Ionizing Radiation-induced Tumor Cell-derived Microparticles Prevent Lung Metastasis By Remodeling The Pulmonary Immune Microenvironment

Objectives:Radiotherapy is an effective tumor treatment method that can achieve local control of the primary tumor,but the complex changes in the tumor microenvironment caused by radiotherapy may also change the risk of tumor metastasis.The effects of radiotherapy on tumor metastasis are complex.On the one hand,after radiotherapy for solid tumor,circulating tumor cells（CTCs）with radiation characteristics are increased in the blood of tumor patients and they have proliferative activity.On the other hand,damaged tumor cells after radiotherapy release tumor-associated antigens（TAAs）and cause damage-related molecular patterns（DAMPs）responses,which can induce anti-tumor immune response both in the primary tumor and in metastatic site.However,whether radiotherapy promotes or inhibits tumor metastasis is inconclusive.Our preliminary study showed that compared with the group without radiotherapy,the number of CTCs and CTC cluster increased in the blood of mice with LLC subcutaneous transplanted tumor after radiotherapy,and they had the ability to proliferate,but the formation of lung metastases was less in the radiation group.Our research aims to explore the causes and related mechanisms of the reduction of lung metastases after radiotherapy of subcutaneous transplanted tumor in mice.Methods:（1）Using DIFF staining to detect the number of single CTC and CTC cluster in the blood of mouse with LLC subcutaneous transplanted tumors after radiotherapy.Clone formation experiment was used to observe the proliferation activity of CTCs after radiotherapy of LLC subcutaneous transplanted tumor.H&E staining was conducted to observe the effects of LLC subcutaneous transplanted tumor receiving radiotherapy on the lung metastases of LLC cells.（2）A single 20 Gy radiotherapy was performed on LLC or B16cells cultured in vitro.After 3 days,differential centrifugation was used to separate the ionizing radiation-induced tumor cell-derived microparticles（RT-MPs）in the tumor cell culture supernatant.Mice were pretreated by injection of RT-MPs through the tail vein,and then tumor cells were injected through the tail vein.The formation of pulmonary metastatic nodules was observed.（3）Flow cytometry was used to detect which cells RT-MPs were mainly ingested by after reaching the lungs,and immunofluorescence was used for in vitro verification.Flow cytometry was used to detect the dynamic change of the proportion of immune cells in lung after RT-MPs injection.（4）The neutrophils or macrophages in mice were removed respectively to observe whether the effect of RT-MPs on the prevention of lung metastasis could be reversed.（5）The chemokine changes in the lung homogenate of mice injected with RT-MPs and the cell culture supernatant of neutrophils stimulated by RT-MPs were detected respectively.The chemotactic effects of corresponding chemokines on specific immune cells were verified by Transwell experiment in vitro.（6）The activation of neutrophils or macrophages after RT-MPs stimulation and their anti-tumor effects were examined in vitro.Results:（1）After radiotherapy of subcutaneous transplanted tumors,the number of CTCs and CTC cluster increased and those CTCs had the ability to proliferate,but the number of pulmonary metastatic nodules in the radiotherapy group was less than that in the non-radiotherapy group.（2）After pretreatment by injection of RT-MPs through tail vein,the formation of pulmonary metastatic nodules was reduced compared with the control group,and the survival was significantly prolonged,indicating that RT-MPs could prevent lung colonization of tumor cells.（3）RT-MPs were mainly absorbed by neutrophils and macrophages in lung,and in vitro experiments also confirmed that neutrophils and macrophages had a strong phagocytic effect on RT-MPs.The proportion of neutrophils in lung significantly increased at 1 h after the injection of RT-MPs,and then decreased gradually;the proportion of monocytes in lung was obviously increased at 4 h;the proportion of macrophages was increased at 6 h,and the proportion of macrophages remained at a high level over time.（4）Elimination of neutrophils or macrophages in mice could reverse the preventive effect of RT-MPs on lung colonization of tumor cells.（5）The concentrations of CCL3 and CCL4 in lung homogenate were significantly increased after 2 h of RT-MPs injection.Besides,the concentrations of CCL3 and CCL4 in the culture supernatants of neutrophils were also significantly increased after 2 h treatment with RT-MPs.CCL3 and CCL4 can recruit monocytes,and after using the corresponding chemokine antibodies to neutralize CCL3 and CCL4,the chemotactic effect of monocytes was significantly weakened.（6）When neutrophils were stimulated by RT-MPs in vitro,the production of H₂O₂ and ROS in neutrophils was evidently increased,and the m RNA expression levels of MPO,i NOS and NOX2 in neutrophils were also significantly increased.Under the stimulation of RT-MPs,macrophages were polarized to M1 type.Besides,the cytotoxic effect of neutrophils and macrophages was remarkably enhanced after stimulated by RT-MPs.Conclusions:（1）After radiotherapy for subcutaneous transplanted tumors in mice,the formation of pulmonary metastatic nodules was reduced.（2）RT-MPs prevented lung colonization of tumor cells by remodeling the lung immune microenvironment.（3）RT-MPs stimulated neutrophils to produce CCL3 and CCL4,which recruited monocytes to the lung,and further differentiated into macrophages.（4）Under the effect of RT-MPs,neutrophils were activated and macrophages were polarized to M1 type,and their anti-tumor effects were significantly enhanced.