| Aims:To isolate TSCs from MB first, and then characterize TSCs including morphology, ratio of CD133+cells, clonogenecity, growth curve and cell cycle distribution. To examine the existence of tumor stem-like cells(TSCs) in primary and recurrent medulloblastoma(MB) and analyze their distribution characteristics and proliferative status. To compare the proliferative index of MB-TSCs before and after recurrence. To analyze the proliferative features of TSCs in human MB after radiotherapy in vitro and explore their radiobiological characteristics in terms of cell cycle distribution, Cyclin expression and DNA damage response.Materials and methods:Five primary MBs obtained from surgical patients and one cell line(D283) were cultured in serum free medium(SFM, containing EGF,bFGF,LIF and B27). The ratio of CD133+cells in D283 and primary MB were analyzed with flowcytometry at different times and different culture condition. Proliferation, expression of stem cell markers and multilineage differentiation of these cells were investigated by clone formation assays and immunofluorescence. CD133+and CD133-MB cells were sorted by fluorescence activated cell sorting. Cell morphology, growth curve, clonogenecity and expressions of stem cell markers in SFM were compared between this two populations. Cells were injected into the armpit of NOD-SCID mouse subcutaneously to determine their tumorgenecity. To characterize the secondary tumor, cells were disassociated again and the ratio of CD133+cells were analyzed with flowcytometry. HE and immunohistochemistry with Ki67, GFAP and p53 were performed.The data used were obtained from five consecutive patients with MBs who subsequently experienced tumor recurrence from 2004 to 2007 in our department and completed the same treatment schedule, including surgery, radiation, and adjuvant chemotherapy. Then we performed the immunohistochemical and immunofluorent assays to analyze the expression of TSC proteins(including CD133,Nestin,DCX,PSA-NCAM and TUC-4) and proliferative features of TSCs in primary and recurrent MBs. Differences were calculated using paired t test for data from the same patient and Student's t test for data from separate groups.The growth curves of unsorted MB cells, CD133+and CD133-cells were drafted using XTT assays. The 6-well palte clone formation assay was used to measure their clonogenecity. The cell cycle of each group was analyzed by flowcytometry using propidium iodide staining. Ionizing radiation(IR) treatment of each group was performed using linear accelerator Varian LINAC 600C at 0.4 Gy/min. The ratios of CD133+cells before and 48 hours after IR were analyzed with flowcytometry. The growth curves and clonogenecity of CD133+and CD133-cells after IR were compared with those before IR respectively. The cell cycle of each group after IR was also analyzed by flowcytometry using propidium iodide staining. The quantity of Cyclin B and Cyclin E before and after IR was measured using Western Blot assay. The IR-induced apoptosis was analyzed by flowcytometry using AnnexinV staining. The DNA damage response of each group after IR was analyzed using comet assay.Results:MB cells cultured in the SFM could propagate and form tumorspheres. Colony efficiency of D283 and primary MB(BT2) was 21.43%±1.69%and 6%±0.58% respectively. The highest CD133 ratio of primary MBs was 4.74%±0.48%from BT2. The CD133 ratio of MB cells cultured in SFM increased with time in the first one week and then remained stationary levels. It began to decrease after MB cells were cultured in serum contained medium(SCM). The CD133 ratio of D283 remained about 20%in SFM and 17%in SCM at different times. Many stem cell markers such as CD133, nestin, SOX2, PSA-NCAM and TUC-4 could be detected in MB cells cultured in SFM. Most MB-TSCs were quiescent while the other were proliferative. MB cells could differentiate to multiple lineages by detecting expression of GFAP,β-tubulin and CNPase after they were cultured in SCM. D283 in SCM could still express stem cell markers and never expressed GFAP,β-tubulin and CNPase. The separated CD133+cells could form tumorspheres in SFM and express kinds of stem cell markers while most CD133-cells were adherent and could not form tumorspheres in SFM. CD133+cells could also be categorized into quiescent and proliferative groups.104 and 105 CD133+MB cells could undergo tumorgenesis subcutaneously in NOD-SCID mouse. Secondary tumors exhibited similar biological features to primary tumors.107 CD133-MB cells also displayed tumorgenesis in NOD-SCID mouse but the secondary tumors were smaller and grew slowly.Of the 5 patients,2 had recurrence at the primary site and 3 had a distant recurrence. TSC markers such as CD133, nestin. DCX, PSA-NCAM, and TUC-4 were expressed no matter in the five primary or recurrent MBs. CD133 and nestin were expressed in a specific region called perivascular niche (PVN). All the 10 tumor specimens had a high proliferative index(PI), regardless of primary or recurrent MBs. In addition, the PI was even higher in the group of patients with recurrent MB at a distant site (p<0.05), while the PI remained almost the same in the group of patients with primary recurrence. Moreover, the Ki67/nestin-, Ki67/DCX-and Ki67/TUC-4-positive cells were significantly increased in recurrent MB at both primary and distant site, whereas TSCs in primary medulloblastoma showed much lower proliferative features(p<0.05).CD133+MB cells grew faster than the unsorted and CD133-cells in terms of their growth curves. Colony efficiency of CD133+cells(32.56%±2.66%) were higher than unsorted and CD133-cells(20.01%±1.49 and 5.89%±6.76% respectively). The G0/G1 ratio in CD133+cell group(61.50%±2.31%) were higher than that in CD133-cell group(54.72%±0.97%). The CD133 ratio increased obviously 24 hours after MB cells were treated with IR in vitro. The proliferative capacity of CD133+cells were inhibited temporarily within 72 hours after IR, but they recovered soon afterwards. Colony efficiency of CD133+cells treated with IR decreased slightly(from 32.56%±2.66%to 30.61%±0.93%). The ratio of G0/G1 phase in CD133+cell group increased after IR(from 61.50%±2.31%to 67.67%±3.34%). The quantity of Cyclin E expressed in CD133+cell group were more than that in CD133-cell group and it decreased after IR. The quantity of Cyclin B expressed in CD133+cells were less than that in CD133-cells and it increased significantly after IR. The ratio of apoptosis induced by IR in CD133+cell group (2.33%±0.06%) was less than in CD133-cell group(9.33%±1.16%). The "comet tails" in CD 133+cell group vanished remarkably 48 hours after IR, while they could still be seen in most irradiated CD133-cells.Conclusions:MB cells in SFM can propagate, differentiate to multiple lineage and undergo tumorgenesis. CD133+cells are enriched with MB-TSCs. Most MB-TSCs are in quiescent status which are not sensitive to IR. The stem cell niche, including cytokines and focal microenvironment is important to the maintenance of TSCs. Present serum free culture condition need to be further explored to accommodate to TSCs'thriving in vitro.The tumorigenesis of MBs and their recurrence might be related to TSCs. TSCs may have strong proliferative capacity and be resistant to radiotherapy. In addition, proliferating TSCs in MB may be associated with its metastases or dissemination. More proliferating TSCs in medulloblastomas denote worse prognosis.CD133+cells are resistant to IR comparing to CD133-cells. This may be attributed to their redistribution of cell cycle, differential expression of Cyclins, anti-apoptosis capacity and preferential DNA damage response. The results may be helpful for the further study of radiobiological characteristics of CD133+cells in the future and improvement of curative effect of radiotherapy.
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