Irradiated Tumor Cell-derived Microparticles Mediate Tumor Eradication Via Cell Killing And Immune Reprogramming:a Translational Research

Part Ⅰ:RT-MPs mediate radiation-induced bystander effect by inducing ferroptosis in tumor cellsObjection: Evidence accumulated over the past three decades has demonstrated that,as well as the direct DNA damage-dependent effects caused by radiation,irradiated cells can also send signals to their neighbors.This phenomenon is called radiation-induced bystander effects(RIBE).Although RIBE has been discovered for more than 30 years,the underlying mechanism has not been fully revealed and not therapeutically exploited.We evaluated the effects of Irradiated tumor cell-derived microparticles(RT-MPs)in cell killing and tested whether RT-MPs could mediate RIBE.Method: Tumor cells were irradiated with 20 Gy by X-ray in vitro,and the cell culture supernatant was separated 3 days later.Irradiated tumor cell-released microparticles(RT-MPs),exosomes(Exo)and the cell supernatant without RT-MPs and/or Exo(CSWPE)were obtained by differential centrifugation.Cell counting Kit-8(CCK-8)and clonogenic assay were performed to test cell viability and proliferative potential.Molecular inhibitors which specifically target ferroptosis,necrosis,autophagy,apoptosis were used to explore the mechanism of RT-MPs-induced cell death.Label-free quantitative proteomics and KEGG analysis were performed on RT-MPs-treated and untreated cells to further elucidate the molecular signaling pathway of cell death caused by RT-MPs.Flow cytometry(FCM)was used to detect cytosolic and lipid reactive oxygen species(ROS),and transmission electron microscopy was used to detect mitochondrial morphology.Results: The clonogenic assay showed that after 48 h of culturing cells with a medium containing RT-MPs,Exo,or CSWPE,the number of colony formation in the RT-MPs group was much less than other groups.CCK-8 test also showed that tumor cell viability was also significantly inhibited by RT-MPs.The cell death induced by RT-MPs was not modulated by inhibitors except for ferrostatin-1(Fer-1)and L-glutathione(GSH)which targeted the molecular pathway involving ferroptosis.Label-free quantitative proteomics and KEGG analysis also demonstrated that ferroptosis pathway was enriched.Moreover,prostaglandin G/H synthase 2(PTGS2)and ferritin(FTL1),which were involved in ferroptosis,exhibited significantly higher expression in the RT-MPs-treated group.In RT-MPs-treated A549 and LLC,TEM images showed shrunken mitochondria.Cytosolic and lipid ROS were significantly increased 4,12,24 h after tumor cells were treated,respectively,and the increased ROS could be reversed by the broad-spectrum iron death inhibitor GSH and the specific iron death inhibitor Fer-1.Conclusion: Taken together,these results indicate that RT-MPs mediate RIBE by causing ferroptosis,which is manifested as increased cytosolic and lipid ROS and mitochondrial shrinkage,and the cell death can be reversed by inhibitors targeted ferroptosis pathway.Part Ⅱ : RT-MPs mediate tumor eradiation via cell killing and immune reprogrammingObjection: Based on the fact that RT-MPs can mediate RIBE by causing tumor cell ferroptosis in vitro,we further explore the effects,safety and underlying mechanisms of RT-MPs in malignant pleural effusion(MPE)and melanoma treatment using murine models.Method: Immunofluorescence(IF)and FCM were used to detect the expression of calreticulin(CRT)on the surface of tumor cells,and luciferin-based ENLITEN? ATP Assay was used to detect the release of ATP.CX3CR1+/GFP mouse window chamber model was used to observe the phagocytosis efficacy of macrophages.PKH-26 was used to label RT-MPs,and the labeled RT-MPs were injected into the thoracic cavity of mice with MPE to identify the subpopulations of cells that uptook RT-MPs in the pleural effusion.RNA sequencing(RNA-seq)and KEGG analysis were used to investigate