Identification And Isolation The Cancer Stem Cells In Hep-2 Cell Line Of Human Laryngeal Carcinoma

Identification and Isolation the cancer stem cells in Hep-2 cellline of human laryngeal carcinomaPARTⅠAnalysis the expression of membrane antigen and original selectionof the marker of cancer stem cells in Hep-2 cell lineBackground Laryngeal carcinoma is one of the most common malignant tumors in thehead and neck. Due to the integration of chemotherapy, radiotherapy, andorgan-preserving surgery, both survival rates and quality of life for patients have beenimproved dramatically. Cause of death can often be attributed to recurrence of thecarcinoma and cervical lymph node metastasis. But some late stage diseases remainincurable by current treatment strategies. New ideas are needed to explain and solve theseproblems. In recent years, there has been overwhelming evidence in some malignanciessupporting the notion that tumors are organized in a hierarchy of heterogeneous cellpopulations. The capability to sustain tumor formation and growth resides exclusively in asmall proportion of tumor cells, termed cancer stem cells or tumor-initiating cells.Tumor-initiating cells have been identified in blood, brain, and breast cancers. Thesestudies have shown that tumor-initiating cells are responsible for tumor formation andprogression. The tumor clone is heterogeneous with respect to proliferation anddifferentiation. These tumor-initiating cells share with stem cells the key feature ofself-renewal. In our study, a Hep-2 cell line of laryngeal cancer was used to analysisdifferent expression of surface markers, and to explore whether its formulation isheterogeneous. The purpose is to reveal the possible existence of cancer stem cell in Hep-2cell line.Methods Immunocytochemical staining technology and flow cytometry were used todetect the expression of the putative stem cell marker CD133, CD44 in a Hep-2 cell line.Results1,Immunocytochemical staining showed only a small proportion of cells in the Hep-2cell line constancy expressed CD 133. On the contrary, almost all Hep-2 cells expressed CD44.2,Consistent with the result of immunocytochemistry, only 3.15%±0.83% of Hep-2cell expressed the membrane antigen, CD133. About 97.12%±1.9% Hep-2 cellsexpressed another membrane antigen, CD44 in flow cytometry.Conclusions Only a small proportion of cells in the Hep-2 cell line constant expressedCD133, a putative marker of cancer stem cells in Hep-2 cell line. PARTⅡIsolation the subpopulations in Hep-2 cell line and Evaluationthe proliferation capability and differentiation capability ofCD133~+ cells in vitro1. Isolating CD133~+ cells from Hep-2 cell lineBackground CD133 is also known as AC133. It is a 5-transmembrane glycoprotein witha molecular weight of 117kD. It localized to membrane protrusions or microvilli.Antibodies to CD133 have been used to enrich for human haematopoietic stem cells,endothelial cells, neurons and glial cells. CD133 is also expressed by the intestine-derivedepithelial cell line Caco-2 where it is down-regulated upon differentiation. CD133 isbelieved to be the human orthologue of mouse Prominin, a protein expressed on the apicalsurface of neuroepithelial cells as well as several other embryonic epithelia, and on brushborder membranes of adult kidney proximal tubules. CD133 has also been foundexpressed on endothelial precursor cells and fetal neural stem cells, as well as humanprostatic epithelial stem cells. Recently, it was identified as stem cell marker in humanbrain tumors and prostate cancer. At present, purification method of cancer stem cell wasmainly fluorescence-activated cell sorting and magnetic activated cell sorting. Only asmall proportion of cells in the Hep-2 cell line constant expressed CD133 was alreadyknown. In this part, magnetic activated cell sorting was applied to purified CD133positive cells.Methods CD133(+) cells were isolated from the Hep-2 cell line using magneticseparation. In order to evaluate the efficiency of magnetic separation, harvested cells weretested by flow cytometry analysis.Results Before isolation with magnetic separation, the CD 133(+) fraction was 3.15%±0.83% by FCM analysis. After isolation, the