Effect Of Radiotherapy Combined With G-CSF On Tumor-Associated Neutrophils In Tumor-bearing Mice

Objective:Radiation therapy is one of the important methods currently used in the treatment of lung cancer.After radiation therapy,some patients still have local recurrence and distant metastasis.The important reason for their recurrence and metastasis is the phenomenon of "radiotherapy resistance" in tumor cells.In a variety of tumors,epithelial mesenchymal transition(EMT)is associated with "radiotherapy resistance".In the process of metastasis formation and development,EMT is not a necessary process.However,the cells that develop EMT significantly promote the recurrence of lung metastases after chemotherapy.Inhibiting EMT may provide a new way to reduce the production of radiotherapy-resistant cells and increase the sensitivity of tumors to radiotherapy.Radiotherapy not only exerts direct killing effect on tumor cells through direct cytotoxicity,but also recruits and regulates multiple cells in the tumor microenvironment(TME)to exert indirect anti-tumor effects.Neutrophils are inflammatory cells that are first recruited to the tumor site after radiotherapy.Locally infiltrating neutrophils release cytokines,and further induce related reactions.Studies have found that compared with neutrophils produced by normal bone marrow,newly recruited tumor-infiltrating neutrophils(TAN)produce reactive oxygen species.(ROS)levels have increased significantly.Several studies have confirmed that ROS can regulate the activation of PI3K/AKT signaling pathway,and the activation of AKT has been found to induce EMT in a variety of malignant tumors.The mechanism of TAN recruitment and regulation after radiotherapy and the effect of newly recruited TAN on tumor cell EMT and radiotherapy sensitivity have not been elucidated.Methods:The first part:The Lewis cells of mice were cultured by standard protocol.LLC cells were incubated in a 6-well plate and RT was administered at a dose of 8Gy on days 1,3 and 5 using 6-MV high-energy radiation.We evaluated chemokines released one day after radiotherapy.40 kinds of cytokines with inflammatory,homeostatic or dual functions were evaluated with mouse cytokine array panel A.In order to confirm the effect of specific cytokines,the supernatant obtained by centrifugation at 2000 rpm for 10 minutes at 12h,1d,2d,3d,5d and 7d after irradiation were regarded as conditioned medium.CXCL1,CXCL2 and CCL5 were evaluated by ELISA using cusabio kits and procedures.Moreover,six week old female C57BL/6 mice were selected,and 1*10 ^ 6 LLC cells were implanted in the right armpit.Until the tumor diameter reached 6 mm,the longest diameter(a)and the shortest diameter(b)of the tumor were measured every other day.The tumor volume was calculated by the formula:volume=0.52*a*b^2.Using CXCL1,CXCL2 and CCL5 neutralizing antibody,the experimental group was divided into control group,radiotherapy group and radiotherapy combined with anti-CXCL1,anti-CXCL2 and anti-CCL5,Immunohistochemistry and flow cytometry were used to detect the degree of neutrophil infiltration;ELISA and RT-PCR were used to detect the expression level of CXCL1,CXCL2 and CCL5;key cytokines regulating neutrophil recruitment were selected in vitro and in vivo.The second part:In vivo experiment,neutralizing antibody with neutrophils leads to TAN ablation and clinical drug G-CSF was used to increase TAN recruitment.Experimental groups:control group,RT group,RT+anti-Ly6G group,G-CSF group,RT+G-CSF group.Collect tumor tissue,spleen and sentinel lymph nodes of tumor bearing mice and prepare single cell suspension to analyze the levels of TAN,CD4+cells,CD8+T cells and Treg cells of tumor tissue,spleen and sentinel lymph nodes of each group by flow cytometry;select Ly6G+granulocytes from single cell suspension by EasySep PE sorting kit,and detect them by flow cytometry The expression of PI3K,p-pi3k,Akt and p-Akt were detected by Western blotting and QRT-PCR.The expression of E-cadherin,N-cadherin,vimentin and snail were detected by Western blotting,QRT-PCR and IHC.Data processing and