Immunological changes and adaptation of the tumor microenvironment during glioblastoma progression
- March 2015 -
Our research project consists of three interrelated parts. Aim of work package 1 is the characterization of the tumor microenvironment and the cellular composition of primary and corresponding recurrent glioblastomas (pGBM, rGBM). The planned study sample of 40 pairs could be completed. These samples were investigated concerning T cell infiltration as well as microglia infiltration by using immunofluorescence staining. We observed a frequent increase of T cells in recurrent tumors. Furthermore, we determined a significant, prognostically relevant decrease of M1-polarized microglia during tumor progression. In addition, 34 pGBM/rGBM pairs were analyzed regarding relevant genes and signaling pathways using microarray analyses. Altered gene expression profiles in recurrent as compared to primary tumors of the same patients could be confirmed in this enhanced study sample. Especially genes, which are involved in immune responses, were found to be differentially expressed. Furthermore, 25 pGBM/rGBM pairs were investigated on the protein level using luminex analyses allowing for the quantification of 27 immune-relevant cytokines and chemokines. We observed a decrease of inflammatory cytokines during tumor progression suggesting a reduced immune response in recurrent tumors, while an increase of M2-related chemokines was found in rGBM.
In work package 2, we isolate tumor and immune cells from tumor tissues postoperatively. These cells will be collected from a substantial number of patients to be used in a final set of functional experiments in order to confirm the observed molecular changes and to attribute them to the cell of origin.
In work package 3, we aim at identifying changes in the repertoire of immunogenic antigens using a combination of protein fractionation (PF2D) and T cell activation assays. Here, we observed both, a decrease as well as an increase of immunogenic fractions in the corresponding recurrent tumor. In total, more than 1500 proteins of all single fractions were identified by mass spectrometry. An intensive filter process resulted in the selection of 16 proteins. Their potential as future vaccine antigens was validated in the patients of origin, healthy donors as well as further GBM patients. Nine of these 16 proteins were able to trigger significant immune responses in more than 40% of tested GBM patients, while showing no immune response in healthy donors.
Finally, we wanted to improve our understanding of peripheral immune responses in glioblastoma patients and if this can be used in order to monitor the efficacy of the identified immunogenic antigens within the frame of an immunotherapy. To this end, a diagnostic tool was developed to check antibody responses in the peripheral blood of GBM patients (Mock et al., Oncotarget 2015 in press). Six GBM-associated antigens were printed as short peptides on glass slides and tested for recognition by patient serum antibodies. By the help of several large study samples we identified and validated a prognostic peptide, whose applicability as an immune monitoring tool will be further analyzed in future studies.