Modulating NOS2 of radiotherapy-recruited CCR2⁺ macrophages enhances radiosensitivity of hepatocellular carcinoma.

Abstract

C-C motif chemokine ligand-2 (CCL2)-C-C motif chemokine receptor-2 (CCR2) axis plays critical roles in hepatocellular carcinoma (HCC) progression and in shaping immune responses following radiotherapy (RT). However, its contribution to radio-resistance and the underlying mechanism remain poorly understood. CCL2 and CCR2 expressions were analyzed in paired pre-/post-RT HCC tumor samples from patients exhibiting differential responses to RT. In parallel, we assessed CCL2 secretion, infiltration of CCR2-expressing myeloid cells, and tumor-associated macrophage (TAM) subsets, including the subpopulations expressing NOS2, in two murine HCC models: radioresistant BNL and radiosensitive Hepa1-6 allografts. Despite similarly elevated CCL2 levels and comparable induction of immunogenic cell death indicated by calreticulin exposure, CCR2⁺ myeloid cells were recruited in significantly greater abundance in RT-resistant than RT-responsive tumors, both in human tumors and murine allografts. Following RT, there was a significantly greater increase in NOS2-expressing M1-like TAMs in Hepa1-6 than BNL tumors. Ex vivo experiments demonstrated that the TAMs isolated from irradiated Hepa1-6 tumors suppressed tumor proliferation, promoted CD8⁺ T-cell infiltration, and improved tumor control compared to TAMs from non-irradiated tumors. In vitro co-culture assays demonstrated CCR2⁺ myeloid cells attenuated NOS2 expression in M1-like macrophages, suggesting a suppressive effect on their pro-inflammatory phenotype. Furthermore, pharmacologic blockade of CCR2⁺ cells using a CCR2 antagonist restored NOS2 expression, enhanced radiosensitivity, and improved tumor control in radioresistant BNL models. CCR2⁺ myeloid cells suppress NOS2-mediated antitumor responses in HCC following RT. Targeting RT-recruited CCR2⁺ myeloid cells may offer a promising radiosensitizing strategy to overcome therapeutic resistance in HCC.