analysis:nonparametric test was used to compare the distribution differences of continuous variables among different groups.The difference of survival as analyzed by K-M survival curve,P<0.05.Results1.Rcruitment of neutrophils depends on cytokines and TAN are the first line of immune responseOn the 3rd day after local RT,the infiltration of CD11b+Ly6G+TAN in tumor and spleen was studied.It was found that TAN in irradiated mice was significantly higher than that in the control group,especially in mice treated with G-CSF.Subsequently,we studied the recruitment of neutrophils and tumor specific CTLs in the tumor after RT in a time-dependent manner.The results showed that the peak value of neutrophils was reached 24h after RT.Then,CTLs began to increase slowly and reached the peak value at 60h.This phenomenon indicates that RT recruits neutrophils as the first line of immune response.In addition,the application of anti-Ly6G antibody in tumor reduced neutrophils in tumor and spleen by 60%and 80%respectively(P<0.05).2.Results in DNA damage of tumor cells and release of cytokinesIn order to verify the effect of CXCL1/CXCL2/CCL5 on TAN concentration.the corresponding antibodies were injected into mice.Compared with the control group,the simultaneous blocking of CXCL1/CXC2/CCL5 may hinder the radiation-mediated neutrophil recruitment(P<0.001).In addition,the use of each neutralizing antibody may also result in down regulation of TAN recruitment,although it is not as effective as blocking all three cytokines simultaneously(P=0.008,0.012 and 0.023 for CXCL1,respectively).CXCL1,CXCL2,and CCL5 are associated with PARC phenomena identified by PRR,thereby activating PAMP.3.TAN raised by radiotherapy reverses EMT and promotes antitumor response The level of CTLs infiltration in tumor,spleen and draining lymph nodes was assessed by IHC.Compared with the control group,CTLs was significantly up-regulated in RT treated mice,especially in mice given G-CSF at the same time(P<0.05).Compared with the control group,the accumulation of CTLs was significantly inhibited in tumor(P=0.03 1)and lymph node(P=0.029)in the mice with decreased TAN.Administration of G-CSF enhanced the CTLs infiltration(P<0.001),spleen(P=0.028)and draining lymph nodes(P=0.033)in tumor.By IHC staining,qRT-PCR and Western blot detected epithelial and mesenchymal markers in a mouse model with LLC.Compared with the control mice,the RT treated mice showed typical reversing EMTphenotypes,including up regulation of E-cadherin and down regulation of Vimentin(P<0.05).The inhibition of EMT was further enhanced by administration of G-CSF.In addition,there was a significant positive correlation between neutrophil level and E-cadherin expression(correlation coefficient=0.624,P=0.041),and a negative correlation with Vimentin(correlation coefficient=-0.673,P=0.032).During the 5 days of RT,the irradiated mice were treated with anti Ly6G mAb.Neutrophils depleted mice showed up regulation of Vimentin and down regulation of E-cadherin,although there was no significant difference between the two groups(P>0.05).4.TAN inhibits EMT via ROS mediated PI3K/Akt/snail signaling pathwayWestern blotting was used to detect the effect of TAN on the activity of PI3K/Akt/snail in tumor tissue:in the mouse model receiving RT and G-CSF at the same time.the phosphorylation level of PI3K/Akt was significantly inhibited.Similarly,the expression level of Snail was also down regulated by WB(P=0.014)and qT-PCR(P=0.002).In addition,RT alone had similar but weaker inhibition on PI3K/Akt phosphorylation(P<0.05).An increase in ROS accumulation was observed in RT-induced TAN compared with normal neutrophils(NNs)from non irradiated tumors(P=0.003).Conclusion1.PAMP induced by CXCL2 is the key mechanism of TAN recruitment by RT;2.TAN can reverse tumor cell EMT to improve the radiosensitivity,and enhance the immune response by inducing CTL cells aggregation and down regulating Treg cells expression,so as to improve the antitumor effect;3.G-CSF can enhance the antitumor effect of TAN after RT by up regulating the recruitment of neutrophils.