RBIO-03. Proteomic profiling of immunogenic molecules following radiotherapy in glioma cells

Abstract PURPOSE This study aimed to characterize the profile of immunogenic molecules released by glioma cells following exposure to radiotherapy which is known as in situ tumor vaccine. METHODS The human glioma cell lines (U87 and U251) were subjected to varying doses 5, 20, 50, or 100 Gy of radiotherapy and the supernatants were collected to generate the in vitro-irradiated cancer vaccine (ICV). Proteomic profiling was conducted using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Damage-associated molecular pattern (DAMP) and DAMP-like candidate proteins were selected based on gene ontology annotation, including cellular localization and functional roles such as chaperones, nucleic acid-binding, ribosomal, and immune-related functions. Differentially released proteins were defined by fold change and statistical significance. RESULTS In the U87 cell line, the ICV supernatant showed increased secretion of DAMP-related heat shock proteins, including HSPA8 and members of the HSP90 family compared to control supernatants. Additionally, molecules typically retained intracellularly and released upon cellular stress or death —such as RNASET2, GULP1, NAP1L1, SMARCA2, PCNA, and NPM1—were detected at higher levels. Despite not being classical DAMPs RNASET2 (a ribonuclease), GULP1 (a phagocytic adaptor protein), NAP1L1 (a nucleosome assembly protein), SMARCA2 (a chromatin remodeling factor), PCNA (a nuclear protein involved in DNA replication), and NPM1 (a nucleolar phosphoprotein) may have potential immunostimulatory roles. On the other hand, the supernatant of U251 cells displayed increased levels of different immunogenic candidates such as HLA-A (an MHC class I molecule), ribosomal proteins (RPL11, RPL28, RPS21), member of heat shock protein family (DNAJA2, DNAJC7, HSD17B4), mitochondrial proteins (NDUFAF6, NDUFS6), and chromatin remodeling factors (CHD2, CHD4). CONCLUSION Radiotherapy-induced release of immunogenic molecules, alongside tumor-derived neoantigens, may contribute to antitumor immune responses. Notably, the profiles of released molecules differed between U87 and U251 cell lines, underscoring the importance of personalized approaches in cancer vaccine development using radiotherapy.