Effect And Significance Of Radiotherapy On VEGFR2 And PD-L1 In Rectal Cancer

Colorectal Cancer(CRC)is a common malignant tumor in humans,of which the incidence of rectal cancer accounts for about 30%.Neoadjuvant chemoradiotherapy(neoCRT)is considered to be the standard treatment for patients with locally advanced rectal cancer(LARC).NeoCRT can significantly improve the local control rate,survival and quality of life in patients with LARC.Studies have shown that about 4060%of LARC patients treated with neoCRT achieve pathologically partial remission,but only 15-20%of patients achieve pathologically free tumor residue after treatment.Therefore,it is an important scientific issue to seek new therapeutic strategies to improve its efficacy.Radiotherapy(RT)could kill tumor cells directly,and trigger anti-tumor immune response and lead to the expression of programmed death ligand 1(PD-L1)and its receptor PD-1 and other immunosuppressive molecules.Various mechanisms could regulate the tumor microenvironment.Immunotherapy has become the standard treatment for some tumors with progression or recurrence.The combination of PD-L1 and PD-1 leads immune escape of the tumor,which facilitates the survival of tumor cells in the host,and further promotes its metastasis and diffusion.Immunotherapy has been applied in clinical practice,which has greatly changed the therapeutic effect of a variety of tumors,such as urinary system malignancy,head and neck malignancy,lung cancer,esophageal cancer,liver cancer and melanoma.Therefore,the effective combination of radiotherapy and immunotherapy may provide a new strategy for improving the efficacy of antitumor therapy.The key factor in the process of tumor cell growth and distant metastasis is angiogenesis.Vascular endothelial growth factor/Vascular endothelial growth factor receptor(VEGF/VEGFR2)is an important factor.VEGFR2 inhibitor Apatinib is considered as an important anti-tumor drug,which inhibits tumor cell proliferation by stimulating apoptosis.It has been approved for the treatment of colorectal cancer,renal cancer,glioblastoma,hepatocellular carcinoma and lung cancer.It can significantly improve overall survival(OS)and progression-free survival(PFS)of patients.Studies have shown that in melanoma and liver cancer,VEGFR2 and STAT3 are inhibited by Apatinib,and STAT3 is downstream of VEGFR2.Apatinib inhibits tumor growth by inactivating STAT3,thereby inhibiting cell proliferation,inducing cell apoptosis and autophagy.Anti-angiogenic drugs have been shown to promote tumor infiltration of CD8+T lymphocytes and enhance the efficacy of immunotherapy for malignant tumors.Moreover,JAK/STAT3 is an important signaling pathway to regulate PD-L1 expression,and PD-L1 expression is inhibited after STAT3 inactivation.In fact,the use of VEGF/VEGFR2 inhibitors in different tumor models resulted in improved hypoxia and immune cell infiltration,and the normalization of tumor blood vessels enhanced tumor oxygenation,thus improving the efficacy of radiotherapy.Therefore,the combination of radiotherapy,PD-1/PD-L1 antibody and VEGF/VEGFR2 inhibitors is expected to be a synergistic treatment regimen for anti-tumor therapy.In this study,we analyzed the relationship between the expression of VEGFR2 and PDL1 in clinicopathological specimens of patients with rectal cancer and the clinicopathological parameters of patients and their clinical significance,so as to provide theoretical basis for prognosis judgment and selection of prognostic markers for rectal cancer.To study and reveal the effects and significance of radiotherapy on the expression of VEGFR2 and PD-L1,and to investigate the effects of radiotherapy,VEGFR2 inhibitors and combined treatment with PD-L1 antibody on rectal cancer tumors and their microenvironment,aiming to provide theoretical basis for the new mode of radiotherapy,anti-angiogenic therapy and combined treatment with PD-1/PD-L1 antibody in the treatment of rectal cancer.Part Ⅰ Expression of VEGFR2 and PD-L1 in rectal cancer tissuesObjective:To investigate the expression of VEGFR2 and PD-L1 in rectal cancer cells and the