| Objective:Radiotherapy has prominent advantages in the treatment of various cancer and has been applied extensively in neoadjuvant treatment of rectal cancer.But about30%of patients still have disease progression due to ineffective treatment.After radiotherapy,the infiltration of numerous tumor-associated macrophages(TAM)and up-regulation of PD-L1 expression may limit anti-tumor effect.Anti-tumor immunotherapy targeting the tumor microenvironment is emerging,but the single-drug regimen has failed to achieve satisfactory results in non-selective colorectal cancer.In colorectal cancer,little is known about whether radiotherapy combined with anti-tumor immunotherapy can achieve synergistic effects and the mechanism are still unclear.Herein,our aims were as follows:1.Identified the predictive value of macrophage-related biomarkers(CD163,CD68,CSF1,and CCL2)in response to neoadjuvant treatment and the survival of locally advanced rectal cancer(LARC).2.Investigated the changes of cytokine secretion in colorectal cancer after radiotherapy and the impact on TAM polarization and PD-L1 expression level within the tumor microenvironment.3.Explore the effect of CSF1-mediated polarization of TAMs on the maturation,differentiation,and anti-tumor cytotoxicity of T lymphocytes.4.Revealed the mechanism of inhibition of CSF-1 combined with radiotherapy and anti-PD-L1 therapy in colorectal cancer.Methods:1.191 patients who received neoadjuvant chemotherapy and radiotherapy(NCRT)and radical resection in the Department of Colorectal Surgery,Fujian Medical University Union Hospital between 2011 to 2015 were included. Preoperative colonoscopy biopsy and postoperative tumor tissues were collected for immunohistochemical.2.CT26 and MC38 murine rectal carcinoma cells were given different doses of radiotherapy.The impact of radiotherapy on the secretion of cytokines and the expression of PD-L1 was assessed at indicated time points base on ELISA and flow cytometry.3.In vitro,mouse bone marrow-derived macrophages(BMDM)were carefully isolated and cultured,then co-culture with murine colorectal cancer MC38 cells,which underwent different doses of radiotherapy.the BMDM surface expression of CD11b,F4/80,CD206 were assayed by flow cytometry,and identified polarization phenotype.Isolate and culture mouse splenic lymphocytes in vitro,and co-culture with both MC38 cells after radiotherapy and BMDM with different polarization phenotypes.Using flow cytometry analyze both lymphocyte membrane molecules(CD4,CD8,CD25,CD127)and intracellular molecules(TNF-?)expression.CSF-1 inhibitor,PLX3397,were used to investigate the effect of blocking CSF1-mediated polarization of TAMs on T lymphocyte differentiation and function in vitro.4.Construct a subcutaneous xenograft model of MC38 cells,and perform 3×4Gy radiation.On the 6th day,peripheral blood,spleen tissue,and tumor tissue were obtained from mice,and by flow cytometry,we accessed the expression of PD-L1on the surface of immune cells,the expression of CD206 on macrophages,the expression of CD4,CD8,CD25,CD127,TNF-?on lymphocyte.Analyzed i NOS,IRF5,Arg1,IL-10,PD-L1 m RNA relative levels by RT-q PCR.5.Construct a subcutaneous xenograft model of murine MC38 cells,and randomly divide them into 5 groups:control group(Ctrl),radiotherapy group(RT),radiotherapy+anti-PD-L1 group(RP);radiotherapy+anti-CSF1 group(RM)and radiotherapy+anti-PD-L1+anti-CSF1 group(RPM).On the 6th day,the peripheral blood,spleen tissue,and tumor tissues of the mice were obtained and tested by flow cytometry.After fixing the embedded tumor tissue,staining with immunohistochemical antibodies CD68,CD206,i NOS,CSF1,PD-L1.6.Construct a subcutaneous xenograft model of murine MC38 cells,and randomly divide them into 5 groups for treatment.Draw the tumor growth curve.Tumor tissue was acquired and measure characteristics.Observe and measure the tumor growth and survival status of mice until the end of the observation,draw the survival curve of mice.Results:1.Compared with the non-pathological complete response(p CR)group,the expression of macrophage-related biomarkers(CD163,CD68,CSF1,CCL2)in p CR group were