the differences in transcriptomics between macrophages treated by RT-MPs and control group.RT-PCR and Western-blotting(WB)were used to verify the expression of major differential genes and activated molecular pathways discovered by RNA-seq.The proportion and phenotypic changes of immune cells in MPE and tumor microenvironment(TME)was quantified by FCM.The pleural effusion of the mice was detected by Bruker In Vivo MS FX PRO Imager.The survival of mice was recorded daily.Mice were identified as dead when tumor burden exceeding 1,000 mm3 in volume in Subcutaneous tumor model.CD8(clone 2.43),CD4(clone GK1.5),NK1.1(clone 108760)monoclonal antibodies(m Ab)and clodronate liposomes(F70101C-AC)were used to deplete CD8+ T,CD4+ T,NK cells and macrophages,respectively.Blood routine,blood biochemistry and hematoxylin-eosin(HE)staining of various organs were used to assess the safety of treatment.Results: IF and FCM revealed that the expression of surface CRT and the release of ATP were significantly increased 24 hours after A549 and LLC cells were treated by RT-MPs.We observed that RT-MPs-treated LLC-RFP cells could recruit more macrophages,and more likely to be engulfed by macrophages in the CX3CR1+/GFP mouse window chamber model.4 h after PKH-26 labeled RT-MPs were injected into the pleural cavity of mice with MPE,F4/80+ macrophages uptook the most RT-MPs qualified by FCM,followed by Gr-1+ cells(representing myeloid-derived suppressor cells,MDSC)and CD45-cells(representing tumor cells),whereas few RT-MPs were taken up by CD3+ T cells.The RNA-seq showed that M1-related m RNAs were higher expressed,and the m RNA expression levels of Nos2,Il-1,Il-6,Il-12,Tnf-α et al were verified by RT-PCR.KEGG analysis also showed that the JAK-STAT signaling pathway and MAPK signaling pathway were enriched in RT-MPs treated macrophages,which was verified by WB.M1 related markers,such as CD86 and MHC-Ⅱ,were significantly increased.Meanwhile,M2 related marker,CD206,notably decreased 24 h after macrophages were treated with RT-MPs.RT-MPs-treated macrophages showed much stronger ability to phagocytose LLC-RFP cells than that of untreated macrophages,which was visualized in the CX3CR1+/GFP dorsal skinfold window chamber model.We also find that macrophages expressed higher level of PD-L1 on the cell surface.So we further combined RT-MPs and PD-1 m Ab,which potently inhibited MPE formation and prolonged mice survival,of which a subpopulation of mice were cured and survived as long as 227 days.FCM revealed that CTL,Th1 cell ratio was significantly increased.Meanwhile,MDSC abundance was robustly decreased in the MPE microenvironment.We further investigated which immune cell subpopulation played the most important role in the MPE restriction by specifically depleting macrophages,NK cells,CD4+ T cells,and CD8+ T cells.The results demonstrated that CD8+ T cell depletion neutralized most of the effects of the combined treatment,followed by macrophages and CD4+ T cells depletion.However,depleting NK cell did not make any differences.The combined RT-MPs and PD-1 m Ab also inhibited MPE formation potently in cisplatin-resistant MPE model.Mice weight,blood routine,blood biochemistry,and HE staining showed no differences between the combined group and untreated group.Conclusion: The combined RT-MPs and PD-1 m Ab potently inhibit MPE formation and cure a subpopulation of mice in MPE models.Furthermore,the combined therapy is also effective against cisplatin-resistant MPE with good security.The possible mechanisms are as follows:(1)Tumor cells killed by RT-MPs exhibited the characteristics of immunologic cell death(ICD),and were more easily phagocytosed by macrophages.(2)Macrophages engulfed RT-MPs polarized to anti-tumor M1 type through JAK-STAT pathway,and had improved capacity to phagocytose tumor cells.(3)Combined RT-MPs and PD-1 antibody therapy improved the immune microenvironment of MPE.