Isolation And Function Study Of CD133 Positive Cells In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC) is a type of cancer common in Southern China and Southeast Asia,especially those of Cantonese origin.The nasopharynx has an abundant supply of lymphatic vessels,as a result,NPC ofen spreads early to local lymph-nodes of the neck.Although the primary tumor is sensitive to radiotherapy,distant metastasis and recurrence after treatment of nasopharyngeal carcinoma is still the leading cause of death.Therefore,the pathogenesis and metastatic mechanism still remains to be clarified.There are two models for tumor propagation.The clonal evolution model postulates that mutant tumour cells with a growth advantage are selected and expanded,with cells in the dominant population having a similar potential for regenerating tumour growth o The cancer stem cell(CSC) hypothesis is an attractive model to account for the functional heterogeneity that is commonly observed in solid tumours.It proposes a hierarchical organization of cells within the tumour,in which a subpopulation of stem-like cells is responsible for sustaining tumour growth.Cancer stem cell theory provides a new description for the origin of tumor,a very small number of cells exists in tumor tissue and serves as stem cells,they possess capacity for self-renewal and multilineage differentiation potency,they are the root causes of tumor occurrence,development,metastasis,recurrence,and resistance to radiotherapy and chemotherapy.Although there are increasing evidence for cancer stem cell,how to separate and purify of cancer stem cells is still a challenge.At present,the most authoritative way for isolating and identifying cancer stem cells is to determine the tumor cell-specific stem cell surface markers,and isolate cancer stem cells based on these markers.In recent years,a variety of tumor cancer stem cells have been identified according to the cell surface specific markers.These cancer stem cells usually express defined surface antigen,such as CD133.CD133 is a cell surface antigen,also known as prominin-1,has a unique five transmembrane domain and two large N-glycosylated of extracellular loops,its expression is a distinctive feature,with the cells divide rapidly downward,which makes it into a separation and identification of stem cells and progenitor cells in a unique molecular markers.CD133 is a traditional molecular markers of hematopoietic stem cells, endothelial progenitor cell,their research began in the 20th century,the 60s.Later, cancer stem cell are found to exist in a variety of tissues,organs and tumor,such as brain,skin,cornea,liver,prostate,B16 melanoma.and so on.Studis found that the CD133~+ cells in brain tumors can differentiate into neurons and glial cells,and formate "neurospheres",that is stem cell clonal growth,when cultured immediately in serum-free medium with growth factors.Days after clutured in serum-containing medium in vitro,the CD133~+ cells can differentiate into a different phenotype of tumor cells.Studies on brain cancer stem cell showed that CD133 could be used as an independent marker of stem cell;Studies on prostate cancer stem cells showed, CD133 together with other surface markers could be used as a marker for stem cell selection;While studies in liver,colon and laryngeal cancer have shown that only a small fraction of stem cells expresses CD133,and it is possible to enrich cancer stem cells according to the surface marker of CD133.CD133 may be a stem cell marker of solid tumor in common.If it is true,we can purify cancer stem cell according to its expression of CD133 and to study its protein expression and signal transduction,or targeted CD133 as a therapy of cancer.However,it was also reported that CD133 negative cells contained some cancer stem cells.So whether CD133 is a cancer stem cell marker of solid tumor in common still needs to be proved.Side populations(SP) cells,as defined by Hoechst exclusion in flow cytometry, have been described a few years ago.While they represent only a small fraction of the whole cell population,their properties confer an important place in several investigations.SP cells express high levels of various members of ABC transporter family,such as MDR1 and BCRP,which are responsible for drug resistance.SP cells have been claimed to be enriched for stem cells in several