Introduction
Cancer immunotherapy based on the immune checkpoint blockade (ICB) strategy has great potential in the therapy of lung cancer, but only 10-20% of patients respond due to the immunosuppressive tumor microenvironment (TME) ^[1]. Although several imaging techniques including MRI, PET, and CT have been widely used in tumor diagnosis, novel protocols to effectively monitor the efficiency of immunotherapy in real-time are precious. Hyperpolarized ¹²⁹Xe MRI with ultrahigh sensitivity has received intense attention for monitoring lung diseases such as evaluating pulmonary function in COVID-19 patients ^[2-3]. Therefore, monitoring immunotherapy of lung cancer by using ¹²⁹Xe MRI has great potential.

Methods
To reverse the immunosuppressive TME for the improvement of the ICB immunotherapy efficacy, a Fe₃O₄ nanoprobe that responds to reactive oxygen species (ROS) was created. Glucose oxidase was loaded into nanoprobe (FG) to convert glucose into hydrogen peroxide. The loading of IR820 endows the nanoprobe with T₂ MR/fluorescence dual-modal imaging ability (FGI). The immune-activating peptide Tuftsin was loaded into the nanoprobe (FGIT) to reverse the immunosuppressive TME. To improve biocompatibility, the Fe₃O₄ nanoprobe was further coated with lipids (FGITL). The successful preparation of FGTL was characterization with TEM, XPS, and XRD analysis. The T₂ MRI/fluorescence imaging was carried out on a 9.4 T imaging system and an IVIS Spectrum imaging system, respectively. The tumor immune activation effect of FGTL was analyzed by flow cytometry. Finally, the ¹²⁹Xe ventilation MRI of the lung was performed on a 7 T imaging system.

Results and discussion
The diameter of the ROS-responsive FGTL nanoprobe with the spherical structure was about 100 nm (Fig 1a). After treatment with H₂O₂, the FGTL was disintegrated into small particles (Fig 1b), promoting the uptake of FGTL by tumor cells. X-ray diffraction (XRD) results demonstrated that the prepared nanoprobe was Fe₃O₄ (Fig 1c). The presence of Fe²⁺ and Fe³⁺ was verified by the XPS analysis of Fe₃O₄ nanoprobe (Fig 1d,e). The results of ¹H MRI revealed that FGTL was equipped with a superior capacity of T₂ MRI and the enhanced T₂-weighted MRI contrast was obtained after treatment with H₂O₂. (Fig 1f-h). After i.v. injection of FGITL into mice, the nanoprobe could accumulate in the tumor region via the enhanced permeability and retention effect, confirmed by the T₂-weighted MR/fluorescence dual-modal imaging (Fig 2). Immunosuppressive TME including low immunogenicity T cell infiltration and insufficient antigen presentation efficacy would hinder the ICB immunotherapy of lung cancer. Thanks to the ferroptosis of lung cancer cells induced by the FGTL nanoprobe under mildly acidic and overexpressed H₂O₂ in TME and the loading of immune-activating peptide Tuftsin, the immunosuppressive TME could be reversed by promoting immunogenic death of tumor cells (Fig 3), finally achieving the enhancement of response rate after i.v. injection of α-PD-L1. After treatment with I) PBS, II) α-PD-L1, III) FGTL, and IV) FGTL plus α-PD-L1, respectively, the immunotherapy efficacy of lung metastatic cancer model mice was evaluated through hyperpolarized ¹²⁹Xe ventilation MRI. Significantly, the ¹²⁹Xe MRI signal intensity of the lung in the FGTL plus α-PD-L1 group was higher than that of groups I-III. The ¹²⁹Xe MRI displayed the complete shape of the lung. In contrast, there were some ventilation defects in groups I-III (Fig 4a). Furthermore, the immunotherapy efficacy of lung metastatic cancer was confirmed by the photos and H&E staining of the lung in all the treatment groups (Fig 4b), indicating the precise evaluation of immunotherapy efficiency by ¹²⁹Xe ventilation MRI.

Conclusion
Compared to CT and PET imaging, ¹²⁹Xe MRI not only possesses the advantage of non-ionizing radiation but also fulfills the high spatial resolution of CT and high sensitivity of PET imaging. Herein, we successfully apply ¹²⁹Xe ventilation MRI to evaluate the immunotherapy efficacy of lung metastatic cancer. Due to the overgrowth of lung metastatic cancer, leading to obvious ventilation defects in the lung were observed by ¹²⁹Xe ventilation MRI. Upon being treated with FGTL plus α-PD-L1, the lung ¹²⁹Xe ventilation MRI revealed the amelioration of ventilation defects, which could be used as a non-destructive imaging method for medication guidance in immunotherapy.

Acknowledgements

This work is supported by National Key R&D Program of China (2018YFA0704000), and National Natural Science Foundation of China (U21A20392, 82127802, 21921004, and 81901737).