Identification and analysis of a minority fraction in a U87 cell line Do side-population cells represent bona fide stem cell-like cancer cells?

摘要

BACKGROUND:Overwhelming evidence suggests that tumor bulks are comprised of differentiated tumor cells and cancer stem cells(CSCs).The stem cell-like side-population(SP) cells account for a minor fraction of the total tumor cells,yet are apparently the cells capable of tumor initiation,growth, maintenance,and recurrence. OBJECTIVE:To identify potential stem cell-like cancer cells in a U87 human brain glioma cell line on the basis of dye efflux,clone formation,and multi-drug resistance capacity. DESIGN,TIME AND SETTING:The cellular and molecular biology experiment was performed at the Laboratory of Shanghai Institute of Hematology and Laboratory of Shanghai Institute of Endocrinology in Ruijin Hospital;in vivo contrast observational animal trial was performed at Experimental Animal Center,School of Medicine,Shanghai Jiao Tong University from June 2007 to May 2008. MATERIALS:The U87 cell line was provided by the Shanghai Institute of Cancer Research, Chinese Academy of Science;DMEM/F12(1:1) and fetal bovine serum were purchased from Gibco, Invitrogen,USA;human recombinant basic fibroblast growth factors were purchased from BD Bioscience,USA;Hoechst 33342,Verapamil,and methyl thiazolyl tetrazolium were purchased from Sigma,USA;phycoerythrin-labeled anti-human-CD133 was purchased from Milteny Biotec, Germany;SYBR(?) PrimeScript^(TM) RT-PCR kit was purchased from TaKaRa Biotechnology,Dalian, China. METHODS:Monolayer cultured cells were harvested by 0.25%Trypsin-EDTA and suspended at a 1×10~6/mL dilution in PBS containing 2%FBS,and were stained with Hoechst 33342 dye,either alone or in combination with Verapamil.Following fluorescence-activated cell sorting,SP and non-SP subsets were cultivated with serum-containing(DMEM plus 10%fetal bovine serum) or serum-free culture medium[DMEM/F12(1:1) + 1×B27 supplement + 10 ng/mL basic fibroblast growth factors + 1×L-glutamine]to determine growth characteristics in vitro.Finally,single free U87 cells and subsets(SP or non-SP cells) were subcutaneously injected into the backs of 5-week-old nude mice for in vivo tumorigenicity. MAIN OUTCOME MEASURES:Cell morphology and clonogenicity were observed under inverted microscope;SP phenotype and fluorescent antibody labeling were analyzed by MoFlo^(TM) flow cytometry;ABC transporter mRNA expression was evaluated by semi-quantitative real-time RT-PCR;efflux capacity for anti-neoplastic drugs from the U87 cell line and subsets was measured with the MTT assay,then detected by enzyme-linked immunosorbent assay at a wavelength of 490 nm;in vivo tumorigenicity in immunodeficient nude mice was evaluated by diameter size. RESULTS:During in vitro passages,human U87 cells maintained a stable SP fraction profile and exhibited the ability to form neurosphere-like clones.SP cell proliferation decreased compared with non-treated U87 cells.CD133 expression was reduced in the SP and non-SP cells.Freshly sorted SP fractions expressed higher levels of ABC drug transporter genes,and exhibited increased potential for cytotoxic drug resistance.The in vivo malignancy of U87 cells was largely dependent on non-SP cells in nude mice,and tumors that formed from the non-SP fraction developed faster and larger compared with tumors from the SP fraction. CONCLUSION:The SP cell component was a key factor that influenced mRNA expression and cytotoxic drug resistance.In particular,cancer stem cells or tumor-initiating cells were not exclusively enriched in the SP subset of the U87 cell line,and non-SP cells were even more tumorigenic.