Isolation And Identification Of Side Population Cells From Human Tongue Squamous Carcinoma Tca8113 Cell Line Under Serum-free Culture Condition

At present, tumor is a great disease of threatening mankind's health. Tumor stem cells theory presumes that tumor is a stem cell disease and it is abnormal tissue of tumor cell which has the capacity of forming tumor proliferating. There are few cells having the capacity of forming tunor in tumor cell. Nowadays, the research of tumor stem cell has become a hot spot.Evidence has accumulated indicating that only a minority of cancer cells with stem cell properties, cancer stem cells (CSCs), are responsible for maintenance and growth of the tumor.Recent advances in stem cell biology enable the identification of CSCs in solid tumors as well as putative stem cells in normal organs. With respect to squamous cell carcinoma of the head and neck (SCCHN), Prince et al demonstrated that a small population of CD44+ cancer cells obtained from fresh tumor tissues, but not CD44- cancer cells, gave rise to new tumors in immunodeficient mice. Interestingly, CSCs have been also identified in cultured oral squamous cell carcinoma (OCC). Ping zhang et al demonstrated that permanent OCC constitutively expressed CD44 and ABCG2, and suggested that those makers might be particularly useful cancer stem cell markers. However, Brigitte Mack and Olivier Gires find that the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision. Therefore, it is necessary to go on to seek for cancer stem cells of human tongue cancer. Goodell et al and Bunting et al found that one specific population of hematopoietic stem cell, the so-called side population (SP) stem cells are being selected for transplantation by their capacity to efflux compounds such as rodamine 123 or Hoechst 33342. Subsequently, SP cells were detected in normal tissue and many cancer tissues, and have been suggested to be a source of cancer stem cells in the tumor tissue.To investigate side population (SP) cells in human tongue squamous carcinoma cell line Tca8113 under serum-free culture medium(SFM) or serum-supplemented medium(SSM) , and to detect proliferative and differential ability, tumorigenic ability of SP cells,and to identify tumor-like stem cells in Tca8113,the present study is designed.Research method and result1. Tca8113 cell line was cultivated in SFM and SSM Method: cultivate the human tongue cancer cell line Tca8113 in the RPMI 1640 (15%CS), put the culture bottle in the CO2 (5%), 37℃incubator. When the cells proliferated to 70%~80% of the culture bottle's bottom, trypsinase(0.125%) digested and passaged.Experimental results indicated that human tongue cancer cell line Tca8113 contained SP cells like some cancer cells. In vitro individual cell culture was employed to observe the proliferating character of Tca8113.2. Side population cells dissociated from human tongue cancer cell line Tca8113Method: Collected 5 bottles of cultivated Tca8113 cells, Prepared about 108 cells for staining. Then used PBS(0.01%) to washout the cells and trypsinase(0.125%) digested them. After broke off diagesting, we used HBSS to washout them, suspended the cells and added Hoechest33342. Use PI to mark dead cells. Detected and sorted SP cells which illuminated slightly through FACS. Cells were incubated (106cells/ml) in pre-warmed DMEM/2% FBS containing 5μg/ml Hoechst 33342 (Sigma–Aldrich, Saint Louis, Missouri, USA) at 37℃for 90 min with intermittent mixing. The control cells were incubated in the presence of 50μM of verapamil (Sigma–Aldrich, Saint Louis, Missouri, USA). After incubation, cells were spun down and re-suspended in ice-cold PBS containing 2% FCS. Propidium iodine (Sigma, 1μg/ml) was added to discriminate dead cells. Analysis and sorting were performed on Becton Dickinson ARIA ?ow cytometry. The Hoechst 33342 dye was excited with the UV laser at 450 nm and its ?uorescence measured with a 675 nm side population filter and a 608 EFLP optical filter. SP and non-SP cells were collected separately for further experiments.Results: After colored by Hoechest33342, SP was positively expressed only 0.2% of the cells in human tongue squamous carcinoma cell line Tca8113, but TSC-SP was 0.4%. Experimental results indicated that SP cells exited in human tongue cancer cell line Tca8113.3. Compared biological features between the SP and TSC-SP cell populations.Method: In vitro individual cell culture was employed to observe the proliferating character of Tca8113. SP were sorted and compared by flow cytometry. Tca8113 cell line was cultivated in SFM and the cancer stem-like cells reforming into floating spheres were isolated, was named TSC-SP. The proliferative ability of SP cells in vitro was detected by self-renewal assays. Sorted SP and TSC-SP cells were incubated (500 cells per well) in 24-well plates or 6-well plates (10,000 cells per well) in triplicates and cultured in complete RPMI 1640 with 10% FCS for 8 days.The hetero-transplant tumor mold wan established using NOD/SCID nude mice by subcutaneous injection of tumor cells. The tumorigenic properties of the different clusters were investigated in vivo. The animal experiments were performed in accordance with the institutional guidelines for use of laboratory animals. 4-week-old NOD/SCID was supplied by the Chongqing Experimental Animal Center, Chongqing Medical University, Chongqing, China. To assess in vivo tumorigene-city of SP and TSC-SP were bilaterally inoculated into subcutaneous of 8 nude mice in 100μl volume medium/ matrigel (1:1) with 1×106,1×105 1×104 and 1×103(two mice/group). Tumor development was monitored every four day until eight weeks after inoculation.Research results indicated that Tca8113 cell line contain a tiny minority of SP cells with stem cell properties. Compared with TSC-SP and SP,TSC-SP cells showed increased proliferative capacity. SP and TSC-SP cells were possessed proliferative, self-renewal and differentiation potential which were responsible for the floating tumor clone.The TSC-SP cells were possessed more clonogenic and in nude mice, only 104 SP cells and TSC-SP cells were needed for tumor development behind 16 days. Tumor spheres in which enrich cancer stem cell can be generated under serum-free culture condition..