Abstract Purpose: Tumor hypoxia is known to cause both radioprotection and immunosuppression, which is associated with repolarization of macrophages from the classical M1 towards an alternative M2 phenotype. Therefore, we asked whether this “skewed” activation may affect tumor cell radiosensitivity, keeping in mind that only M1 macrophages were reported to produce nitric oxide (NO), an inhibitor of mitochondrial respiration. Methods and Materials: To activate M1 and M2 polarization, peritoneal macrophages from BALB/c mice were exposed to IFN-γ/LPS or IL-4 respectively. The expression of inducible nitric oxide synthase (iNOS) was examined by western blotting, and iNOS-mediated NO synthesis by nitrite production. In addition, the M1 markers iNOS, IL-6, IL-12α and IL-12β, and the M2 markers arginase-1, Ym1, Fizz1 and CCL17 were quantified by RT-PCR. Colorectal cancer (CRC) cells and macrophages were incubated in a tissue-mimetic co-culture system (TMCS) that featured diffusion-limited oxygenation, as monitored by an oxygen-sensitive fluorescent probe. Hypoxic radiosensitivity of CRC cells in the TMCS was assessed at doses of 0, 4, 8 and 12 Gy by an 8-day colony formation assay. Results: Exposure of macrophages to IFN-γ/LPS triggered M1 polarization with the iNOS, IL-6, IL-12α and IL-12β mRNA up-regulation by 49700, 220, 260 and 740-fold respectively, and with the NO/nitrite output of 42 μM. The alternative activation of M2 macrophages by IL-4 resulted in up-regulation of arginase-1, Ym1, Fizz1 and CCL17 by 20, 499, 118 and 72-fold respectively and only marginal changes in M1 markers including iNOS. Mouse CT26 and human HCT116, HT29 and DLD-1 CRC cells showed distinct respiratory profiles in the TMCS, wherein the half-level of metabolic hypoxia was induced (within 30 min) at 5, 5, 10, and 20x10^6 cells/ml respectively. M2 macrophages consumed oxygen as fast as CT26 cells, while M1 macrophages displayed a profound respiratory arrest up to 60 min. Addition of M2 macrophages to the TMCS (at 1:1 ratio) uniformly contributed to oxygen consumption and further sustained hypoxic radioprotection up to 2.5-fold, as compared with well-oxygenated CRC cells. Contrasting, iNOS(+) M1 macrophages were able to profoundly arrest oxygen consumption and thereby restore radiosensitivity of CRC cells. Oxygen sparing and radiosensitization was abrogated by the metabolic iNOS inhibitor aminoguanidine, thus implying the role of the iNOS/NO pathway, a hallmark of M1 polarization. Conclusion: Our study provides evidence that M2 macrophages contribute to impaired radioresponse of CRC cells in a model of tumor-relevant metabolic hypoxia. Reprogramming of macrophages towards the iNOS(+) M1 phenotype efficiently counteracts hypoxic radioprotection through NO-induced oxygen sparing, and suggests a promising radiosensitizing strategy. Citation Format: Heng Jiang, Femke Steenbeke, Wim Leonard, Inès Dufait, Kalun Law, Dirk Van den Berge, Valeri Verovski, Storme Guy, Mark De Ridder. Hypoxic radioresistance of tumor cells is sustained by M2 macrophages but can be counteracted by M1 activation resulting in nitric oxide-induced oxygen sparing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 421. doi:10.1158/1538-7445.AM2013-421