| CHAPTER ONEMethodological Investigation on Serum-free Suspension Culture for Cancer Stem Cells and Identification of Cancer Stem-like Cells in Nasopharyngeal Carcinoma Cell Line CNE-2ZObjectives:To investigate the methodology for culturing and enriching cancer stem cells and to identify cancer stem-like cells in nasopharyngeal carcinoma cell line CNE-2Z.Methods:(1) Nasopharyngeal carcinoma cells were collected from the cultured cell line CNE-2Z when they grown into logarithmic gowth phase by a common culturing procedure. Then, cell number was adjusted to the densities as 1×103/ml, 1×104/ml, 1×105/ml and 1×106/ml respectively and cultured in serum-free culture medium.0.3% trypan blue was used to count the percentage of living cells at day 0, day 1, day 3, day 5, day 7 and day 9 during the culturing period respectively for each density of cell culturing system; (2) Coverslips crawled with living cells were prepared at the 9th day in serum-free and common culture system respectively. Then, the procedures of immunohistochemical staining for CD 133 and CD44 were carried out on these coverslips with anti-mouse monoclonal antibody; (3) Samples for electron microscopic observation were prepared with these cells cultured in serum-free culture system at the 9th day and observed under electron microscope.Results:(1) Nine days after following serum-free suspension culture, the living cells ratio in these culturing system, with the added cell concentration being 1×103/ml, 1×104/ml, 1×105/ml, and 1×106/ml, were 0.00%,0.05%,3.10% and 0.27% respectively, with the highest living cells ratio in the culturing system at a cell density of 1×105/ml; (2) The positive cell percentages of CD 133 and CD44 on the coverslips crawled with living cells in common culture system were 2.71% and 3.00%, while they were 66.7% and 67.00% respectively on such coverslips in serum-free culture system, with statistical significances (P<0.01) and a positive correlation (P<0.01) among them; (3) It was found through scanning electron microscopy and transmission electron microscopy that the cancer stem-like cell in nasopharyngeal carcinoma cell line CNE-2Z held surface scales and budding like protrusions. Under transmission electron microscope, these cells showed greater heterogeneity, in round or oval shape, with nuclear pyknosis, extremely condensed cytoplasm and severely reduced organelles. And seen also were extracellular discharged apoptotic body from these cells.Conclusions:(1) Cancer stem-like cells in nasopharyngeal carcinoma cell line CNE-2Z can be enriched from a serum-free suspension culture system, and the cell concentration of 1×105/ml should be the best suspension cell culture concentration for nasopharyngeal carcinoma cell line CNE-2Z. Serum-free suspension culture is a more economical culturing procedure with such advantage as easy to carry out for the cultivation of tumor stem cells in practice. This procedure should be promoted into use widely.(2) CD133 and CD44 can be considered as their surface markers of nasopharyngeal cancer stem-like cells during the period of serum-free suspension culturing. These cells can grow in two ways as self-replication and proliferation but grow slowly.(3) Under electron microscope, nasopharyngeal cancer stem-like cells can be observed in greater heterogeneity, with nuclear condensation, cytoplasmic extremely condensed, severely reduced organelles as well as apoptotic bodies discharged from their cytoplasm extracellularily. These changes may be associated with their growth in serum-free culturing environment leading to such variation in these cells’biological characteristics.CHAPTER TWOBiological Characteristics of Tumor Stem-like Cells in Nasopharyngeal Carcinoma Cell Line CNE-2Z and Differentiation-Inducing Effect of. Qi-Boosting Toxin-Resolving Formula on ThemObjectives:To observe the biological characteristics of tumor stem-like cells in nasopharyngeal carcinoma cells line CNE-2Z and to investigate the differentiation-inducing effect of Qi-Boosting Toxin-Resolving Formula on these cells.Methods:(1) Tumor stem-like cells balls were enriched in the cells of nasopharyngeal carcinoma cell line CNE-2Z by the method of serum-free suspension culture at first; (2) These cell balls were cultured continuously in serum-free medium or in a common culture system respectively, and then, observed were their cloning capacity, proliferating activity and differentiating potentiality. The cell balls at different densities were injected subcutaneously into nude mice to observe their implanting tumor growing capacity; (3) Cancer stem cell-like cell balls were added into incubation solution