess some characteristics of CSCs, namely high proliferation ability, chemoresistance and radioresistance, which may be helpful to elucidate novel targets for effective clinical treatments of cervical cancer in the future.

Highlights

Several reports have revealed that cancer stem cells (CSCs) exist in many types of solid tumors
The characteristic of Side population (SP) cells to rapidly extrude Hoechst 33342 is based on the expression of ABCG2/BCRP1, a breast cancer resistance protein (BCRP) of the ATP-binding cassette (ABC) transporter family
SP cells among HeLa cells After excluding dead cells and cellular debris based on scatter signals and propidium iodide (PI) fluorescence, the SP and non-SP cells were sorted

Summary

Introduction

Several reports have revealed that cancer stem cells (CSCs) exist in many types of solid tumors. Some studies have demonstrated that side population (SP) cells isolated from diverse cancer lines harbor cancer stem-like properties, but there are few reports examining the characteristic of SP cells in human cervical cancer. The aim of this study is 1) to find out a feasible way to detect the tumor stem-like cells in cervical cancer, and 2) to analyze the properties of the SP cells being sorted. The characteristic of SP cells to rapidly extrude Hoechst 33342 is based on the expression of ABCG2/BCRP1, a breast cancer resistance protein (BCRP) of the ATP-binding cassette (ABC) transporter family. The expression of ABCG2/BCRP1 shows a strong positive correlation with the phenotype of SP cells in a series of studies, which is the molecular basis of the phenotypic characteristics of SP cells [12]. High expression of ABCG2/BCRP1 in SP cells is contributed to drug resistance and tumor recurrence [10]

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