NR4A3 functions as an immuno-metabolic checkpoint in tumor-associated macrophages to mediate radiotherapy sensitivity in rectal cancer​

Background: Radiotherapy resistance remains a major therapeutic challenge in rectal cancer. Tumor-associated macrophages (TAMs) are pivotal components of the tumor immune microenvironment. The nuclear receptor NR4A3 is a key regulator of TAM activation. This study aimed to investigate the role of macrophage-expressing NR4A3 in radiotherapy sensitivity in rectal cancer. Methods: Transcriptomic RNA sequencing analyses were used to identify key differential genes and pathways. Interfering RNAs were applied to modulate NR4A3 expression in macrophages. Signaling pathways and metabolic alterations were assessed by Western blot, qPCR, ELISA. Functional effects were evaluated in macrophage-colorectal cancer cell co-culture models. Results: High NR4A3 expression in TAMs correlated with improved pathological response and prolonged survival in rectal cancer patients receiving neoadjuvant radiotherapy. Transcriptomic analysis revealed marked alterations in NR4A3 expression and lipid metabolism pathways in post-radiation cells. NR4A3 expression was induced in macrophages following radiotherapy, which was consistently validated in vitro (THP-1, RAW264.7), in murine MC-38 xenograft models, and human rectal cancer samples. NR4A3 knockdown promoted an M2-like polarization state (evidenced by elevated CD163, IL-10) and enhanced expression of immune checkpoint molecules (PD-L1, siglec-15), while suppressing M1 markers (CD86, IL-1beta, TNFalpha). Functional assays revealed that NR4A3-deficient macrophages lost tumor-suppressive capacity in co-culture systems. On the other hand, NR4A3 promoted cholesterol synthesis and efflux in macrophages, with ABCG1 identified as a critical downstream cholesterol exporter. Macrophage-derived cholesterol contributed to tumor cell proliferation. Inhibiting ABCG1 rescued tumor suppressive effect of radiation-activated macrophages. Conclusion: ​ NR4A3 acts as a radiation-induced "immuno-metabolic checkpoint" in TAMs, enhancing M1 polarization and antitumor immunity, while concurrently driving ABCG1-dependent cholesterol efflux and a pro-tumor metabolic milieu. These results illuminate the dual role of NR4A3 in mediating the paradoxical effects of radiotherapy on the immune microenvironment and underscore its potential as a therapeutic target for overcoming radiotherapy resistance in rectal cancer.