CD133(+) fraction was raised to 90.26±7.61%. Subsequent experiments proved that cells grew well after isolation.Conclusion MiniMACS was an ideal cell sorting device in purification of cancer stem cell.Key words CD133(+) cells; Magnetic cell sorting2. Evaluation the proliferation capability and differentiationcapability of CD133~+ cells in vitro Background Stem cells are functionally defined as self-renewing and multipotent. Theyhave the capability to generate mature cell types through differentiation. The tumor stemcell hypothesis states that only a rare, phenotypically distinct subset of cells has thecapacity to form new tumors. Compared to other cells, extensive proliferation andself-renewal potential are fundamental characteristics of cancer stem cells. Cancer stemcells cannot proliferate without the microenvironment on which they live. The nichenourished the cancer stem cells and kept them in stable state. From the relationshipbetween normal stem cell and its niche, it can be inferred the survival, proliferation anddifferentiation of cancer stem cell was supposed and regulated by its niche. In cancer stemcell, it seemed that their growth and development could not leave away from its niche.Under normal conditions, they remain dormant, usually in the G0/G1 phase. Only wheninduced by correlated cytokine can they enter into the cell life cycle and proliferate. Thepurpose of this part was to observe the proliferation and differentiation ability of CD133positive cells in Hep-2 cell line in vitro.Methods Sorted CD133(+) tumor cells, CD133(-) tumor cells, and unsorted tumor cellswere plated in 96-well microwell plates at a density of 2000 cells/well. Medium withoutcells was added as a control. Proliferation assays were performed on days 1, 3, 5, and 7(post-plating) using Roche 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.Quantification of viable cells through reading of UV absorption spectrums at 490 nm wasperformed on a versamax microplate reader. The number of days in culture and averagedata of different groups were used to draw growth curves to compare the proliferationabilities of the three groups. After isolation, CD133(+) tumor cells were cultured inRPMI1640 supplemented with 10% FCS. To observe differentiation ability, thepercentage of CD133(+)tumor cells was detected using flow cytometry on days 0, 4, 8,and 12 (post-plating). Average data of different groups were used to draw curves ofdifferentiation.Results Compared with the control, CD133(+) cells demonstrated increased proliferationcapacity in vitro than unsorted Hep-2 cells and CD133(-) cells (P＜0.05). In days 1-3 and5-7, the curve became steep. Proliferation of the control Hep-2 cells was relatively low,and the reproductive activity of CD133(-) cells was the lowest with a plane curve. Thisillustrates that the poor reproductive capability of CD133(-) cells. After isolation, singleCD133(+) cells were cultured in RPMI1640 medium supplemented with 10% FBS. Theexpression levels of surface marker CD133 were observed to estimate the differentiation of CD133(+) cells. The results displayed the proportion of CD 133(+) cells that decreasedin culture as days passed. It was 90.37%±2.37%, 45.18%±4.51%, 23.50%±3.69%,4.76%±0.75% on days 0, 4, 8, and 12 respectively.( P＜0.05, compared 0 days with 4days, 4 days with 8 days, 8 days with 12 days). In twelve days of culture, the percentageof CD133(+) cells decreased to 4.76%±0.75%, similar to unsorted Hep-2 cells.Conclusion CD133(+) cells showed a greater degree of cell proliferation than bothCD133(-) cells and unsorted tumor cells under the same conditions. This demonstrates theproliferation ability of CD133(+) cells had characteristic of stem cells. CD133(+) cellsalso exhibited a highly differentiated potential in vitro. With the daily culture addition, thepurity of the CD 133(+)cell populations decreased rapidly, especially in the first four days.This infers that only small numbers of Hep-2 cells retain their own character. Theremaining majority differentiated to terminal cells which did not express the CD133phenotype. In other words, CD133(+) cells have the capacity of self-renewal and thecapacity to give rise to daughter cells