Gadolinium-based nanoparticles AGuIX and their combination with ionizing radiation trigger AMPK-dependent proinflammatory reprogramming of tumor-associated macrophages

Background: Tumor-associated macrophages (TAMs) are essential components of the inflammatory microenvironment of tumors and are associated with poor clinical outcomes in the majority of cancers. TAMs mainly exhibit anti-inflammatory functions that promote and support the tissue remodeling, the immune suppression and the tumor growth. Regarding their plasticity, the functional reprogramming of anti-inflammatory TAMs into proinflammatory phenotype recently emerged as a therapeutic opportunity to improve the effectiveness of anticancer treatments such as radiotherapy. Results: Here we show that gadolinium-based nanoparticles AGuIX alone and in combination with ionizing radiation (IR) induce in a dose-dependent manner, the accumulation of DNA double strand breaks, an Ataxia telangiectasia mutated (ATM)-dependent DNA-damage response, an increased expression of the Interferon regulatory factor 5 (IRF5) and the release of proinflammatory cytokines from targeted macrophages, thus directing their proinflammatory reprogramming. This process is associated with the activating phosphorylation of the Adenosine Monophosphate (AMP) activated protein kinase on threonine 172 (AMPKT172*) and the fragmentation of mitochondria. Furthermore, we demonstrate that the inactivation of AMPK reduces the mitochondrial fragmentation and the proinflammatory reprogramming of macrophages detected in response to AGuIX and their combination with IR. These results reveal that the AMPK-dependent regulation of mitochondrial fragmentation plays a central role during the proinflammatory reprogramming of macrophages. Accordingly, a positive correlation between AMPKT172* and proinflammatory activation of TAMs is detected following IR+AGuIX combination in syngeneic mouse model of colorectal cancer. Conclusions: Altogether, our results identify a novel signaling pathway elicited by AGuIX and their combined treatment with IR, that targets macrophage polarization, skews macrophage functions toward the proinflammatory phenotype and may enhance the effectiveness of radiotherapy. ### Competing Interest Statement J.-L.P. reports research grants from NH TherAguix SA and Wonna Therapeutics and is founder of Findimmune SAS, an Immuno-Oncology Biotech company. S.D., T.D. and G.L.D. are currently employees of NH TheraAguix SA. A.A. was employed by NH TheraAguix SA to develop research activities related to the current work. D.T. was recipient of a CIFRE contract in partnership with NH TherAguix SA. J.-L.P. and A.A. are co-inventors on patents (WO2016185026 and WO2018050928), relating to macrophage reprogramming. J.-L.P., A.A., F. L. and O.T are co-inventors on patents (WO2019008040A1; WO2021053173A1), relating to AGuIX and relevant to the current work. J.-L.P., D.T., S.D. and G.L.D. are co-inventors on patent (EP23173710.7), relating to immunomodulatory properties of AGuIX and combinatorial strategies. G.L.D., F.L. and O.T. are co-inventors on patent (WO2011135101), relating to AGuIX. G.L.D., S. D. D., F. L. and O. T. possess shares in NH TheraAguix SA. The remaining authors declare no other competing financial interests.