lower.The lower scores of CD163,CD68,CSF1,CCL2 before NCRT and post operation were related to the improvement of DFS.COX regression analysis showed that the pre-n CRT CD163 score(HR=1.008,95%CI1.003-1.013,P=0.003)and CSF-1(HR=2.187,95%CI 1.343-3.564,P=0.002)was independently associated with DFS.2.We developed a risk scoring model with a strong ability to predict the p CR of LARC patients.By Cox regression analysis,tumor size(HR=1.291,P=0.041),poor pathological TNM staging(HR=1.789,P=0.005),and higher risk score(HR=1.084,P=0.001)were highly correlated relatively poor disease-free survival.Based on above results,a nomogram and decision curve analysis were generated.3.It was showed in vitro experiments that radiotherapy promoted the up-regulation of CT26 and MC38 PD-L1 expression in murine colorectal cancer cells,especially MC38 cells after receiving 20Gy radiotherapy for 72 hours.Radiotherapy caused CT26 and MC38 cells to secrete more cytokines CCL2 and CSF-1,especially MC38 cells 72 hours after receiving 12Gy radiotherapy.4.The results of cell co-culture showed that MC38 cells after radiotherapy induced-macrophages to polarize to M2 type and expressed massive PD-L1.After72 hours of receiving 12Gy radiation,the BMDM,co-culture with MC38 cells,showed a markedly increase in the proportion of CD11b+/F4/80+CD206highcells,and the PD-L1 expression rate was higher(93.43±2.57 vs 4.21±1.99,p<0.0001)CSF1R inhibitor PLX3397 inhibited the M2 polarization of BMDM induced by MC38 cells after radiotherapy in vitro,revealing the mediating effect of CSF1..5.In the experiment of co-cultivation with splenic lymphocytes,it showed that MC38 cells after radiotherapy induced BMDM polarized to M2,inhibiting the activation of CD8+T cells and the function of secreting TNF-?And PLX3397 in vitro experiments can eliminate the resulting inhibition of CD8+T cell function and reactivate its anti-tumor effect.6.The subcutaneous xenograft model of MC38 cells showed that radiotherapy induced the up-regulation macrophages PD-L1 expression in peripheral blood,spleen tissue,and tumor,and the obvious polarizing to M2 of macrophages in mice tumor tissues after radiotherapy.CD4+T lymphocytes infiltrated in tumor tissue after radiotherapy increased markedly,CD8+T lymphocytes relatively decreased,and the ratio of CD4+/CD8+markedly increased.The proportion of CD4+Treg cells with anti-tumor immunosuppressive function in tumor tissues increased after radiotherapy.7.The infiltration of CD8+T cells in tumor tissues were not observed after radiotherapy combined with anti-PD-L1 therapy.But radiotherapy combined with anti-CSF-1 and anti-PD-L1 triple therapy can reverse the tumor microbes of mouse colorectal cancer environmental TAM M2 polarization,decrease the expression of PD-L1 in tumor tissues,impact the differentiation of CD4+Treg cells,increase the proportion of CD8+T cells and activate TNF-?secretion,remodel the tumor immune microenvironment.8.In the subcutaneous xenograft model of MC38 cells that radiotherapy combined with anti-CSF-1 and anti-PD-L1 triple therapy powerfully inhibited tumor growth,prolonged the survival of mice.Conclusions:1.The expression levels of macrophage-related biomarkers(CD163,CD68,CSF-1 and CCL2)were correlated with the chemotherapy and radiotherapy resistance and prognosis of LARC patients after NCRT.A predictive tool was eventually established for LARC.2.Radiotherapy can promote the up-regulation of PD-L1 expression and the secretion of cytokines CCL2 and CSF-1 in murine colorectal cancer cells.Radiotherapy induced CSF-1-mediated macrophages to polarize to M2 type,and express massive PD-L1,inhibiting the function of CD8+T cells.3.Radiotherapy combined with anti-CSF-1 and anti-PD-L1 triple therapy can reverse the TAM M2 polarization in the tumor microenvironment of colorectal cancer,decreased PD-L1 expression,activate CD8+T cell,and remodel the tumor immune microenvironment.4.Anti-CSF-1 combined with radiotherapy and anti-PD-L1 triple therapy can markedly inhibit the growth of transplanted tumors of colorectal of mice and improve survival. |
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