Identifizierung von Tumor- und Stromaantigenen zur Behandlung von HNSCC
Immunologie
Zusammenfassung der Projektergebnisse
Epithelial-derived tumors are histopathologically characterized by a desmoplastic stromal compartment which surrounds malignant cancer cells. This tumor stroma mainly consists of cancer-associated fibroblasts (CAFs) which are characterized by an irreversibly activated phenotype that is assumed to be associated with the expression of CAF-associated antigens. In the current project, we focused on the identification of immunogenic CAF-associated T cell target antigens (CAF-TAs) using a novel approach which combines the PF2D-ELISPOT method with a microarray data-based filter process. CAFs and corresponding tumor cells (TUCs) were isolated from freshly resected HNSCC tumor tissues using primary cultures and established cell lines following several in depth characterization steps including verification of the CAF activation status. We detected significant T cell (TC) responses against CAF and TUC lysates and showed the presence of CD4+ and CD8+T cell populations in HNSCC patients that can directly recognize CAF. Interestingly, the amount of CAF-reactive TCs was comparable to TUC-reactive TCs, indicating that CAF-TAs might also play a role in tumor immune responses. By applying the PF2D-ELISPOT method the CAF proteome was fractionated and then analyzed for immunogenic proteins. Subsequent mass spectrometric analysis of immunogenic fractions resulted in 852 potential immunogenic candidates. The intersection-based filter process resulted in 36 potential CAF-associated antigens. Immunogenicity of candidates was verified by the use of long synthetic peptides in peptide ELISPOTs. Thereby we have identified 15 novel CAF-associated target antigens of spontaneous TC responses in a cohort of 19 HNSCC patients as compared to nine healthy individuals. Furthermore, we have shown that five of these newly identified CAF-TAs (DNAJB11, NANS, HIST2H2AC, TXNDC17, THBS2) triggered TC responses in 16 to 40 % of cancer patients, whereas no reactive TCs were detected in healthy individuals. In addition, mRNA expression analysis of the TCGA data set confirmed overexpression of these antigens in HNSCC tissues as compared to dysplasia-free normal mucosa samples. Immunohistochemical staining further corroborated a stromaassociated expression which in case of THBS2 and DNAJB11 was entirely limited to the stromal compartment. In conclusion, we have successfully shown that CAFs might serve as additional source of TC target antigens, since we identified a set of novel CAF-TAs that are highly immunogenic and expressed in the stromal compartment of HNSCC tumors.
