Study On The Regulatory Effect And Mechanism Of Heavy-ion (¹²C⁶⁺) Irradiation On The Immune Microenvironment Of Non-small Cell Lung Cancer

Background:In recent years,immunotherapy has made breakthroughs and has even become the standard of treatment for patients with advanced Non-small cell lung cancer（NSCLC）.The development of multiple pathways of combined immunotherapy has become an effective solution to break the immune escape dilemma and synergize the effectiveness of the current treatment.Radiation therapy is known to be one of the effective treatments for NSCLC.However,the synergistic effect of conventional radiotherapy is greatly reduced by the presence of radiation resistance.Therefore,the search for a new radiotherapy treatment that can break through the tolerance of radiotherapy due to hypoxia and cell cycle dependence while providing superior radiobiological effects has become an urgent task.In this context,heavy ions(¹²C⁶⁺)have become an ideal way to complement conventional X-rays due to their powerful tumor-killing ability and superior biological effects.Objective:（1）Analysis of the radiobiological advantages of carbon ions(¹²C⁶⁺)over conventional X-ray and the differences in immunogenicity changes induced by both in NSCLC at different dose gradients and time windows.（2）Exploring the effects of carbon ion(¹²C⁶⁺)irradiation on NK cell infiltration and function in the immune microenvironment of lung cancer and related pathways.（3）Exploring the role and pathways of carbon ion(¹²C⁶⁺)irradiation on Treg cell infiltration in the immune microenvironment of lung cancer and the mechanisms involved in enhancing NK cell function and improving the immune microenvironment of lung cancer.Methods:（1）To explore the radiobiological differences of different lung cancer cell lines A549,H520 and Lewis at different dose gradients and time windows using colony-forming unit assays,Cell Counting Kit-8（CCK-8）and Annexin V-FITC/PI double staining,respectively;（2）The expression of Damage Associated Molecular Patterns（DAMPs）such as HMGB1,IL10 and TGF-βin lung cancer cells at different irradiation doses and time windows was detected by enzyme-linked immunosorbent assay（ELISA）and bioinformatically validated based on the sc RNA-seq results of CD8⁺T-cell clusters in the GEO database;（3）Constructing the C57BL/6tumor-bearing model and screening the differentially expressed genes after ¹²C⁶⁺irradiation based on RNA-seq analysis and biomimetic analysis,and exploring the signaling pathways NKG2D/NKG2DLs,which were significantly up-regulated in lung cancer after ¹²C⁶⁺irradiation,and JAK2/STAT3,which were significantly enriched in down-regulated DEGs,by enrichment analysis;（4）The expression of core genes and target proteins on the NKG2D/NKG2DLs axis and the JAK2/STAT3pathway were verified at the gene and protein levels using Real-time PCR（q PCR）,Western Blot（WB）and ELISA,respectively;（5）Exposure levels of NK cell function-related factors（IL-12,IL-18,IL-2,CD16,CD107a,IFN-γ,IL-10 and TGF-β）and IL-6,a cytokine upstream of the JAK2/STAT3 pathway,in the serum of C57BL/6mice after ¹²C⁶⁺irradiation were measured using ELISA and the expression of genes and proteins related to the JAK2/STAT3 pathway and the main NK cell ligand MICA/B was analysed using recombinant human IL6 intervention（rh IL6）.（6）The survival status and tumor changes in mice were observed via ¹²C⁶⁺irradiation combined with Treg inhibition pathway（anti-CD25&Niclosamide）intervention.Flow cytometric analysis of NK cells,Treg cells,and CD8⁺T-cells infiltration in Lewis tumors following ¹²C⁶⁺irradiation combined with Treg inhibition pathway.（7）Bioinformatics analysis of the NKG2D receptor marker gene KLRK1 and the core gene for the downstream JAK2/STAT3 pathway,FOXP3,for correlation with prognosis,clinicopathological features,and functional status of NK/Treg cells in NSCLC patients.