Immunological changes and adaptation of the tumor microenvironment during glioblastoma progression
2nd funding period, November 2017
Within the funding period we focused on the characterization of the tumor microenvironment in primary glioblastoma (pGBM) and corresponding recurrent glioblastoma (rGBM). We were able to collect and study a unique and meaningful study sample including more than 60 pairs of corresponding pGBM and rGBM tumor tissues. In these samples we observed strong differences in the composition and activation of different immune cell populations (microglia/macrophages, T cells, NK cells, and B cells). By means of transcriptomic analyses we could further confirm the observed changes and identified genes associated with anti-inflammatory tumor-associated microglia and macrophages (TAMs). In order to investigate the influence of TAMs on the functional properties of T cells, different cell types such as tumor cells, TAMs, and tumor-derived endothelial cells have been isolated from fresh tumor tissues and conditioned TAM-medium has been collected. By conducting transmigration assays and Immune Cell Killing assays in an autologous setting, we observed an inhibitory effect of TAMs on T cells. First experiments revealed an improved T cell functionality after TAM targeting suggesting TAMs as a therapeutic target to reduce immunosuppression in GBM.
Moreover, by the combined PF2D-IFN γ-ELISpot method we identified novel immunogenic T cell target antigens which are highly expressed in pGBM and rGBM tumor tissues. We further ensured that these antigens induce immune responses against quiescent and therapy-resistant glioma stem-like cells (GSCs) as well as their differentiated counterpart. Among more than 700 potential T cell target antigens identified by mass spectrometry 32 proteins were selected through a thorough filtering process for further validation. Of these antigens, especially five peptides corresponding to HSPD1, PPIA, FSCN1, ANXA1, and CSTA induced significantly increased tumor-specific T cell responses in a large study sample of GBM patients but not in healthy donors. A comprehensive characterization of these antigens provided evidence for a consistently high mRNA and protein expression during tumor progression. The relevance of these tumor-associated antigens (TAAs) was further corroborated by their expression in quiescent, slow-cycling GSCs as well as differentiated tumor cells and by quantifying a considerable number of antigen-specific T cells.
Finally, by means of printed peptide arrays we could show that the newly identified TAAs do not only induce T cell responses but also robust antibody responses in the serum of pGBM and rGBM patients in a large study sample. In addition, some of these peptide responses showed an association with patient survival. Thus, this method will enable to test immunogenicity of potential TAAs in a less invasive setting.