human normal tissues, cancers and cell lines,and thus may be useful for the identification and isolation of cancer stem cells.However,with further research,it is also proved that not all SP cells were stem cells,SP cells is not always consistent with stem cells.CD133,as an important molecular marker of epithelial and mesenchymal stem cells in normal and tumor cells.At present,the biological function of CD133 in nasopharyngeal carcinoma,whether CD133 positive cells is consistent with SP cells in nasopharyngeal carcinoma or not,is still unknow.The purpose of the study is(1): to detect the expression of CD133 in nasopharyngeal carcinoma cell line,isolate CD133 positive and negative cells and identify their biological function of self-renewal,proliferation,migration in vitro;(2) to analyze the expression of CD133 in SP and non-SP cells by flow cytometry.Methods1.Detection of expression of CD133 in nasopharyngeal carcinoma cell line by flow cytometry2.Isolation of CD133~+ cells and purity examination1) CD133~+ cells were isolated by flow cytometer2) purity examination of CD133~+ cellsa) flow cytometerb) laser scanning confocal microscope3.Identification of CD133~+ biological function in nasopharyngeal carcinoma cell linesIdentification of proliferation,differentiation and migration capacity of CD133~+ and CD133~- cells by MTT assay,Flat colony formation,Transwell assay in vitro. 4.Analyze the expression of CD133 in SP and non-SP cells by flow cytometer.Results1.Detection of expression of CD133 in nasopharyngeal carcinoma cell line by flow cytometerExpression of CD 133 in nasopharyngeal carcinoma cell line 5-8F,6-10B,CNE1, CNE2 were detected by Flow cytometry,results show that CD133 expression in nasopharyngeal carcinoma cell lines ranged from 0.1%to 0.2%.2.Isolation of CD133~+ cells and purity examination1) CD133~+ cells were isolated by flow cytometer2) The purity of isolated CD133~+ cells were identified by flow cytometer and laser scanning confocal microscopea) flow cytometerCD133~+ cells sorted by flow cytometer were in a considerable purity of more than 98%and can be used for the follow-up experiment.b) laser scanning confocal microscopeObserved under confocl microscope,more than 98%of the CD133 positive cell showed fluorescence,while fluorescence were difficult to detect in CD133 negative cells.3.Identification of CD133~+ cell biological function in nasopharyngeal carcinoma cell1).in order to show whether CD133~+ cells were capable of extensivelly proliferate when compared to CD133~- cells,we observed their growth by MTT assay in vitro. Our data showed that the tumour cultures derived from CD133~+ cells display higher proliferative potential with respect to CD133~- cells.(F=115.545, P=0.001).2).Flat colony formation results showed that the number and speed of colony formation in CD133~+ cells were higher than that in CD133~- cells.(t=19.069, P=0.000). 3).Scratch assay results showed that the number of CD133~+ cells migrate to the scrape area of was significantly more than that of CD133~- cells.4).Transwell results also showed that migration of CD133~+ cells significantly enhanced as compared with that of CD133~-5).After cultured in serum-containing medium,CD133~+ cells could differentiate into a different phenotype of CD133~- tumor cells,the percentage of CD133~+ cell population decreased rapidly as detecded by laser scanning confocal microscope and flow ytometer.4.SP cells were detected in CNE2(0.8%) and markedly decreased by treatment with verapamil(0.1%);while CD133~+ cells were not found in the SP cells,the CD133~+ population of CNE2 cells does not overlap with SP.Conclusions1.CD133 was expressed on a small fraction of cells in nasopharyngeal carcinoma cell line,the percentage of CD133~+ cells ranged from 0.1%to 0.2%.2.CD133~+ cells could be effectively obtained high-speed by Flow cytometer (regular purity＞98%).3.CD133~+ cells displayed stem cell-like properties of self-renewal and differentiation compared with the CD133~- cells in vitro.4.CD133~+ cells posessed stronger motility than CD133~- cells in vitro,CD133~+ cells may contribute to the metastsis of nasopharyngeal carcinoma cell.5.SP cells were detected in CNE2(0.8%) and markedly decreased by treatment with verapamil(0.1%);while CD133~+ cells were not found in the SP,the CD133~+ population of CNE2 cells does not overlap with SP.