Immunological changes and adaptation of the tumor microenvironment during glioblastoma progression
2nd funding period, November 2016
In the first funding period we have been focusing on the characterization of the tumor microenvironment in primary glioblastoma (pGBM) and corresponding recurrent glioblastoma (rGBM). Some of our major findings consisted of changes in the amount and the polarization of microglia cells as well as an altered infiltration of different T cell subtypes during the course of the disease. To further corroborate our results, we expanded our study sample from 40 to 64 patients with available material of primary and corresponding recurrent tumor tissues and thereby could amply confirm our previous observations. In addition, we could strengthen our recent findings on the differential gene expression occurring between pGBM and rGBM. Interestingly, some of the identified differentially expressed genes are involved in immune-relevant signaling pathways such as the chemotaxis of immune cells. These data were further complemented on the protein level by Luminex analyses indicating a decrease of positive immunemodulatory cytokines during tumor progression. To get a more complete understanding of the immune infiltrate landscape in primary and recurrent glioblastoma, just recently we started to analyze intratumoral infiltration of natural killer and B cells in this unique study sample.
Moreover, in the first funding period we aimed at identifying immunogenic antigens in primary tumors as well as in corresponding recurrent glioblastoma by using a method which combines a protein fractionation method (PF2D) with a T cell activation assay (IFN-γ ELISpot Assay) followed by a rigorous validation process. These analyses revealed marked changes of the repertoire of immunogenic antigens during tumor progression. To account for the extensively described intratumoral heterogeneity of glioblastoma which seems to be caused among others by glioma-stem like cells (GSCs), we extended our study design to the analysis of well-characterized GSC lines by the PF2D-IFN-γ-ELISpot method. Mass spectrometry of immunogenic GSC fractions identified more than 3000 potential T cell target antigens. Applying an extensive filtering process, 20 peptides were chosen for further validation steps which included testing the immunogenicity in a larger study sample of glioblastoma patients as well as healthy donors. The before obtained data from the microarray analysis were used to learn more about the mRNA expression level of potential T cell target antigens in pGBM and rGBM and thus their potential relevance. Altogether, this resulted in the identification of several so far unknown immunogenic target antigens in glioblastoma present on undifferentiated GSCs as well as on differentiated cells of primary and recurrent glioblastoma.