Cancer Stem Cells In Nasopharyngeal Carcinoma

BACKGROUND & OBJECTIVEStem cells were classified as embryonic stem cells(ESCs) and adult stem cells(ASCs).ES Cs are totipotent,precursors of all cells of the organism.Tissuespecific ASCs reside in adult tissues,which are capable of self-renewal and multipotent differentiation.The ASCs divide asymmetrically,whereby one daughter cell retains as the stem cell,whereas the other daughter cell begins the process of differentiation.On the basis of nomal stem cells(NSCs),cancer stem cells(CSCs) were recognized.The hematopoetic cancer stem cells were described first.Acute myelogenous leukemia(AML) were categorized into two subtypes with phenotypes CD34+,CD38+ and CD34+,CD38-.These two types of cells were injected to NOD/SCID mouse.Only a fraction of CD34+,CD38-cells could transfer human AML to mouse.These cells were named as leukemic initiating cells which are necessary and sufficient to maintain the leukemia.AL-Hajj et al discovered that CD44⁺CD24^-/low subtype in breast cancers could generate the same first and second generation of heterogenous tumors as the original tumor.These cells featured as the stem cells,therefore were named as cancer stem cells.CSCs in brain tumors could differentiate into neuron and neuroglia and clonally proliferated into "neurosphere". CSCs were also discovered in other solid tumors,such as lung,colon and prostate cancers.Except of high tumorigenity,there are a lot of similarities between NSCs and CSCs,including self-renewal,the ability of unlimited proliferation,differentiation, resistance against drug,and similar surface biomarkers.Therefore,it is generally accepted that CSCs might be derived from NSCs due to mutation or epimutation,or from progenitor cells and differentiated cells which have acquired the ability of dedifferentiation and self-renewal as the results of oncogenic mutations.Generally,division of NSCs are considered as asymmetrical.However,Division ways of CSCs are conjectured not only asymmetrical but also symmetrical division. In recent years,some researchers have raised another type of division—neosis. Neosis still is a new concept and needs advanced research.At present,ASCs were isolated and identified mainly according to the following characteristics.1.tumors contain a subpopulation of cells that excludes the DNA binding dye,Hoechst 33342,out of the cell membrane.These cells,with stem cell characteristics are named as side population(SP) cells.2.Because of the asymmetrical division feature,ASCs can be labeled by 5-bromo-2-deoxyuridine (BrdU) and ³H-TdR for a long time,so the ASCs are also called as label-retaining cells(LRCs).3.Compared with other cells,stem cells have distinctive surface biomarkers.For example,CD133,a mouse prominin-1 homologous analog,has been verified as a biomarker for normal and tumor stem cells of many tissues and organs such as hematological system,renal,colon and prostate et al.A dilemma for stem cell research is that though important,NSCs or CSCs are very low in number.That fact hinders the study of stem cells.Therefore,it is very important to enrich stem cells in vitro. Up to now,there are only two articles which are involved in the investigation of nasopharyngeal normal and cancer stem cells.Zhang found long-term BrdU-labeled LRCs in nasopharyngeal epithelia of adult mice and subcutaneous xenografts of human NPC cell lines in nude mice.Wang isolated SP cells in nasopharyngeal carcinoma cell line CNE2 and considered that CK19 is a biomarker of nasopharyngeal stem cells.CD133,an important biomarker of normal and tumor stem cells of many epithelial and mesenchymal tissues,is still not applied to stem cell researches of nasopharyngeal carcinoma.Our purpose of this study is to clarified whether cytokeratin 19 and CD133 are possible surface biomarkers of NPC stem cells and then try to look for a way to enrich CSCs of NPC.Methods1.Exploration of the possibility of several cytokeratins and CD133 as biomarkers of nasopharyngeal cancer stem cells.Cytokeratins comprised