| Background:Malignant glioma represents about 40%of all adult primary brain tumors.The prognosis of human malignant glioma remains poor with an overall 5-year survival rate of less than 3%and a median survival time of about 1 year for higher grade tumors such as glioblastoma.Gliomas present as diffuse tumors with invasion into normal brain tissue,but frequently recur or progress after surgery,radiation or chemotherapy as focal masses,suggesting that only a fraction of tumor cells is responsible for regrowth.Recently,there is accumulating evidence that malignant solid tumors may contain their own stem cells,termed cancer stem cells(CSCs).Despite their small quantity,this subpopulation within tumor possesses the ability of infinite proliferation and multipotency thus may play a crucial role in the initiation,progression and recurrence of cancer.The concept of cancer stem cells was intuitively suggested by clinical experience with leukemia,and also evident in teratocarcinomas and other solid tumors,such as breast cancer,prostate cancer and pancreas cancer. These studies have provided insights into the understanding of glioma cancer biology and paved the way for thefuture CSCs-targeted theropy.CSCs in nervous system were first reported by Ignatova and Singh.They cultured glioma cells in serum-free neural stem cell medium and obtained a group of cells that were dramatically different from common glioma cells in growth conditions,cell markers,proliferating and differentiating capacity and tumor-initiating ability in vivo.Singh separated CD133- and CD133+ cells with immunomagnetic beads and planted them separately to the front lobe of 6-week-old nude mice.12-24weeks later,he found only 102 CD133+ cells could generate a tumor,but 105 CD133- cells only made a glial scar. Importantly,elevated expression of transporters that pump out chemotherapeutic agents and an increased capacity to repair DNA damage may also contribute to CSCs' ability to survive conventional modes of therapy.Based on the ability of tumorgenesis and to survive conventional modes of therapy,Singh proposed a tumorgenic pattern based on CSCs.In the cancer stem cell model of tumors,there is a small subset of cancer cells,the cancer stem cells,which constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and maintain the tumor.These cancer stem cells have the capacity to both divide and expand the cancer stem cell pool and to differentiate into the heterogeneous nontumorigenic cancer cell types that in most cases appear to constitute the bulk of the tumor.If cancer stem cells are relatively refractory to therapies that have been developed to eradicate the rapidly dividing cells,they are unlikely to be curative and relapses would be expected.The rat glioma cell line C6 is one of the widely used cell lines in the studies of glioma.However,it remained controversial about the culturing methods and fraction of CSCs in this cell line.For instance,Toru Kondo et al. found only 0.4%of C6 cells acted as cancer stem-like cells in a complicated serum-free DMEM medium.However,Xuesheng Zheng et al.pointed out that most C6 cells were cancer stem cells from clonal and population analyses. Given the difference of tumorigenic activity and resistance to treatment between CSCs and normal tumor cells,CSCs could act as a new target for cancer therapy.However there is a problem:where do they locate? Until now, there is little research on the location of CSCs in tumor bulk.In our study,we cultured C6 cell line in a simplified serum-free neural stem cell medium and built up C6 subcutaneous xenografts model in nude mice.CD133 and Nestin marked immunohistochemistry and immunofluorescence confirmed the existence of CSCs both in C6 cell line and xenografts.Nestin marked flow cytometry demonstrated that 4.02%cells in C6 cell line presented as CSCs.Immunohistochemistry, immunofluorescence and flow cytometry were applied to detect the location and distribution characteristics of CSCs in subcutaneous xenografts.We proposed the hypothesis of "explosive distribution" of CSCs in tumor bulk and believed it helpful in explaining how CSCs participate in angiogenesis, tumor initiation,invasion and recurrence in a histological view.Objective1.To find a simple and effective method to culture,isolate and characterize cancer stem cells and confirm the fraction of cancer stem cells in C6 cell line.2.To build up C6 subcutaneous xenografts model in nude mice and detect CSCs in C6 xenografts.3.To study the distribution characteristics of brain cancer stem cells in C6 glioma.Methods:1.Tumor sphere cultureC6 glioma cells were firstly seeded in a 100ml culture flask containing 5ml 1640 culture solution,supplemented with 10%fetal bovine serum,100 U/ml penicillin G and 100μg/ml streptomycin.Cells which were in exponential phase of growth were collected and transplanted to a new culture flask with an equal volume of serum-free neural stem cell medium containing DMEM/F12,B27 supplement,recombinant human epidermal growth factor(rhEGF,20 ng/ml), and basic fibroblast growth factor(bFGF,20 ng/ml).Cells were incubated at 37℃with 