Separation And Characterization Of Cancer Stem Like Cells In Human Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, is the most important type of head and neck squamous cell carcinomas. OSCC is the 6th malignant tumor all over the world,which is one of the most common tumor types worldwide with more than 500,000 new cases each year,and about 211,000 people died from OSCC each year. the five-year survival rate after diagnosis for HNSCC remains low, approximately 50%, which is considerably lower than that for other cancers, such as those of colorectal, cervix and breast origin. The limited survival of OSCC patients is likely due to a high proportion of patients presenting with advanced disease stages, lack of suitable markers for early detection, and failure to respond to available chemotherapy. The poor prognosis may be also a reflection of the fact that while many of the most common risk factors involved in OSCC development, such as alcohol and tobacco use, betel nut chewing, and infection with the human papillomavirus (HPV) are recognized, we still have an incomplete knowledge of the mechanisms underlying the malignant progression of this cancer type. Multidisciplinary and general treatment is the clinical treatment principle nowadays, which is conventional surgical treatment assistanted by chemotherapy and radiotherapy. Otherwise,the immunotherapy has become important treatment way for OSCC.The high recurrence rate and development of second primary tumors in advanced clinical stage is the most important factor determining the survival of OSCC patients. There is no effective treatment for those with distant metastasis and recurrent tumors. Therefore, a better understanding of the biologic features of OSCC is essential in development of novel strategies to prevent and treat the disease. The theory of Cancer stem cells (CSCs) is presented, which brings out first light of morning to cure of cancer.Cancer stem cells (CSCs) are tumoral cells which have stem features such as self-renewal, high migration capacity, drug resistance, high proliferation abilities.The study of cancer stem cells in OSCC is in the initial stage, and there are a lot of basic works to be done. CD44 is the putative cancer stem cell markers of head and neck squamous cell carcinoma (HNSCC).The CD44 protein is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. This protein participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis. CD44 mediates cell-cell and cell-matrix interactions. Adhesion with hyaluronic acid plays an important role in cell migration, tumor growth and progression.In this study, we establish the model of primary OSCC, sorting CD44~+ subpopulation group of SCC-9 by flow cytometry and compared biological features between the CD44~+ and CD44- cell populations.1 A reaserch on primary culture and identification of oral squamous carcinoma cells and carcinoma- associated fibroblasts in vitro1)Objective:To cultivate,purify and identify oral squamous carcinoma cells(OSCCs) and carcinoma-associated fibroblasts (CAFs),and investigate their bionomics preliminarily. To summarize the successful experience on OSCCs primary culture model. Preliminarily to conclude that there are the cancer stem cells in OSCCs by detecting the specific surface marker CD44.2)Methods:The primary oral squamous carcinoma tissues were obtained from clinical fresh specimen tissue. Then cells were dissociated by mechanical separating method and collagenase typeⅣdigestiving method.Cells were digestived by 0.25% trypsin and were purified by adherence again and again.Morphological characteristics were observed under light microscope. Oral normal fibrolasts and oral squamous carcinoma cells line(Tca8113) were as the control groups. The proteins including vimentin and cytokeratin were examined by immunohistochemistry. To draw proliferative curve used MTT method. The expression of CD44 in OSCC was detected by immunohistochemistry using S-P staining.3)Results:The purified oral CAFs and OSCCs were maintained.The biological characteristics of OSCC and CAFs were expounded by comparing to the control. The oral CAFs showed negative staining for cytokeratin, and positive staining for vimentin,and the OSCCs showed positive staining for cytokeratin and CD44, and negative staining for vimentin and CD44.4)Conclusions:The advantage of primary culture cells includes that the first is keeping the OSCCs bionomics and losing the antigens little; and the second is harvesting all kinds of the cancer and profiting the research of the cancer microenvironment ;and the third is that there is important significance at individualized treatment because of more individuality and more specificity; and the forth is that there is important significance at individualized chemotherapy and drug screening in cancer stem cells; and the fifth is the experiment tissue is extensive from clinic specimen. The difficulty of primary culture cells includes that the first is that the oral flora is complicated,the oral strain is multiplicity in oral environment, which increase contaminated risk; and the second is the cancer cells are purified difficultly because there are many kinds of cells; and the third is that the cancer microenvironment has changed after the cells are in vitro, the primary culture needs growth factors that can promote cells growth; and the forth is that the culture time in vitro is not suitable longer, along with the time being keep longer the variant may be increased, and long culture can increase the opportunity of losing antigens which is disadvantageous of series research. The purified OSCCs and CAFs were obtained in these empirical study. There are significant differences of the morphological characteristics and growth feature between the CAFs and NFs. Positive staining of CD44 in OSCCs is the evidence that tumor stem cells are directly sorted among squamous cell carcinoma tissue.2 Separation and identification of the cancer stem cells of the tongue squamous cell carcinoma line SCC-91)Objective:To characterize the biological features of CD44~+ cell populations in oral squamous cell carcinoma(OSCC), CD44~+ and CD44- cell populations were sorted from SCC-9 line.2)Methods:①To sort CD44~+ subpopulation group of SCC-9 by flow cytometry;②To detect the cell cycle of the CD44~+,CD44- cell populations;③To detect the expression of CD133,Oct-4,BIM-1,ABCG2,Ezrin gene and protein,comparing differential gene and protein expression in the CD44~+ cell population,CD44- cell population and ESCs;④To detect cloning efficiency of the CD44~+ cell population,CD44- cell population;⑤To inoculate nude mouse and compare tumorigenesis ratio.3)Results:①We show that a minority population of CD44~+ cancer cells, which comprise about 30% of the cells in SCC-9.②70% CD44~+ cancer cells lie in G0/G1 period;③CD44~+ cells differentially express the CD133,Oct-4,BIM-1,ABCG2 genes, at both the RNA and protein levels, and the CD44~+ cells in the tumor express high levels of these nuclear genes and protein(P<0.01).The expression of oct-4 gene has no deffrence between the CD44~+ OSCCs and ESC(P>0.05),and the expression of BMI-1 gene has no deffrence between the CD44~+ OSCCs and ESC(P>0.05), and the expression of oct-4 gene has no deffrence between the CD44~+ OSCCs and ESC(P>0.05),and the expression of Ezrin gene has no deffrence between the CD44~+ OSCCs and ESC(P>0.05);④The clone formation rate of the CD44~+ OSCCs is higher than the CD44- OSCCs;⑤CD44~+ and CD44- OSCCs can form the tumor,and the volume has obviously deffrence.4)Conclusions:①CD44 can become specificity surface marker for sorting cancer stem cells in lingua squamous cell carcinoma line SCC-9.②We separate the minority population of CD44~+ cancer cells, which comprise about 30% of the cells in SCC-9.③The expression of CD133 is higher in CD44~+ cells and CD133 may become other specificity surface marker for sorting cancer stem cells in lingua squamous cell carcinoma line SCC-9.④The expression of stem gene Oct-4 and protooncogene BIM-1 is higher in CD44~+ cells,thses genes can play important role in maintain the cancer stem cells of OSCC.⑤The expression of Ezrin raise in CD44~+ OSCCs, Ezrin as a bridge can links CD44 and with cytoskeleton,so Ezrin can play aan important role in metastasis and infiltration in OSCC.⑥The complex of CD44/Ezrin maybe become new target point for radically curing OSCC because it correlates close with the cancer stem cells of OSCC.⑦What has happened when the complex of CD44/Ezrin for example of modification and cell signaling,which may be our next work.