| Cancers are heterogeneous tissues composed of various cancer cells differing in proliferation, differentiation, invasion potential and therapeutic resistance. In vitro, clonogenicity also varies among different cancer cells, and this heterogeneity may be embodied by different tumorigenicity in vivo. When classified according to their tumorigenicigy, cancer cells can be divided into many subpopulations, and the subpopulation endowed with high tumorigenicity is defined as tumor stem cells or cancer stem cells, compared with those non-tumorigenic cancer cells. As far, cancer stem cells have been isolated and identified within leukemia and several types of solid tumor. The cancer stem cells are of self-renewal and infinite proliferation, initiating the genesis, relapse and metastasis of malignancies, although they merely take a low proportion in the total cancer cell number.Heterogeneity is also an outstanding feature of glioma, and accumulating evidence proved the persistence of glioma stem cells (GSCs) within this tumor. Although great achievements have been acquired in the research of GSCs, sorting GSCs remains one prior and critical problem. For the purpose of harvest GSCs, two methods are employed nowadays. One method is sorting CD133+ cells through magnetic beads or flow cytometry, since CD133 has been regarded as the specific marker of GSCs. Another method applies the the biological feature of stem cells that they highly express ATP-binding cassette transporter and sort side population(SP) cells by flow cytometry, this subpopulation has been proved to be enriched with cancer stem cells. Effective as these two methods may be, it is a pity that they also show some weakness, for instance, sorting GSCs by these methods always need specific apparatuses, and sometimes the activity of the sorted cells is greatly influenced, except for questionable sensitivity and specificity of these methods. Therefore, new strategies or methods for sorting GSCs are urgently required. Accounting for heterogeneous differentiaton status and proliferation potential, cancer stem cells and non-tumorigenic cancer cells may vary in clonogenicity ability in vitro, as well as the phenotypes of their colonies. Recently, it was reported that long-term-cultured cells derived from head and neck squamous cell carcinoma, breast carcinoma and prostate carcinoma could form heterogeneous colonies, and certain colonies featured with a tight and regular shape contain stem-like cancer cells which were highly tumorigenic, growing xenografts in NOD-SCID mice when merely 100 cells were transplanted, whereas other colony types were non-tumorigenic. Thus colony heterogeneity, especially morphology heterogeneity, may be applied to recognize colonies containing cancer stem cells, and this provides us with an easy and simple method to collect cancer stem cells, which also may be more consisted with functional definition to cancer stem cells. However, it remains unclear whether such colony heterogeneity exist in glioma and can be used to harvest GSCs. What's more, mechanisms through which the colony morphology heterogeneity predicts the"stemness"of a colony is also beyond understanding.To clarify the problems mentioned above, we employed malignant glioma cell line U251 as a model, characterizing and classifying its colonies, through which we isolated and identified GSCs colonies. Furthermore, the mechanisms of recognizing cancer stem cell colonies through colony morphology were partially elucidated. In addition, we isolated and identified cancer stem cells from primary anaplastic oligoastrocytoma, and determined their ability of recapturing the heterogeneity of primary tumors. Our research was divided into three parts:In the first part, we observed the morphology and differentiation heterogeneity of U251 cell colonies. U251 cells were plated in a low density, and its clonogenic efficiency was 7.88%. According to morphology phenotype, colonies were classified into three types: tight colonies, intermediate colonies and loose colonies, which took a percentage of 11.9%, 61.0% and 27.1% respectively. Immunofluorescent staining was employed to assay expression of differentiation markers GFAP, vimentin and nestin by different colonies, and the tight colonies showed the strongest staining for vimentin and nestin, while GFAP was weakest in this type. On the contrary, the loose colonies were most positive for GFAP, and weakest for Vimentin and nestin. As for the intermediate colonies, they exhibit a moderate expression for these differentiation markers.In the second part, we isolated and identified GSCs colonies derived from U251 cell line. Futhermore, the mechanism of morphological genesis of various colony patterns is analyzed. Here cloning cylinders were used to isolate the tight colonies which were most poorly differentiated, and singles cells derived from tight colonies were plated in low density for a second time. Intriguingly, singles cells of the tight colonies could reconstruct colonies similar to the primary tight colonies, moreover, they also give rise to other two types of colonies. When these cells were cultured in serum-free medium supplemented with growth factors, they could form floating spheres resembling neural stem cells, and the spheres expressed CD133, a specific marker for brain tumor stem cells. Then the tumor spheres were plated in medium containing 10% fetal bovine serum. After 2 weeks, they attached to the bottom and differentiated, generating cells respectively expressing three lineage markers of CNS, namely GFAP, MBP andβ-tubulinⅢ, and some of them simultaneously expressed both GFAP andβ-tubulinⅢ.In addition ,we also employed wound healing assay and transwell assay to compare motility of cells from different colonies, and the distribution and arrangement of F-actin were observed by immunofluorescent staining. As a result, U251 cells derived from tight colonies in vitro exhibit low motility and immature development of cytoskeleton relating cell motility, while the cells from loose colonies showed higher motility. Furthermore, the tight colonies were strongly positive for CD44, compared with a lower CD44 expression within intermediate and loose colonies, and this may constitute one important molecular basis for the invasion of tight colony cells. In the third part, GSCs isolated from human anaplastic oligoastrocytoma were transplanted into SCID mice and the phenotypes of xenografts were observed. We found GSCs were highly tumorigenetic, recapturing the phenotypes of the primary tumor, and the xenografts were composed of heterogeneously differentiated tumor cells. Moreover, undifferentiated tumor cells are enriched within the xenografts..Our results can be concluded as following:1. U251 cell colonies possess heterogeneity relating to morphology and differentiation, and can be classified into tight colonies, intermediate colonies and loose colonies morphologically. The tight colonies are most poorly differentiated.2. U251 cells of tight colonies can self-renew and differentiate into cells expressing markers of three cell lineages within CNS. What's more, tight colony cells are of low motility, constituting one of the mechanisms through which they form colonies with a tightly packed morphology.3. GSCs can form xenografts composed of differentiation heterogeneous glioma cells, and undifferentiated tumor cells are enriched within the xenografts.In summary, cell colonies heterogeneity can be used to recognize and isolate GSCs, and this may provide an easy and simple way to collect GSCs.
|
PDF Full Text Request