of a different phenotype, just like cancer stem cells. PARTⅢEstablishment the SCID mouse model and investigation thetumorigenicity of subpopulation in Hep-2 cell lineBackground Cancer stem cells are clonogenic cells with self-renewal and differentiationpropefity. Recent evidence has demonstrated that cancers can be viewed as an abnormalorgan in which tumor growth is driven by a population of cancer stem cells (CSCs), Inboth breast cancers and central nervous system tumors, cancer cells differ in their abilityto form tumors. While the majority of the cancer cells have a limited ability to divide, apopulation of cancer stem cells that has the exclusive ability to extensively proliferate andform new tumors can be identified based on marker expression. They have been identifiedin blood, brain, and breast cancers through an experimental strategy that combines thesorting of tumor cell subpopulations, identified on the basis of different expression ofsurface markers, with functional transplantation into appropriate animal models. AlthoughOnly a small proportion, cancer stem cells possess a marked capacity for tumor formationin vivo. When injected into immunologic deficit animal, its tumorigenicity was strongerthan ordinary tumor cells. In tradition, athymic mouse was selected as heterogenictransplantation animal. This mice still have immunologic function which influenceddetection of cancer stem cells. In this part, SCID (Severe Combined Immune Deficiency) micewas used to observe tumorigenicity of CD133(+) cells in vivo. Lack of T and B cellsimmunologic function, SCID mice was ideal animal model to investigate the tumorformation ability of cancer stem cell. To determine whether the difference intumorigenicity in subpopulation of Hep-2 cells was due to differences in cell growthactivity and cell cycle, we observed the growth of sorted cells in vitro with HE stain andanalyzed cell cycle of CD133(+) cells and unsorted cells by flow cytometry.Methods After isolated from the Hep-2 cell line using magnetic separation, CD133(+)cells and cultured unsorted cells were all stained with a routine method HE to observegrowth activity in vitro. The cell cycle status of sorted and unsorted cancer cells were alsocompared by flow cytometry. To compare the tumorigenic potential with control, sortedCD133(+) cells, CD133(-) cells and unsorted Hep-2 cells were injected subcutaneouslyinto abdomen of SCID mice. unsorted and CD133(-) subpopulation. Each injectionconsisted of 5×10~5 cells. Nodules grown in mice were counted and immediately removed and stained with HE. Positive ratio of two groups was analysis by Statistical analysis.Results Both unsorted cells and sorted cells grew with large nuclei and prominentnucleoli. All cells were large with fusiform shapes. Appearance of pathologickaryokinetic division could be seen in field of vision. These qualities were all consistentwith the character of malignant tumor cells. Comparison of the cell cycle status of sortedand unsorted cancer cells after magnetic sorting revealed that both cells exhibited asimilar cell cycle distribution. Sorted CD133(+) cells and unsorted Hep-2 cells wereinjected subcutaneously into the SCID mice with twenty sites each group. In period of sixweeks, 16 sites contained tumor in CD133(+) group, whereas only 10 sites contain tumorin unsorted group and 7 sites in CD133(-) group. Statistic analysis demonstrate CD133(+)cells have strong ability to forming tumor in vivo than unsorted cells (P＜0.05).Conclusions The result suggested that a hierarchy exists in Hep-2 cell line. CD133(+)cells have strong ability to forming tumor in vivo than unsorted cells. In other words, asmall subset of cells is enriched for clonogenic capacity. Compared with CD133(-) cells,CD133(+) cells encompassed a small fraction of the cell line (3.15%±0.83%). Neitherphase of cell cycle nor non-tumor cell or migrating normal stem cells affected orincreased tumor-forming ability. Our results suggest that CD133 is a marker of cancerstem cells in the Hep-2 cell line. Identification of this marker provides a powerful tool toinvestigate the tumorigenic process in the larynx and to develop therapies targeting thecancer stem cell.