Construction Of Tongue Cancer Stem Cells Enrichment Model Based On Cancer Drug Resistance

Background:The theory of cancer stem cells have proposed that there is a small account of cells named cancer stem cells (CSC) in cancer tissue with the ability of self-renewal, differentiation potential and high-tumorigenic,they play a decive role of cancer formation,growth,recurrence and metastasis. Treatment targeted at CSC is the future direction of cancer treatment.However, CSC in cancer tissues account for very small quantities,to find out the effective enrichment and purification of CSC is the first step of CSC research,it is the foundation for finding out CSC markers and subsequent experimental of CSC. There are some researches for tongue CSC, CD44+CD24-cell is thought to be the stem cells in tongue cancer.Our team find out that CD44expresses highly in tongue cancer cell line TCA-8113,which does not match the character of CSC, so we need to add more cell surface markers to prove the existence of tongue cancer stem cells.Objecitve:Establish different enrichment models for tongue cancer cells in vitro and vivo,according to the character of tongue CSC of drug-resistance, to explore weather these modles can enrich CSC.Methods:Took TCA-8113cell line as our research object.In vitro,①TCA-8113cells were selected by0.02μg/ml adriamycin for short term(4days), named TCA-8113/ADM-s.②Drug-resistant cell line of tongue cancer TCA-8113was established by adding increasing doses of adriamycin for8months,named TCA-8113/ADM-L.In vivo,TCA-8113cells were inj ected into and passaged in nude mice,injecting adriamycin(8mg/kg,once a week) and Cyclophosphamide(100mg/kg,3times every8days),then,took out the transplated tumor for primary culture when it came to1.5cm.Compared the biology characteristics of self-renewal. abilities of propagating and high-tumorigenic, including①single cell clony-forming assay,②Clony-forming assay,③Sphere-forming assay,④detected the expression of cancer stem cell related markers CXCR4by FCM.Resylts:The probabilities of clone formation of TCA-8113/ADM-S and TCA-8113/ADM-L are lower than parental cell line.The single cell clone formation rates of cell TCA-8113,TCA-8113/ADM-S and TCA-8113/ADM-L are(77.71±3.74)%,(4.55±0.25)%and(1.30±0.74)%,the clone formation rates of cell TCA-8113,TCA-8113/ADM-S and TCA-8113/ADM-L are(258.67±54.04)‰,(32.004±9.00)‰and(20.004-5.29)‰.The expressions of CXCR4of TCA-8113.TCA-8113/ADM-S and TCA-8113/ADM-L CUltured in RPMI-1640mudium are(32.31±18.69)%,(33.34±8.71)%,(7.15±2.14)%, the expressions of CXCR4of TCA-8113, TCA-8113/ADM-S and TCA-8113/ADM-L cultured in stem cell mudium are(32.74±14.43)%,(29.90±5.26)%,(17.77±8.51)%.The single cell clone fomation rates of cell TCA-8113/ADMP1+,TCA-8113/ADMP2+are (20.10±4.1)%,(19.18±2.03)%, the clone formation rate of cell TCA-8113/ADM PI, TCA-8113/ADM P2are (166.00±45.99)%o,(111.67±8.50)%o=The single cell clone formation rates of cell TCA-8113/CTX P1+, TCA-8113/CTX P2-, TCA-8113/CTX P3, TCA-8113/CTXP4are (24.67±10.60)%,(48.55±19.16)%,(31.67±6.03)%,(68.93±17.09)%, the clone formation rates of cell TCA-8113/CTX P1+, TCA-8113/CTX P2, TCA-8113/CTX P3and TCA-8113/CTX P4are (192.75±48.03)‰,(398.25±15.92)‰,(361.00±59.11)%o,(444.00±14.76)‰。 All of TCA-8113, TCA-8113/ADM-S and TCA-8113/ADM-L can not form spheres cultured in low adhesion96well plates with mTeSRTM1medium Just only TCA-8113/CTX P4cells can form spheres when culturing in low adhesion96well plates with mTeSRrM1medium,the formation rate is (8.25±3.41)%.Conclusions:In vitro,drug selected tongue cancer cells have not acquired cancer stem cell characters.In vivo, TCA-8113cell under pressure of chemotherapy enrich for cancer stem-like cells. CXCR4may not be the specificity sorting marker of tongue cancer stem cell.