The Radiobiological Mechanism And Clinical Significance Of Non-small Cell Lung Cancer After Hypofractionated Radiotherapy

Although hypofractionated radiotherapy(HFRT)in early non-small cell lung cancer(NSCLC)patients achieved good curative effect,there are still certain local and regional recurrence rates,and this needs to explore the mechanism of cancer radiotherapy resistance,provide new strategy for improving the efficacy of HFRT.Cancer has ten hallmarks to promote its development and resist therapy,and the professional scholars tried to find the way to interfere with the development,invasion and metastasis of tumor in severy ways,so as to improve the curative efficacy of cancer.Among them,resistance to cell death and neovascularization are two important hallmarks of cancer,and it is one of the common reasons that many tumors have no significant response with chemotherapy and radiotherapy.Importantly,the HFRT resistant mechanism has not been fully elucided.After radiotherapy,many kinds of cell death form can occur.And apoptosis resistance is one of the reasons for the poor efficacy of radiotherapy,but there is no obvious benefit by the large number of studies on apoptosis resistance.High dose irradiation can induce cell necrosis,so it is urgent to explore the role of other forms of cell death in the radiotherapy.Neovascularization is another important hallmark of cancer,and is the inevitable stage of tumor progression.It provides nutrition for the unrestricted growth of cancer,and also promotes the invasion and metastasis of tumor.Radiotherapy has a variety of effects on tumor microenvironment,and previous studies have found that HFRT promotes endothelial cell apoptosis and homing of mesenchymal stem cells.While the potential mechanism and the subsequent effect is not clear,so the changes of HFRT on tumor microvasculature will be of great significance to improve the radiosensitivity.This study is divided into two parts:the effects of hypofractionated radiotherapy on the tumor cell death types and tumor microvasculature remodeling.Part IPurpose:To investigate how necroptosis(ie,programmed necrosis)is involved in killing of NSCLC after ablative HFRT.Methods:The death forms of NSCLC cells were assessed after RT.The overexpression and silencing of receptor-interacting protein kinases 3(RIP3,a key protein involved activation of necroptosis)-stable NSCLC cell lines were successfully constructed.The forms of cell death,the number of colonies,and the regulatory proteins of necroptosis were characterized after SBRT in vitro.Finally,NSCLC xenografts and patient specimens were used to examine involvement of necroptosis after SBRT in vivo.Results:SBRT at>10 Gy/fraction preferentially stimulated necroptosis in NSCLC cells and xenografts with high RIP3 expression,as characterized by induction and activation of RIP3 and mixed-lineage kinase domain-like protein(MLKL)and the release of HMGB1.In contrast,RNA interference of RIP3 attenuated ablative HFRT-induced necroptosis and activation of its regulatory proteins.Among early-stage central NSCLC patients receiving stereotactic body radiation oncology(SBRT),high expression of RIP3 was associated with improved local control and progression-free survival(all p<0.05).Conclusions:SBRT at>10 Gy/fraction enhances killing of NSCLC with high RIP3 expression via preferential stimulation of necroptosis.RIP3 may serve as a useful biomarker to predict favorable response to SBRT.Part ?Objective:To investigate the molecular mechanisms behind SBRT stimulating MSC migration and differentiation.Methods:In vitro,ELISA detected the expression and release of stromal cell-derived factor-la(SDF-la)and platelet-derived growth factor subunit B(PDGF-B)from LLC and B16F10 cells after SBRT and Transwell evaluated the migration of MSC.In vivo,bone-marrow derived MSC were identified and harvested from green fluorescent protein(GFP)-expressing transgenic male mice and transplanted into sub-lethally irradiated recipient female mice to establish a model of bone marrow transplantation.LLC and B16F 10-bearing recipient mice were treated with SBRT,14 Gy/1fraction,or with the inhibitors AMD3100 and Imatinib.ELISA detected serum chemokines,and immunofluorescence test bone marrow-derived MSC migration,microvascular density and pericytes in xenografts.And we did statistical analysis of the data and drew mice transplanted tumor growth curve.Results:SBRT increased the release of SDF-1? and PDGF-B in LLC and B16F10 tumor cells;and these ligands bound to C-X-C chemokine receptor type 4(CXCR4)and Beta-type platelet-derived growth factor receptor(PDGFR-?)on circulating bone marrow-derived MSC respectively,resulting in recuitment of the MSC into the tumor parenchyma.The newly homed MSC differentiated into pericytes,which induced the tumor vasculogenesis,and promoted tumor regrowth.Targeted therapies with AMD3100 and imatinib abrogated MSC homing,vasculogenesis,and tumor regrowth.Conclusions:Bone-marrow derived MSC migrate to the tumor parenchyma and differentiate into pericytes via SDF-1?/CXCR4 and PDGF-B/PDGFR-? signaling pathway,inducing tumor vasculogenesis after SBRT,and promoting tumor recurrence.MSC migration and maturation may be abrogated with AMD3100 and imatinib.This novel treatment strategy warrants clinical investigation.