of CK19,CK18,CK8,CK14 and CK5/6 were detected by immunohistochemistry(ISH) in specimens of NPC and chronic nasopharyngitis, human NPC cancer cell lines and xenograft tumors of those cell lines.CD133 was also detected by ISH and other methods in 39 cases of NPCs with atypical hyperplasia and normal epithelia and cancer cell lines.2.Isolation of CD133+ cells and purity examination1) MACS isolation of CD 133+ cells2) purity examination of CD133+cellsa.Flow cytometry detected pecentage of CD133+cellsb.CD133 immunofluorescence c.The Quantitive Real-time PCR3.detection of LRCs and numerical comparison between LRCs and CD133+ cells in 5-8F1) LRCs in vitroThe 5-8F cells were grown in standard medium,and pulsed with 10ng/ml BrdU for 7 days,and then washed out Brdu for 14 days.Brdu immunhistochemistry and immunofluorescence were proceeded on the second,the senventh day and the fourteenth day after Brdu washed out,respectively.LRCs were numbered under the light microscope.2) Tumorigentiy in nude mice After being cultivated with Brdu for 48 hours,5-8F cells were inoculated into nude mice subcutaneously.After chasing 8 weeks,xenograft tumors were cut off and LRCs were displayed by ISH and numbered under the light microscope.3) Percentages between LRCs and CD133 were compared,to confirm that CD133 is an important surface marker ofNPC CSCs.4.Identification of CD133+ cells features1) sphere formation assayCD133+cells and CD133- Cells(1000 cells/ml) were cultured in serum-free DMEM-F12 supplemented with some growth factors,and observed everyday under light microscope.2) Test of colony formating efficiency(CFE)3) CD133+ and CD133-cells were observed under light microscope,SEM and TEM. 5.The effect of TPA and Brdu on expression of CD133We classified 5-8F cells as four groups:5-8F,5-8F-Brdu,5-8F-TPA,5-8F-Brdu+TPA to culture 48 days,and then took the following tests:1) Quantitive Real-time PCR2) Western blotting3) flow cytometry4) Boyden chamber testsResults:1.Exploration of cytorkeratins and CD133 as biomarkers of nasopharyngeal stem cells.1) Expression of CK19 and other cytokeratinsCK19 was positively expressed in cancer cell foci of 39 NPC specimans and in pseudostratified ciliated columnal epithelia of all NPCs and chronic nasopharyngitis.Some metaplastic squamous epithelia were also positive for CK19. CK19 expression almost could not be found in cell lines 5-8F,6-10B and CNE2 while xenograft tumors of those cancer cell lines were positively stained with ahti-CK 19.CK18 and CK8 are a pair of partner.The expression ways of them were similar with CK19 in human tissues and xenograft tumors of cancer cell lines.CK14 and CK5/6,another pair of partner,are generally expressed in basal cells of squmaous and columnar epithelial layers and squamous cell carcinomas.In this study,the whole squamous epithelial layer and basal cells of columnar epithelia were stained with anti-CK14 in all cases.However,in NPCs,only 6 cases were sporadically stained with anti-CK14.Though they are a pair of partner,expression of CK5/6 were not coincided with CK14 in nasopharyngeal tissues.The positive signals of CK5/6 were mainly presented in mature squamous cells and ciliated columnar cells while basal cells were negative for CK5/6.In 58 NPC cases,CK5/6 were weakly and sporadically expressed in 22 cases.The results suggest that CK19 and the other cytokeratins studied are not proper biomarkers for nasopharyngeal stem cells.2) CD133 expression in NPCs biopsies and cell linesa.CD133 detection in NPCs biopsies.In 39 cases of NPC specimens,only 8 cases showed CD133+ signals in some cells just in margin areas of a few cancer nests,and 2 cases were CD133 positive in basal cells of epithelial layer.b.CD133 expression in normal epithelial cells,immortalized cell line NP69 and cancer cell linesSeveral CD133 positive cells could be found in normal epithelial cells and NP69.But we could not find positive cells in 5-8F,6-10B and CNE2.Gel electrophoresis of PCR amplification with CD133 showed that 5-8F and 6-10B and CNE2 had distinct