| BackgroundGliomas, especially glioblastoma (GBM) multiforme, are the most common brain tumors in adults and have a grim prognosis despite the combination of various treatment. There has been great progress in understanding the molecular genetics of these tumors, however, the origin of the tumor is still controvertial. A better understanding of the cellular origin and molecular pathogenesis may help to identify new targets for treatment of these tumors.Regarding the origin and progression of tumors, different kinds of hypothesis have been proposed, the most important are Clonal Evolution Model and Cancer Stem Cell Model. In the Clonal Evolution Model, during the progression cancer cells aquire different muations which are selected to give them a growth advantage. For each cycle, some cells will aquire new properties such as resistance to apoptosis and different kinds of treatment or increased invasiveness; This model could explain intratumoral heterogeneity based on the mutitude of different clones that are produced. In the Cancer Stem Cell Model, tumors are organized in a hierarchy in which only a minor populaton, the cancer stem cells, have the capacity to sustain tumor growth; during the progression cancer stem cells could differentiate into various specialized cells, form the intratumoral heterogeneity; targeting cancer stem cells could cure cancers.Until recently, gliomas were believed to arise from glial cells in the brain parenchyma, however, several articals reported that gliomas of different grades expressed neuronal markers; cells derived from GBM expressed neuronal-type ion channels, they even exhibited action potential similar to neuron. Normally neuronal markers are not expressed in glial cells. However, the expression of neuronal markers could be detected in gliomas, we assume that glioma cells are from neural stem cells which have the ability to differentiate into neurons. We also assume that gliomas cells arise from glial cells residing within brain parenchyma, they achieve the ability of neuronal differentiation when they form neoplasms. Since glioneuronal tumor cases have been frequently reported. The term'glioneuronal tumor with neuropil-like islands'was introduced in the2007WHO classification as a pattern of anaplastic astrocytoma, the Working Group felt that this kind of tumors constituted a distinct pattern of differentiation but more cases with genetic analysis and clinical follow-up were needed to consider them as a distinct new variant or entity. Furthermore, the neuronal marker class Ⅲ β-tubulin has been linked to dedifferentiation in glial tumors. In contrast, gene expression analysis of GBMs has identified a subgroup with neuronal differentiation patterns correlating with longer survival. The function and prognostic value of neuronal markers in gliomas are still controversial.Before CD133was regarded as the marker for brain tumor stem cells, only cells expressing CD133could develop tumors in vivo. Recently more and more data suggest that tumor-initiating cells exist among CD133-cells; these studies questioned the use of CD133as the stem cell marker, or challenged the cancer stem cell theory. Malignant brain tumors often display a striking cellular heterogeneity, besides CD133and glial cell markers, several other markers are expressed in gliomas, for instance, neuronal markers. We assume brain tumors may contain several subpopulations with different functional properties. Here we developed lentiviral vectors expressing green fluorescent protein (GFP) under different cell lineage promoters, with which we could select tumor cells expressing different markers. We will construct lentiviral vectors expressing GFP under nestin, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and compare the characteristics of these cell subpopulations.ObjectiveIn this study, we would explore the expression of neuronal markers (class III (3-tubulin, neurofilament protein (NFP), microtubule-associated protein2(MAP2) and NSE), stem cell marker (nestin) and glial cell marker (GFAP) in gliomas. Then we aimed to figure out the regulation of these markers under different culture conditions. Then we investigated the influence of NSE on characteristics of gliomas and the role it might played in mediating tumor progression and resistance to different treatment. We investigated the expression of NSE on a cohort of human glioma biopsies of different grades and correlated the expression levels with survival data. In the CD133-glioma cell subpopulations, we compared the characteristics of different subpopulations grouped by nestin, GFAP and NSE, including the proliferation in vitro and tumor development in vivo. Methods1. The