containing drug serum of Qi-boosting toxin-resolving formula for culturing, compared with a common culture and a control culture. Then, changes’in the ratio of CD133/CD44 positive cells in these cell balls were counted at the culturing time points of 24 hrs,72 hrs and 5 days respectively.Results:(1) After dispersed following a period of time culturing, cell balls in CNE-2Z nasopharyngeal carcinoma cells present in serum-free suspension medium could still form such cell balls again. When these balls of cell were transferred into a common culture medium, cells separated in a single way were visible growing in a way of adhered to the wall of culture flask 24 hrs late. After 7 days, these cell balls disappeared in the medium and they grow on the culture flask wall in the same way as the cells of original nasopharyngeal carcinoma cell line CNE-2Z. When detected for the positive expression of CD133+/CD44+ among them by immunohistochemical staining at various intervals of time, the percentage of cells was gradually declined day by day with statistical significance (P <0.05). Once alternatively cultured in another medium system, these cells could change their living form between cell balls as grown in serum-free medium and adherent cells onto the wall of culture flask as grown in common medium, but with cell morphology unchanged; (2) When cell balls were injected subcutaneously into nude mice with the densities of 1×103, 1×105 and 1×107, implanted tumors could grow, with a positive correlating tendency of tumor growth rate with cell density. The proliferating rate of cells from the primarily cultured cells implanted tumor induced by cell balls was significantly higher than that of the cells from the original nasopharyngeal carcinoma cells line CNE-2Z (P<0.05). (3) CD133/CD44 positive cell ratio was declined when cells were co-cultured with drug-containing serum of Qi-boosting toxin-resolving formula, with a rate faster than that of common culture group and control group, with statistical significances among various points of time intervals (P<0.05).Conclusions:1) Cell balls obtained from CNE-2Z NPC cells through serum-free suspension culture hold such biological characteristics of tumor cancer stem cell as self-renewal capacity, active differentiation and proliferation activities, and they can be considered as nasopharyngeal tumor stem cell-like cell populations2) In vitro, Qi-Boosting Toxin-Resolving Formula can induce such cancer stem-like cells, which come from nasopharyngeal carcinoma cells line CNE-2Z, to differentiate in the direction of reduced malignant biological behaviors.CHAPTER THREEPreliminary Study on the Intervening Effects of Qi-Boosting Toxin-Resolving Formula on Cancer Stem-like Cells in the Tissue of Implanted Tumors among Nude Mice Induced by Nasopharyngeal Carcinoma Cell Line CNE-2ZObjectives:To investigate the intervening effects of Qi-Boosting Toxin-Resolving Formula on tumor stem-like cells in the tissue of implanted tumors among nude mice induced by nasopharyngeal carcinoma cell line CNE-2Z and to compare their intervening efficiency of different treating regimens to these cells.Methods:Routine procedures were used to prepare implanted tumor model with nasopharyngeal carcinoma cell line CNE-2Z among nude mice at first. Then, these modeling nude mice were randomly divided into four groups, i.e. model group, Qi-Boosting Toxin-Resolving Formula (QBTRF) treating group, cisplatin treating group and combined treatment group with Qi-Boosting Toxin-Resolving Formula and cisplatin, with normal saline, QBTRF, cisplatin and QBTRF plus cisplatin given to each group respectively. After that, careful observation was made on the changes in animal’s body weight, skin color, the size of implanted tumors and general living status among all these mice of four groups, with other related indicators measured and recorded before and after the treatment. Two weeks late following the treatment, all animals were put into death and tissue samples were taken from implanted tumors to detect the expressive activities of CD 133 and CD44 with immunohistochemical staining procedures and to determined protein expressing levels of CD 133 and CD44 with Western blotting method. These results were statistically analyzed and compared at last.Results:1) Body weight changes in these four groups of nude mice as compaired before and after treatment being as follows. Body weight of model group and QBTRF group had statistical significance (P<0.01), no statistical significance in the combined treatment group and DDP group (P>0.05). Changes in implanted tumor size being as follows:QBTRF group, combined