| Gliomas, accounting for40-50%of intracranial neoplasms in adults, are the most common primary neoplasms of central nervous system (CNS), of which, the glioblastoma multiforme (GBM) is the one with the highest malignancy and is characterized by high mortality rates. The median survival of GBM patients from the time of diagnosis is no more than3months without effective treatment, with97%of patients dying in12months. Even though they were treated with standardized treatment, the median survival will still no more than12months. Over the past decades, although advances in surgical excision and adjuvant radio-and chemotherapy for GBM have shown some limited promise, these tumors inevitably recur in the majority of patients and are nearly uniformly fatal.A key feature that underlies the malignant behavior of the GBM is its exceptional migratory nature and ability to integrate seamlessly and extensively into normal neural tissues. Glioma cells often migrate great distances from the main tumor mass, making it extremely difficult to treat by surgical resection or radiotherapy, which contributes to recurrence and a very poor prognosis. Therefore, to figure out the mechanisms and characteristics of GBM migration will be great significant for the clinical treatment. However, there is still no specific marker for indicating the direction of cell migration. The golgi apparatus, which is responsible for the modification process of protein in eukaryocyte, has been found to take part in the formatiom of microtubule organizing center and the formation and maintenance of cell polarity during cell migration.It is reported that dispersal of GBM cells occurs along characteristic pathways of anatomical structures in the brain including the meninges, ventricular lining, perivascular regions, and especially the myelinated fiber tracts. More over, the chemokine family is considered to play a significant role in cell migration. Zagzag et al found that the SDF-la and its receptor CXCR4, which are associated with directional cell migration, express at the perivascular space and white matter fibers. Therefore, we hypothesized that the chemotactic guidance would be required during the tumor cells migration along white matter fibers and their congregation to the microvascular niche in the white matter fibers.The present study included two parts:The first part was to investigate the relationship between the position of golgi apparatus within cell and the direction of tumor cell migration both in vitro and in vivo. Cell migration assays were carried out with rat C6glioma cells, human U251and SNB19glioma cells respectively. The immunofluorescence was used to detect the position of golgi in migrating cells. The percentage of cells with the golgi facing towards the wound edge was calculated. Cell pseudopodium was stained with TRITC-phalloidin, and the relationship between golgi position and pseudopodium was detected. Immunohistochemistry was used to locate the golgi in tumor tissue sections, and the percentage of cells with golgi apparatus facing opposite to the necrotic zones was calculated. The result showed that the golgi was facing towards the wound in the majority of cells locating at the wound edge (C6:83%±6%, U251:80%±7%, SNB19:82%±6%). In U251and SNB19cells, the golgi apparatus situated in the same direction with cellular pseudopodium. Immunohistochemical staining showed that the golgi was found facing opposite to the necrotic zones in majority of cells locating around the necrotic zone (C6glioma tissue sections:80%±7%, human glioma tissue sections:82%±6%).In the second part of this study, immunofluorescence and western blotting were performed to detect the expression of SDF-1a and CXCR4in C6cells, U251cells, U87cells and HUVECs; immunohistochemistry was used to detect the expression of SDF-1α and CXCR4in tumor tissues. The transwell migration assay was conducted to analyze whether the SDF-1/CXCR4was involved in the chemotactic movement of tumor cells. For each glioma cell line, cells were treated with five different regimens in the lower chambers in Boyden chamber assay:600μl of DMEM without serum (control group);600μl of serum-free DMEM with SDF-1α(100nmol/ml)(SDF-1a induction group);600μl of media collected from cultured HUVECs supernatant without serum (HUVECs supernatant induction group);600μl of media collected from HUVECs supernatant media without serum but with AMD3100(25μg/ml)(H+AMD3100group);600μl of serum-free DMEM containing both SDF-1α (100nmol/ml) and AMD3100(25μg/ml)(S+AMD3100group). Cells that had migrated to the bottom of the cellulose acetate membrane were calculated. For each cell line, every treatment was repeated triply. C6glioma cells were stereotactically inoculated into the wistar rat brain at callosum. The immunohistochemical staining, Hematoxylin and Eosin (H&E) staining and Luxol Fast Blue (LFB) staining were conducted to show the distribution patterns of tumor cells in rat brain, and the relationship among tumor cells, vessels, and white matter fibers. Results: immunofluorescence and western blotting showed that CXCR4was expressed in C6cells, U251cells, and U87cells, and SDF-la was expressed in HUVECs. The immunohistochemical staining revealed that CXCR4expressed in tumor cells while SDF-la expressed in endothelium cells. In the Boyden chamber assays, very few cells migrated to the bottom of cellulose acetate membrane in the control groups (C6:2.9±1.8; U251:3.1±1.8; U87:2.3±1.8). In comparison with the control groups, much more of cells were calculated in both SDF-1α groups (C6:107.5±15.6; U251:110.7±13.1; U87:109.5±13.5)(P<0.05) and HUVECs supernatant media groups (C6:99.6±13.2; U251:104.7±13.1; U87;106.9±12.7)(P<0.05). Compared to the SDF-la groups and HUVECs supernatant media groups, the migration capacity of cells was dramaticlly inhibited in both S+AMD3100groups (C6:2.8±1.5; U251:2.1±1.0; U87:1.7±1.2)(P<0.05) and H+AMD3100groups (C6:20.1±4.5; U251:15.6±3.8; U87:15.1±4.3)(P<0.05). H&E staining and LFB staining showed tumor cells migrated along the white matter tracts and assembled in the perivascular niches. The Ki-67staining revealed that tumor cells assembling in the perivascular niches had high proliferation activity.Conclusions:1. The golgi apparatus showed close association with cell migration, and could be considered as a direction indicator of cell migration both in vitro and in vivo.2. The glioblastoma cells could migrate along the white matter fibers while invading into the mormal brain tissues, and part of which would assemble around the perivascular niches.3. Migrating glioblastoma cells would scatter around the perivascular niches for proliferation.4. The SDF-1/CXCR4signaling played an important role in glioma cell migration. |
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