expressions of class III (3-tubulin, NFP, MAP2, NSE, nestin and GFAP were investigated both in glioblastoma multiforme (GBM) cell lines and GBM patient biopsies with immunocytochemistry (ICC) and Western blot. After adding BrdUrd, we detected the proliferation of U251expressing different cell type markers with immunocytochemistry.2. We assessed the expression levels of the same markers under different culture conditions using real-time qPCR.3. After NSE siRNA treatment, we investigated the growth and migration of GBM cells using Electric cell-substrate impedance sensing (ECIS) and cell survival in hypoxia or after irradiation and temozolomide treatment with MTS assay.4. We investigated the expression of NSE in a series of human glioma biopsies with immunohistochemistry (IHC) and quantitated NSE expression using a morphometry software, after that the survival data for the corresponding patients were collected by a clinician. We then grouped the biopsies according to NSE expression level in two halves and correlated the expression with survival using the log-rank test.5. We developed lentiviral vectors expressing GFP under under the cell lineage promoters regulating the expression of Nestin, NSE and GFAP, after infecting tumor cells with lentivirus, we analysed the proportion of CD133+and GFP+cells; we sorted GFP cells expressing different cell markers and performed the cell cycle analysis under different culture conditions.6. We investigated the tumor initiating ability of cell subpopulations expressing different cell markers with animal experiment.Results1. ICC and Western blot showed that class Ⅲ β-tubulin, MAP2, NSE, nestin and GFAP were expressed in all the GBM cell lines and patient biopsies, whereas NF was hardly detectable.2. Real-time qPCR demonstrated that NSE was upregulated in GBM cell lines in cellular stress conditions, such as serum-starvation and hypoxia; MAP2was upregulated in serum-starvation medium, but downregulated in hypoxia; class III P-tubulin was downregulated in hypoxia. 3. Since NSE was consistently upregulated in different cellular stress conditions, we hypothesized that it may promote cell survival and mediate tumor progression. We therefore investigated how knockdown of NSE impacted on cell growth, migration and the response to hypoxia, radiotherapy and chemotherapy, after confirming siRNA-mediated knockdown of NSE by western blotting. NSE knockdown significantly reduced the migration of GBM cells, sensitized them to radiotherapy, temozolomide and increased cell death under hypoxia.4. From IHC, we found that NSE was expressed in all the glioma patient biopsies. Dramatically increased cytoplasmic and nuclear stainings were found in pseudopalisading cells surrounding areas of necrosis. Glioma patients belonging to the group with NSE expression above the median lived significantly shorter. Moreover, after excluding all non-GBM patients from the two groups, we found that GBM patients in the group with the highest NSE expression lived significantly shorter than GBM patients in the low-expresson group.5. After culturing in serum-containing DMEM medium and SCM, from cell cycle analysis we found that all subpopulations (nestin, NSE, GFAP) contained cell fractions in the G1,G2, and S phases. From BrdUrd experiment we found all cell subpopulations had the proliferation ability in vitro.6. Animal experiment revealed that different cell subpopulations grouped by nestin, NSE and GFAP had similar tumor initiating ability in mice, no significant results were found between different groups regarding tumor development and overall survival.Conclusion1. Neuronal markers, stem cell marker and glial cell marker are aberrantly expressed both in GBM cell lines and patient biopsies, and their expressions are altered by cellular stress.2. NSE is consistently upregulated in different cellular stress conditions. Knockdown of NSE reduces the migration of GBM cells, sensitizes them to hypoxia, radiotherapy and temozolomide.3. From IHC NSE is expressed in all glioma patient biopsies, dramatically increased cytoplasmic and nuclear stainings were found in pseudopalisading cells surrounding areas of necrosis.In addition, glioma patients (including all grades) with high NSE expression lived significantly shorter than patients with low NSE expression, GBM patients in the highest NSE expression group also had significantly shorter survival than patients in low NSE expression group.4. Stem cell marker nestin and glial cell marker GFAP were expressed in gliomas, different cell subpopulations grouped by nestin, NSE and GFAP could proliferate in vitro and have similar tumor initiating ability in vivo.
|
PDF Full Text Request