Exploring the immune enhancement effect of radiotherapy combined with NK cells on non-small cell lung cancer based on proteomics technology.

Background: Although radiotherapy (RT) and natural killer (NK) cell-based therapy have shown promising potential in the treatment of non-small cell lung cancer (NSCLC), the therapeutic efficacy and safety of their combination remain to be fully elucidated. In this study, we evaluated the efficacy and safety of combined RT and NK cell therapy and performed preliminary proteomic profiling to characterize its potential effects on the tumor immune microenvironment (TIME), providing new insights and a theoretical and clinical basis for the development of NK cell-based therapeutic strategies for NSCLC. Methods: C57BL/6-derived subcutaneous Lewis lung carcinoma models were established to determine the minimal effective dose of both radiotherapy and adoptively transferred natural killer cells. Based on the MED-derived findings, the mice were further randomized into five experimental groups for therapeutic efficacy evaluation: the Control group, the RT group, the NK group, the combination therapy group (RN group), and the repeated combination therapy group (RN2 group). Tumour volume and body weight were monitored longitudinally. Differentially abundant proteins were interrogated by Gene Ontology enrichment and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analyses to systematically describe the synergistic antitumor efficacy of differentially expressed proteins. Results: Compared with RT or NK cell-based immunotherapy as monotherapies, the RN group exhibited markedly enhanced tumor growth inhibition, with no overt treatment-related toxicities observed. Following the second cycle of combined therapy, mice in the RN2 group displayed a more pronounced reduction in tumor growth rate with those in the single-cycle combination cohort; notably, no significant adverse events associated with the combined regimen were detected throughout the entire experimental duration. Proteomic profiling unraveled that RT disrupted the core regulatory networks governing intracellular DNA replication and repair processes in tumor cells in a therapy-specific manner, whereas NK cell immunotherapy potently and selectively activated the classical pathway of the host complement system. Conclusion: Collectively, RT and NK cell therapy demonstrate superior synergistic efficacy and no overt toxicity was observed, with repeated treatments further delaying tumor progression. Mechanistically, proteomics shows that RT disrupts DNA replication/repair networks, whereas NK cell therapy was associated with upregulation of multiple complement components, including those belonging to the classical and lectin pathways, suggesting a potential involvement of these pathways.