Carbon ion radiotherapy (CIRT) is an emerging type of
radiotherapy for the treatment of solid
tumors. In recent years, evidence accumulated that CIRT improves the therapeutic outcome in patients with otherwise poor response to
immune checkpoint blockade. Here, we aimed at identifying the underlying mechanisms of CIRT-induced
tumor immunogenicity and treatment efficacy. We used human U2OS
osteosarcoma cells for the in vitro assessment of immunogenic cell death and established several in vivo models of
melanoma in mice. We treated the animals with conventional radiation, CIRT, PD-1-targeting
immune checkpoint blockade or a sequential combinations of
radiotherapy with checkpoint blockade. We utilized flow cytometry,
polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis, immunofluorescence, immunohistochemistry, as well as
enzyme-linked
immunosorbent assays (ELISA) to assess
biomarkers of immunogenic cell death in vitro. Treatment efficacy was studied by
tumor growth assessment and the
tumor immune infiltrate was analyzed by flow cytometry and immunohistochemistry. Compared with conventional
radioimmunotherapy, the combination of CIRT with anti-PD-1 more efficiently triggered traits of immunogenic cell death including the exposure of
calreticulin, the release of
adenosine triphosphate (
ATP), the exodus of high-mobility group box 1 (
HMGB1) as well as the induction of type-1
interferon responses. In addition, CIRT plus anti-PD-1 led to an increased infiltration of CD4+, and CD8+ lymphocytes into the
tumor bed, significantly decreased
tumor growth and prolonged survival of
melanoma bearing mice. We herein provide evidence that CIRT-triggered immunogenic cell death, enhanced
tumor immunogenicity and improved the efficacy of subsequent anti-PD-1
immunotherapy.