positive bands.The expression model of CD133 in NPC tissues and cell lines suggested that CD133 maybe a potential surface marker of NPC cancer stem cells.2.Isolation of CD33+ cells and detection of puritya) CD133+cell percentage detected by Flow cytometry:Due to very low quantity of CD133 positive cells,the detection was not successful with several times.b) Immunofluorescence:Observed under fluorescence microscope,almost all CD133+cells showed fluorescence,while fluorescence were difficult to be seen in CD133-cells.c) Quantitive real-time PCR The quantity of mRNA of CD133+ cells is 7.656 times higher than that of CD 133-cells.2.Detection of LRCs and numerical comparison between LRCs and CD133+ cells in 5-8Fa) LRCs in vitroBrdu immunohistochemistry discovered that almost all cells were positive on the second and the seventh day after Brdu added.Only sporadic cells were positive on the fourteenth day after Brdu washouted,and these cells were thought of LRCs.The average percentage of LRCs is 0.67±0.32%.b) 5-8F xenograft tumors in nude miceOnly a small quantity of cells in margin area were Brdu positive.These positive cells were considered as LRCs.The average percentage of LRCs under ten high magnified powers is 0.55±0.36%.c) the percentage of CD133+cells(0.5%) were similar to LRCs.This result confirms once again that CD133 is an important marker for NPC CSCs.4.Identification of CD133+cells features1)Sphere assay:From the fourth day CD133+ cancer cells formed spheres,the case in CD133-tumor cells was different.CD133-tumor cells failed to form spheres and most of cells died within several days.2) colony forming efficiencyA significant difference was found in clony formation efficiency among different cells(F=68.045,P=0.000).The CFE of CD133+ cells is higher than that of 5-8F (P=0.000) and CD133-cells(P=0.000).3) Observation of CD133+and CD133- cells under light microscope,SEM and TEM a.Observation under light microscopeThe most distinguished characteristics of CD133+ cells is that a lot of big cells are dispersed among small cells.Under high magnification,polykaryon could be found in some big cells.On the other hand,most of CD133- cells are similar in size.b.Observation under SEMUnder SEM,appearances of CD133+ and CD133- cells are different.In CD133+ cells,we can see some little spherical bodies on a big mother cell,like budding phenomenon.The other cells are generally small,round and with short villi. With long villi,CD133-cells are bigger,polygonal,and tightly sticking to slides.In CD133- cells,we almost can not find budding phenomenon.b.Observation under TEMMost of CD133+ cells appeared regular and round nuclei,whereas nuclei were beteromorphic and nuclear grooves could be seen in CD133- cells4) The effects of TPA and Brdu on CD133 expression and CD133+cell quantity1) The quantitive real-time PCRCD133mRNA and ABCG2mRNA were significantly higher in the groups of 5-8F-Brdu and 5-8F-Brdu+TPA than 5-8F(CD133:P=0.037 and P=0.003,respectively; ABCG2:P=0.000 and P=0.006 respectively).CD133 mRNA was decreased in 5-8F-TPA.It proved that Brdu can promote CD133mRNA expression.2) The percentage of CD 133+cells by Flow cytometryThe percentage of CD133+cells in 5-8F-Brdu and 5-8F-brdu+TPA were significantly higher than that of 5-8F(P=0.000).CD133+ cells in 5-SF-TPA was also higher than 5-8F.However,no statistics difference was found.2) Boyden chamber invasion test in vitroThe invasion ability of four groups is significantly different(F=127.360,P=0.000). Among them,the ability of 5-8F-Brdu+TPA was the strongest,the combined effects of Brdu and TPA promote the invasion.Conclusions:1.The presence of CSCs in NPC was confirmed once again;CD133+ but not CK19 maybe an important surface marker of NPC CSCs.2.MACS isolation of CD133+ cells is an effective method.3.Biological and morphological characteristics are different between CD133+ and CD133-cells.4.Brdu,besides as a marker of LRCs,can increase the mRNA expression of CD133 and ABCG2,and promote CD 133+ cell proliferation and invasion ability.