Enhanced tumor immune cell infiltration and antigen presentation after carbon ion radiotherapy correlates with curative outcomes

Limited immune cell infiltration and deficient presentation of neoepitopes and tumor-associated antigens (TAA) via major histocompatibility complex class I (MHC-I) represent key barriers to effective immunotherapy. This study investigates whether particle radiotherapy (RT) can modulate tumor antigen presentation and T cell recruitment. By evaluating protons, helium, carbon, and oxygen ions, we assessed how distinct biophysical properties-particularly ionization density (linear energy transfer, LET)-influence immune priming. To this end, we evaluated the induction of MHC-I expression in a comprehensive panel of human tumor cell lines (CaSki, SK-MEL-37, A549, PC3, LNCaP), as well as in B16-F1 melanoma, GL261 and 005 genetically engineered glioblastoma models in syngeneic C57BL/6 mouse. Flow cytometry revealed MHC-I upregulation is time-, dose-, and LET-dependent. The role of the cGAS-STING pathway was confirmed using isogenic STING-KO B16 cells, which exhibited reduced RT-induced MHC-I expression. In vivo, high-LET carbon ion irradiation (CIRT) resulted in enhanced tumor MHC-I expression and increased intratumoral infiltration of CD3⁺ and CD8+ T cells. Adoptive transfer of Superparamagnetic Iron Oxide Nanoparticles (“SPION”)-labeled T cells, followed by high-field MRI tracing, confirmed enhanced intratumoral influx after fractionated (5 × 3 Gy) and single ultra-High Dose Rate (uHDR, FLASH) CIRT. Further characterization of tumor-infiltrating lymphocyte (TIL) composition and immunological memory was performed using single-cell RNA sequencing and mass cytometry (CyTOF). Collectively, these data indicate that modulating RT quality can improve neoantigen expression, MHC-I presentation, and T cell recruitment, highlighting the potential of particle radiotherapy to reprogram the tumor immune microenvironment.