Targeting Tumor Cell Secreted MCSF To Improve Post Radiation Immune Microenvironment

Objective: Radiotherapy is one of the main means of tumor treatment.In recent years,with the development of tumor immunology,the immunomodulatory effects of radiotherapy have gradually attracted attention.The immunomodulatory effect induced by radiotherapy is a double-edged sword,which also promotes the release of inhibitory immune factors while activating antitumor immunity.Through screening with flow cytometry multi-factor kits,this study found that macrophage stimulating factor(MCSF),an immunosuppressive cytokine,was elevated early and persistently elevated after radiation.Blocking the early increase of MCSF after radiation may help to shift the immune balance towards immune activation,which is of great significance for activating the post-radiation tumor immune microenvironment,improving the efficacy of radiotherapy,and improving the prognosis of cancer patients.Methods: 1)Lewis lung carcinoma subcutaneous tumor tissues after 8 Gy irradiation were examined by multi factor kits(LEGENDplex Custom Mouse 7-plex Panel),looking for inhibitory cytokines elevated at the early post radiation stage.Explored the tumor suppressive effects of radiotherapy combined with MCSF receptor inhibitor PLX3397 in murine Lewis lung carcinoma and SCC7 head and neck squamous cell carcinoma subcutaneous tumor models.H&E staining in important organs of mice was used to verify the biosafety of the combination treatment.2)Flow cytometric detection of altered immune cell subsets and functions within the tumor microenvironment and spleen,following radiotherapy combined with PLX3397 of murine Lewis lung carcinoma and SCC7 head and neck squamous cell carcinoma subcutaneous tumor models.3)Single-cell transcriptome analysis of Lewis lung carcinoma subcutaneous tumor tissue after 8 Gy irradiation was used to search for the source of elevated MCSF after radiation.The results of single-cell transcriptome analysis were validated by RT-qPCR,flow cytometry,and ELISA assays.4)Prediction of transcription factors regulating MCSF gene expression and validation of the transcription factor regulation of elevated MCSF after radiation using inhibitors.ChIP-qPCR was used to validate the direct role of the transcription factor with the gene of MCSF.5)The transcript levels of colony stimulating factor 1 receptor(CSF1R)in each cell were detected by RT-qPCR.Co-culture experiments were performed to examine the effect of cytokines released by tumor cells after irradiation on macrophages.Macrophage clearance experiments validated by which immune cells the inhibitor of CSF1 R acts through.Results: 1)The secretion of MCSF,an inhibitory cytokine,was elevated early and persistently elevated after radiation,and blocking the MCSF pathway at the early stage after radiation significantly inhibited tumor growth and promoted tumor cell death,and radiation combined with CSF1 R inhibitor had a favorable biosafety profile.2)Blockade of the MCSF pathway did not affect local inhibitory cytokine secretion,but activated local and systemic T cell immunity and improved immunosuppression by myeloid cells.3)Single cell transcriptome analysis combined with RT-qPCR,flow cytometry and ELISA suggested that elevated MCSF after radiation was mainly secreted by tumor cells.4)Radiation enhanced tumor cell p65 binding to the promoter region of the MCSF gene to promote MCSF secretion.5)Blocking the MCSF pathway at the early stage after radiation exerted tumor suppressive effects through macrophages.Conclusion: After ionizing radiation,p65 protein within tumor cells was rapidly phosphorylated and binded to the MCSF gene promoter to promote its transcriptional expression,secreting MCSF to the tumor microenvironment.MCSF acted on macrophages,inducing a large number of inhibitory cytokine secretion.The use of CSF1R inhibitors early in radiotherapy blocked the negative immunomodulatory effects initiated by MCSF,further enhanced the immune activating effects of radiotherapy,activated local and systemic antitumour immune responses and improved the post radiation immune microenvironment.