95%air,5%CO2,and 100%humidity.When the tumor spheres reached the size of approximately 100-200 cells per sphere,culture propagation was performed at the ratio of 1:2.2.Characterization of cancer stem cells from C6 glioma cell lineCD133 or Nestin marked immunohistochemistry and immunofluorescence were used for characterization of cancer stem cells.Cells were plated onto poly-L-lysine coated glass coverslips in 1640 culture solution with 10%FBS or serum-free neural DMEM medium for 12 h.Cells were washed and then fixed in 4%paraformaldehyde and processed for immunofluorescence of CD133 or Nestin.The staining was detected under laser confocal scanning microscope3.The detection of cancer stem cells in C6 glioma cell lineNestin marked flow cytometry was used to detect the fraction of cancer stem cells in C6 cell line.Cells which were in exponential phase of growth were collected and cell density was adjusted to 1×107/ml.Each sample contained 100μl single-cell suspension.Ten C6 cell line samples were taken and incubated with anti-nestin antibody.Flow cytometry was then performed to test the fractions of CSCs in C6 cell line.4.Xenografts assaysC6 cells(1×105 cells ) were implanted subcutaneously to both right and left thighs of 4-week-old female immunocompromised mice(n = 20).Xenograft tumors were harvested and examined 6 weeks after implantation.5.The distribution characteristics of brain cancer stem cells in C6 glioma.(1) Xenografts specimens were fixed in 4%paraformaldehyde,and sected into serial sections(5μm in thickness).For each specimen,20 slides were selected with uniform distribution along the tumor macroaxis. Immunohistochemistry and immunofluorescence were performed on the tissue slides for CD133 and Nestin.(2) For any fresh tumor bulk,a section(0.5cm in thickness) was taken along its macroaxis.The section was equally divided into 5 parts of ring-like tissue from the center to the periphery and these 5 parts were incubated with anti-CD133 antibody.Flow cytometry was then performed to test the percentage of CSCs in each part.Results:1.Formation of tumor spheres in serum-free mediumWhen cultured in traditional serum-supplemented medium,C6 cells grew as single cells attached to the culture fask,while presented as single cells suspending in medium 1 hour after reseeded in serum-free neural stem cell medium,as small tumor spheres 24 hours later and as typical tumor spheres after 48 hours.The tumor spheres were globular,different in size,and floating in medium.2.Expression of cancer stem cell markersCD133 and Nestin marked immunofluorescence showed that:sparsly distributed CD133+ and Nestin+ cells could be seen in C6 cells cultured in serum-supplemented medium;in tumor soheres formed in serum-free medium, CD133 and Nestin were strongly abundantly expressed.3.The fractions of CSCs in C6 cell lineNestin marked flow cytometry demonstrated that about 4%cells in C6 cell line presented as CSCs.t tests demonstrated the fraction of CSCs in C6 cell line was 4.02±0.04%and 95%CI was 3.93-4.10%.4.Formation of subcutaneous xenografts in nude miceAll the 20 experimental nude mice formed subcutaneous xenografts 4-6 days after injection.When nude mice were harvested 6 weeks after implantation,the maximum diameter of the tumor bulks was 1.7-2.0cm.5.The distribution characteristics of brain cancer stem cells in C6 glioma.(1) Immunohistochemistry and immunofluorescence resultsCD133+ or Nestin+ cells could be found in xenografts of C6 glioma with three patterns of distribution:spot-like,nest-like,and zone-like.Interestingly, these three patterns of distribution were found in different parts of tumor bulks. At the center of a tumor bulk,very few CSCs could be found,which usually emerged in a spot-like pattern around capillaries.At the margin of a tumor bulk, CSCs displayed a zone-like pattern of distribution.In the area between the center and the margin,both nest-like and zone-like pattern could be seen,and the density of positivity showed a stepwise increase from the center to the margin.In some typical visual fields,CSCs displayed as cocenric citcles at the margin of the tumor bulk,forming a package of high density of CSCs for the tumor.(2) Flow cytometry resultsAt the tumor centre,the fraction of CSCs was 0.90±0.01%.In contrast,at the margin of the tumor,the fraction was as high as 9.81±0.04%.Oneway-ANOVA analysis of the fractions showed that the fraction of CD133+ cells presented a stepwise increase from the center of tumor bulk to the margin.Conclusions:1.The fraction of CSCs in C6 cell line is 4.02%and the simplified serum-free neural stem cell medium can act as a simple and effective method for isolation and detection of CSCs to study the initiation and progression of human glioma.2.C6 subcutaneous glioma in nude mice is a reliable animal model for studying glioma.3.There are three distributing patterns of CSCs in C6 glioma:spot-like, nest-like and zone-like and their distribution shows a tendency of radioactive enhancement from the center to the margin of tumor bulk as "explosive distribution" pattern.
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