Abstract
BACKGROUND: Glioblastomas are the most prevalent malignant brain tumors in adults, with a frequency ranging from 0.59 to 3.69 in the western world. They are accompanied by a fatal prognosis and a median survival of 12.2 to 18.2 months. Their significant intratumoral and intertumoral heterogeneity is the reason for this. Tumor associated macrophages including microglia can represent up to 30-40% of the cellular component of the tumor and are thought to have an impact on the tumor's behavior. The MGMT promotor methylation is the most significant clinical prognostic factor. Patients with a methylated promotor have a better prognosis and a better response to Temozolomid treatment. This experimental study should determine if the microglia or the tumor cells are responsible for the MGMT status. Moreover, it should show how the MGMT-protein is distributed intracellularly in both cell-populations. MATERIAL AND METHODS: Therefore, the MGMT-Status of 13 samples of patients with glioblastoma from the division of neurosurgery of the TU München were analyzed. Using magnetic cell sorting and an anti-CD11b antibody, tumor cells and microglia were separated in each sample. A MGMT-assay was carried out for each cell population to demonstrate the proportion of methylated MGMT promotor regions. 25 histological sections underwent immunofluoresence double staining to get a better look at the MGMT protein's intracellular distribution. Two sections of each sample were stained with anti-MGMT antibody and anti-GFAP antibody or anti-IBA1 antibody, respectively, to determine if the MGMT protein is present either in the nucleus or in the cytoplasm of the tumor cells and microglia. A minimum of 200 cells of every section was analyzed manually. Statistical analysis was performed using Mann-Whitney-U-test and a p-value <0.05 was defined as significant. RESULTS: The separarte MGMT-assay of microglia and tumor cells showed methylated MGMT promotors in 3 samples in both cell populations. Tumor cells as well as microglia demonstrated intracellular MGMT Protein, with no significant difference (0.122 MGMT+ Microglia vs. 0.152 MGMT+ Tumor Cells; p=0.101). The intracellular distribution of the MGMT protein strengthened this result. Tumor cells demonstrated a significantly higher percentage of nuclear MGMT protein (0.602 nuclear MGMT vs. 0.203 cytoplasmic MGMT; p<0.0001), whereas microglial cells showed a significantly higher expression of cytoplasmic MGMT protein (0.608 cytoplasmic MGMT vs. 0.157 nuclear MGMT; p<0.0001). CONCLUSION: According to the findings of this experimental study, both the microglia and the tumor cells are responsible for the clinical MGMT status. This might fit into the theory of macrophage polarization in the microenvironment of heterogenous tumors. The MGMT protein distribution pattern implies that tumor cells and microglia express their MGMT protein in different cellular compartments.