treatment group and DDP group showed statistical significance when compared with that in model group (P<0.05), with the inhibitory rates of QBTRF group, combined treatment group and DDP group being 18.7%,58.4% and 43.8% respectively. Observation on the changes in skin color and living status as compaired before and after the treatment showed that no statistical significance was seen among model group and QBTRF group, while there was statistical significance in the combined treatment group and DDP group, manifested as darken skin color and decreased vigor of life, especially obvious among the mice in DDP group.2) The results of Western blotting assay on the expressive activities of CD 133/CD44 proteins among these four groups of nude mice showed that, near to the levels of markers with the molecular weight being 120 kDa and 80～95 kDa, the corresponding electrophoresis strips could be seen but with differences in the brightness of these strips among the different groups. The brightest strip was seen in the gel of model group, the less bright were those of QBTRF group and the combined treatment group, and that of DDP group was the darkest one. There were significant differences with statistical significance in the brightness when compared among the combined treatment group and DDP group, QBTRF group and model group.3) The results of immunohistochemistry on CD 133 and CD44 showed that, among the four groups of nude mice with implanted tumors, there were significant differences in CD 133 expressive activities of all treating groups with QBTRF, DDP or both, when compared with that of model group (P<0.05). Furthermore, there existed statistically significant difference between the combined treatment group and QBTRF group or DDP group (P<0.05), when compared among the treating groups still. However, there was no statistical significance present in the difference as compared between QBTRF group and DDP group (P>0.05). The results of immunohistochemical assay on the expressive activity of CD44 were shown in the similar tendency as the expressive feature of CD 133 among these groups, i.e. statistical significance present in the differences of all treating groups when compared with that of model group (P<0.05). Also, there was statistical significance in the differences between the combined treating group and QBTRF group or DDP group as a result of comparison among them (P<0.05), and no statistically significant difference seen when compared between QBTRF group and DDP group (P>0.05). As a whole, the positively expressive level of CD44 was significantly higher than that of CD 133 except the one of combined treatment group. Moreover, both indicators were shown with highly positive correlation analysis each other as a result of correlative analysis (P<0.01).Conclusions:1. It is suggested that the presence of a small amount of nasopharyngeal cancer stem-like cells in the implanted tumors induced by nasopharyngeal carcinoma cell line CNE-2Z should be true because a certain ratio of CD133+ and CD44+ cells and their expressive products are present in the tissues of each group of implanted tumors induced by these cells, as shown from the results of immunohistochemical staining and Western blotting assay on these specimens. However, the number of CD44+ cells is higher than that of CD 133+ ones in each group of specimens. Therefore, it should be demonstrated further which is more specific as the surface marker for identifying nasopharyngeal cancer stem-like cells.2. Anticancer drug cisplatin may holds the potentiality to enrich nasopharyngeal in the tumor focus and QBTRF may be able to induce such cancer stem-like cells to differentiate, further, as it has been determined that the positive expressive rates of CD133/CD44 are significantly higher in the specimens of DDP (cisplatin) group than that of combined treating group.3. To compose a more practical therapy with better therapeutic effect against cancer, combined treatment strategy with Chinese herbal medicines and chemical drugs should be more effective on malignant tumors and in improving the quality of life for patients with such a lesion, because simply used herbal medicines hold limited direct killing effect on tumor cells while chemical drugs used in an alone way may give rise severe side effects on host’body, even to obstruct further use of chemical therapy for such a case. Still more, a combined therapy with herbal medicines and chemical drugs will cause a better synergistic effect on cancer cells and/or cancer stem cells as shown in this study for implanted tumors induced by NPC cells. |
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