correlation between the expression and clinicopathological characteristics.Methods:We detected the expression of PD-L1 and VEGFR2 by IHC in rectal tumor tissues.SPSS 22.0 software and GraphPad Prism 8.0 were used for statistical analysis.Results:The results of tissue microarray staining showed that 54 cases(71%)had high expression of PD-L1 and 43 cases(56%)had high expression of VEGFR2.The OS of patients with high PD-L1 expression and low VEGFR2 expression in tumor tissues was significantly higher than that of patients with low PD-L1 expression and high VEGFR2 expression.Patients with high TNM stage had a poor prognosis.Those with low PD-L1 and high VEGFR2 expression had a significantly increased risk of death.Conclusion:PD-L1 and VEGFR2 expression may be independent prognostic factors for rectal cancer.This can provide evidence for the selection of prognostic markers for rectal cancer,and provide theoretical basis for anti-tumor therapy.Part Ⅱ Effect of radiotherapy on the expression of VEGFR2 and PD-L1 in rectal cancer cellsObjective:To investigate the effect of radiotherapy on the expression of VEGFR2 and PD-L1 in rectal cancer cells.Methods:Colorectal cancer cell SW480,HCT116 and CT26 were divided into blank control group and experimental group.The experimental group included X-ray radiotherapy alone group,RT+Apatinib,RT+AG490 group,RT+Apatinib+AG490,and RT+Apatinib+AG490 group.RT was treated with 4 Gy or 6 Gy X ray.The protein and mRNA expressions of VEGFR2,p-VEGFR2,STAT3,p-STAT3 and PD-L1 were detected by Western blot and qRT-PCR.The proliferation and apoptosis of tumor cells were detected.Results:The expressions of p-VEGFR2,p-STAT3 and PD-L1 in tumor cells were significantly increased by RT.The protein and mRNA expression of p-STAT3 and PD-L1 did not increase after adding Apatinib and AG490.The proliferation intensity of the RT+Apatinib+PD-L1 antibody group was the weakest.After irradiation,the apoptosis of tumor cells was increased.And the apoptosis of the RT+Apatinib+PD-L1 antibody group was the strongest.Conclusion:RT can further activate the JAK/STAT3 signaling pathway by activating VEGFR2 to p-STAT3,thereby increasing the expression of PD-L1.RT-induced elevations of p-VEGFR2,p-STAT3,and PD-L1 were inhibited after blocking with Apatinib,a VEGFR2 inhibitor.RT can inhibit the proliferation of tumor cells and promote their apoptosis,and RT+Apatinib+PD-L1 can synergically reduce the proliferation of tumor cells and enhance the apoptosis of tumor cells.Part Ⅲ Interventional mechanism of radiotherapy,VEGFR2 inhibitor and PD-L1 antibody on the immune microenvironment of rectal cancerObjective:Study the effect mechanism of radiotherapy,VEGFR2 inhibitor and PD-L1 antibody on the microenvironment of rectal cancer tumors.Methods:The expressions of VEGFR2 and PD-L1 in tumor tissues of mice were tested by IHC.We detected the expressions of VEGFR2,p-VEGFR2,STAT3,p-STAT3 and PDL1 by qRT-PCR and Western blot.We detected the infiltration of CD8+T cells in tumor tissues by flow cytometry.Results:Compared with control group,radiotherapy group of tumor growth in mice significantly inhibited.The tumor tissue in the RT+Apatinib+PD-L1 antibody group was the lightest.RT could increase the expression of p-VEGFR2,p-STAT3 and PD-L1,and Apatinib could inhibit the increasing expression of PD-L1 induced by RT.The infiltration of CD8+T cells in tumor tissues of each radiotherapy group was significantly higher than that of the control group,and the infiltration level was further increased after RT combined with Apatinib and PD-L1 antibody.Conclusion:RT combined with Apatinib and PD-L1 antibody could significantly inhibit the growth of rectal cancer in mice.RT can activate VEGFR2 to p-VEGFR2 in tumor tissue to activate the JAK/STAT3 signaling pathway,and activate STAT3 to p-STAT3,and ultimately lead to PD-L1 expression increasing.However,the application of Apatinib will reduce the increasing of PD-L1 by RT.X-ray irradiation increased the infiltration of CD8+T cells in tumor tissues,and RT combined with Apatinib and PD-L1 antibody further increased the CD8+T infiltration level.