| Background: Cancer stem cells (TSCs) are rarely part of existing in thetumor tissue of tumor cells with stem cell properties，which play an importantrole in tumor growth, resistance, metastasis and recurrence. Theproportion of cancer stem cells in tumor tissue is rarely. Effective separation andenrichment of these cells is the first step in research, also is the difficulty. In ourprevious study, Tca-8113/ADM-P4cell lines (human tongue squamous cellcarcinoma cell line TCA-8113fourth generation with low dose of adriamycinintervention) were established in nude mice by the use of drug-resistant cancerstem cells. Follow-up tests showed that this enrichment methods still needfurther improvement. Therefore, in this study separation and enrichment oftongue cancer Tca-8113and Tca-8113/ADM-P4cells of stem cells is formed byunicellular culture differentiation to form the holoclone. Recent studies havefound that the three different types of cloning namely: full clone (Holoclone),part of the clone (Meroclone) and the next clone (Paraclone), may occur in avariety of tumor cells with single cell culture in vitro. Among them, theholoclone have the biggest volume which can reach10-30mm2and have therule of round or oval, smooth edge, good refraction, the cloned cells small,uniform, closely connected, proliferation ability, with a strong tumorigenicity; However, paraclone was the smallest clone and cell proliferation worst; Volumeof meroclone is between the holoclone and meroclon. Therefore, holoclone maybe a form of tumor stem cells cloned formation.Objective: Unicellular culture was used to observe the human tonguecancer cell lines (Tca-8113and Tca-8113/ADM-P4) cells whether them canproliferate to three different forms of cloning. And then detect the stem cellcharacteristics of three different kinds of clone.Methods: In the first part, three different clonal stem cell characteristics,including the clone formation rate, constituent ratio, growth curve, these will beobserved in the single cell clone culture in Tca-8113and Tca-8113/ADM-P4celllines by limited dilution system. The second part, the cloning of continuous sub-culture, serum-free suspension culture, serum-free suspension balloonsubculture, Hoechst33342efflux experiments, RT-PCR and immunodeficiencytumor formation in nude mice, the cancer stem cell characteristics detection onthe first part of the formation of three clones, were used for the threecharacteristics of clonal formation of cancer stem cell detection, these clonesare formed in the first part of the experiment.Results:The first part of result:After the single cell clone culture Tca-8113cells and Tca-8113/ADM-P4cells can formed three different kinds of clone andthey clone formation rate were (68.36±1.78)%and (30.72±8.03)%.After thesingle cell culture Tca-8113cells can formed holoclone, meroclone, paraclonewhich were expressed as Tca-8113-HC,Tca-8113-MC, Tca-8113-PC and theirconstituent ratio were (45.60±8.87)%,(30.83±9.15)%,(24.57±0.61)%;In thesame way,after the single cell culture Tca-8113/ADM-P4cells can formed whichwere expressed as Tca-8113/ADM-P4-HC,Tca-8113/ADM-P4-MC,Tca-8113/ADM-P4-PC and their constituent ratio were(31.87±1.03)%,(42.66±1.79)%, (25.47±6.97)%.The second part of result:Using the Tca-8113-HC,Tca-8113-MC,Tca-8113/ADM-P4-HC, and Tca-8113/ADM-P4-MC by single cell subculture tofurther observe its passage formation rate and constituent ratio.After the singlecell subculture the Tca-8113-HC and Tca-8113/ADM-P4-HC clone formation ra-te were (89.12±15.17)%,(48.14±1.75)%.After the clone subculture theTCA-8113-HC can formed holoclone, meroclone, paraclone and theirconstituent ratio were (5.54.±5.47)%,(62.81±2.05)%,(31.6±2.07)%. After theclone subculture the TCA-8113/ADM-P4-HC can formed holoclone, meroclone,paraclone and their constituent ratio were (18.07±2.76)%,(48.21±3.02)%,(33.72±2.07)%, After the clone subculture the Tca-8113/ADM-P4-MC can onlyformed meroclone and paraclone and their constituent ratio were (79.32±4.80)%,(20.68±3.64)%.In our prophase research,the Tca-8113cells in serum-freemedium balloon formation rate was (8.25±3.41)%.In this experiment, theTca-8113-HC in serum-free medium balloon formation rate was(10.83±4.15)%.The Tca-8113-HC suspension ball after the serum-free passage suspensionculture whose balloon formation rate was (14.32±3.20)%,bur the mereoclonecannot form the ball.In addition,Tca-8113/ADM-P4-HC and Tca-8113/ADM-P4-MC can not form the ball.In the fluorescent dye Hoechst33342experiment,the Tca-8113-MC,Tca-8113-PC,Tca-8113/ADM-P4-HC,Tca-8113/ADM-P4-MCand Tca-8113/ADM-P4-PC were all emits blue fluorescence.Only the Tca-8113-HC was no stained or low pollution with the fluorescent dye Hoechst33342.Real-time PCR test results show that: in the holoclone the expression level ofCD44,CXCR4and ESA which is the most relevant stem markers with humantongue squamous carcinoma who is the highest. Immunodeficiency tumorformation in nude mice display: injected the number of5×104Tca-8113-HC cellsinto the Immunodeficiency nude mice in5weeks can appeared1.2×1.5mm size of tumor. In8weeks, the tumor reached the size of9×11.5mm,While the othergroups were no tumor tissue.Conclusions:1.Single cell culture in vitro was found in two cell lines wereable to proliferate for three kinds of different forms of cloning, TCA-8113pare-ntal cells than TCA-8113/ADM-P4cells have a stronger total colony formingability.2. Cancer stemness related feature detection indicated that TCA-8113holoclone may contain a higher proportion of tongue cancer stem cells. |
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