Fig. 4: Carbon ion radiotherapy enhances the anti-tumour immune responses in vivo.

The C57BL/6 mice (Ctr) bearing subcutaneous B16 melanoma (model) were locally irradiated with the physical dose of 5 Gy X-ray (XR) or 5 GyE carbon ions (CIRT) on the tumour sites. On the 7^th day after radiation, the tumour was excised, and the tissue was embedded with paraffin and stained with haematoxylin-eosin A or specific antibodies to detect the composition of the tumour immune infiltrate. The abundance of CD3⁺, CD4⁺, CD8⁺, Granzyme B⁺ and F4/80⁺ was assessed carbon ions irradiated vs X-ray irradiated and non-treated models B–F. Also, the quantity of CD3⁺ was assessed by flow cytometry in the dissociated tumours after the radiation in the models G. The expression of ARG1 and iNOS as downstream effector proteins of JAK2/STAT3 signalling pathway was assessed by immunoblot H and the expression of iNOS was decreased significantly both in MelanA I and S91 J melanoma-bearing mice in the group that received carbon ion radiation. The serum levels of the inflammatory cytokines-TGF-β, IL-6, VEGF were measured by ELISA K–M. The representative images and quantifications are shown (mean ± SD of triplicate assessments, Student’s t-test, *p < 0.5, **p < 0.01, ***p < 0.001).