（8）The correlation heat map between the KLRK1&FOXP3 genes and major immune cell infiltration in lung cancer was obtained by six algorithms,including TIMER,x Cell,MCP-counter,CIBERSORT,EPIC,and quan TIseq,using the R software"immunedeconv"and validated by the ss GSEA immune infiltration algorithm.Results:（1）Compared with the same physical dose of X-ray,the proliferation of A549,H520 and Lewis lung cancer cells was more strongly inhibited by ¹²C⁶⁺;the killing capacity of ¹²C⁶⁺for the three lung cancer cells was approximately 2-3 times that of X-ray under the same conditions;（2）Both ¹²C⁶⁺and X-ray irradiation promoted the exposure of HMGB1,IL10 and TGF-βin lung cancer in a time-dependent manner;unlike X-ray,the induction of the above DAMPs by ¹²C⁶⁺peaked at 4Gy irradiation;besides,the induction of the immune effector HMGB1 by¹²C⁶⁺was more significant;（3）Different functional states of CD8⁺T cells showed different gene enrichment variations as revealed by principal component analysis（PCA）and TSNE clustering analysis;（4）Based on RNA-seq analysis,up-regulated DEGs such as KLRK1 induced by ¹²C⁶⁺irradiation were significantly enriched in the NKG2D/NKG2DLs axis while down-regulated DEGs such as FOXP3 and STAT3were significantly enriched in the JAK2/STAT3 pathway;correlation biomaterial analysis showed that high expression of KLRK1 could be a marker gene for good prognosis in NSCLC and the expression of FOXP3 gene was significantly higher in tumour tissues than in normal tissues（P<0.001）,while high expression of FOXP3predicted lung cancer invasion and progression;（5）With six immune infiltration algorithms,including TIMER,x Cell,MCP-counter,CIBERSORT,EPIC,and quan TIseq,we found that the TME of lung cancer with high KLRK1 gene expression was enriched with more immune effector cell infiltration,such as CD8⁺T and activated NK cells.In contrast,low KLRK1 gene expression was accompanied by a decrease in immune effector cells and an increase in infiltration of major immunosuppressive cells such as Treg cells and M₂ macrophages.There was a significant positive correlation between FOXP3 and the infiltration of immune cells,especially Treg cells,in the immune microenvironment of lung cancer.Mostly immune effector cells such as CD8⁺T cells and NK cells were negatively correlated with FOXP3 gene expression;（6）After ¹²C⁶⁺irradiation,the exposure levels of NK cell function-related factors such as IL-12,IL-18,IL-2,CD16,CD107a,and IFN-γand major ligand proteins such as MICA/B and ULBP2-3 were significantly increased;whereas the expression of IL-6,an upstream protein of the JAK2/STAT3 pathway,was lower after ¹²C⁶⁺irradiation compared to the same dose of X-ray;The inhibition of the JAK2/STAT3 pathway by ¹²C⁶⁺was attenuated by the addition of recombinant human interleukin 6（rh IL6）,and the expression of the NK cell ligand-protein MICA/B and the transcriptional cofactor FOXP3 in Treg cells showed a shift towards the inhibition of NK cell function.（7）By comparing the survival status and tumor changes of C57BL/6 mice after ¹²C⁶⁺irradiation and combined immunotherapy（Treg inhibition）,we found that ¹²C⁶⁺irradiation,anti-PD1,anti-CD25,and Niclosamide groups significantly inhibited Lewis lung cancer growth compared with lg G2bκcontrol group（P<0.001）,and the inhibition was particularly significant in the carbon ion combination immunotherapy group.（8）By flow analysis of NK cell infiltration in the tumor-bearing model,we found that compared to the control group（lg G2bκ）,¹²C⁶⁺irradiation,the anti-CD25 group,and the Niclosamide group all significantly promoted NK cell infiltration in the tumor with a statistically significant difference in the proportion of NK cells of 4.5%,4.8%,and 5.6%,respectively（p<0.001）.In contrast,the induction of NK cell infiltration by the carbon ion combined with the Treg inhibition pathway was more significant,with infiltration proportions of 12.6%and 14.1%,respectively（P<0.001）;compared to the control group（7.8%）,Treg cell infiltration was reduced after ¹²C⁶⁺irradiation（3.9%）,and especially in the ¹²C⁶⁺combined with Niclosamide group（0.2%）;in addition,CD8⁺T-cell infiltration was increased after ¹²C⁶⁺irradiation compared to control and X-ray.Conclusion:（1）Carbon ion(¹²C⁶⁺)has an advantage over X-ray for immunogenic changes in lung cancer;（2）Carbon ion(¹²C⁶⁺)irradiation induces KLRK1 gene expression through the NKG2D/NKG2DLs axis to increase NK cell infiltration and function in lung cancer;（3）Carbon ion(¹²C⁶⁺)irradiation inhibits the IL6/JAK2/STAT3 axis to alter FOXP3/NKG2DLs expression levels to enhance NK cell infiltration and function and improve the immunosuppressive microenvironment;（4）Carbon ions(¹²C⁶⁺)combined with Treg inhibitors exerted synergistic effects in improving survival status and increasing NK cell infiltration.