| Background:Tumor cells and immune cells regulate and restrict each other in tumor immune microenvironment.Myeloid-derived suppressor cells from the immune microenvironment perform a function of immune negative regulation and inhibitory effects on anti-tumor immune response.Our initial clinical datas confirmed that the spleen deficiency Zheng was the common type syndrome.Clinically,(Jianpitiaoqifang,JPTQ-Formula)JPTQ was commonly used in breast cancer patients with "spleen deficiency",and the effect was obviously clear.Therefore,we take the tumor microenvironment as a breakthrough point to study the effectiveness of the JPTQ Formula and to clarify the mechanism of breast cancer anti-metastasis,that will provide a scientific basis for traditional Chinese medicine preventing brest cancer metastasis.Objective:Exploring the effect of JPTQ in the tumor microenvironment and tumor growth and metastasis through the 4-T1 burdened mice,clarifying the regulation of inhibitory immune cells to the tumor microenvironment by the cell sorting of MDSC in tumor tissue,analyzing the mechanism of the effect of JPTQ in the accumulation and amplification of MDSC.Methods:Part one:Normal Balb/c mice were obtained for the preparation of JPTQ medicated serum.Part two:Take the containing medicine serum as the object,the proliferation of 4-T1 under the different drug concentration and retention time of drug was tested using MTT,choosing the best experiment condition for the next step;the cell cycle of 4-T1 with the medicine serum was detected using flow cytometer;the transfer ability of 4-T1 with the medicine serum was detected using transwell chamber.Part three:Primary tumor growth and lung metastasis of 4-T1 tumor bearing mice under the different drug concentration of JPTQ were tested using the Luc-4T1 tumor bearing mice model and the living fluorescence imaging system.Part four:The accumulation of MDSC in blood\spleen and tumor tissue under the different drug concentration of JPTQ were tested using flow cytometer through the Luc-4T1 tumor bearing mice model;the genes related to the function of MDSC in tumor tissue under JPTQ treatment by flow sorter were detected using the real-time PCR detection.Part five:The accumulation of T cells\TAMs\Tregs in the 4T1 tumor bearing mice model under JPTQ treatment were tested using flow cytometer.Part six:The cytokine secretion in the tumor microenvironment of primary tumors and pulmonary metastasis was tested using cytokines chip technology through the 4T1 tumor bearing mice model with and without JPTQ treatment;The expression of VEGF\MMP-9 in the tumor microenvironment of primary tumors and pulmonary metastasis were detected using immunohistochemical method.Part seven:The signaling pathway in primary tumor tissue and MDSC in tumor tissue were detected using protein phosphorylation signaling pathway chips through the 4T1 tumor bearing mice model and MDSC cells sorting from tumor tissue with and without JPTQ treatment;the signaling pathway in primary tumor tissue and MDSC in tumor tissue were verified using the western blot method.Results:Part one:Normal Balb/c mice were treated with or without JPTQ for the preparation of medicated serum marked "JPTQ" group and "Control" group.Part two:The MTT showed that the inhibition ratio at 24h(16.6%)was significantly higher than the 48h(4.7%)under the 1%medicated serum(P=0.000);the inhibition ratio at 24h(22.5%)was significantly higher than the 48h(4.5%)under the 2%(P=0.000);the inhibition ratio at 24h(17.6%)was significantly higher than the 48h(11.5%)under the 6%(P=0.006);the inhibition ratio at 24h(25.8%)was significantly higher than the 48h(11.8%)under the8%(P=0.000);the inhibition ratio at 24h(21.9%)was significantly higher than the 48h(17.5%)under the 10%(P=0.001);the inhibition ratio at 24h(21.9%)was lower than the 48h(17.5%)under the 12%(P=0.021).The cell cycle results showed that the blocking effect of JPTQ medicated serum occurred in G2 stage.The transwell showed that the migration of 4-T1 in CK group was 32.67±4.16,48.67±3.06 in 8%Control group,18.67±4.73 in 8%JPTQ group(lower than the 8%control group,P=0.0026;higher than the normal group,P<0.05),32±9.85 in 12%Control group and 12±5.57 in 12%JPTQ group(lower than the 12%control group,P=0.0487).Part three:In the seventh tumor-burdened day,the tumor volume in JPTQ 0.8g/ml group and 1.6g/ml group were smaller than the control group(P<0.01),then the tumor volume between JPTQ and control group had no difference.Only in JPTQ 1.6g/ml group had no distant metastases using the living fluorescence imaging system.Part four:In the sixteenth tumor-burdened day,the accumulation of MDSC in spleen of JPTQ group was lower than the control group(P<0.05);In the twenty-second tumor-burdened day,the accumulation of MDSC in tumor tissue of JPTQ group was lower than the control group(P<0.01);The expression of arginase-1 and iNOS were lower in JPTQ group(P<0.01).Part five:In the fifteenth tumor-burdened day,the CD4+and CD8+T cells in blood/spleen/tumor had no difference between the JPTQ and Control treatment.In the twenty-second tumor-burdened day,the CD4+T cells in JPTQ group from the spleen was higher than control group(P<0.05);the CD4+and CD8+T cells in JPTQ group from the tumor was higher than control group(P<0.01;P<0.05);the Tim3+in T cells in JPTQ group tended to decrease compared with the control group;the F4/80+CD11c+and F4/80+CD206+in blood/spleen/tumor had no difference while F4/80+CD206+in the JPTQ group tended to increase.In the fifteenth tumor-burdened day,the Treg cells in blood/spleen/tumor in the JPTQ group tended to decrease(P>0.05).Part six:There was 15 cytokines common lower expression in both primary tumor and metastasis using cytokines chip with the treatment of JPTQ or not,including:MGF-E8,gp130,SDF-1a,IGF-1,CRP,CD40-L,Eotaxin,Eotaxin-2,CCL28,MIP-1b,MMP-10,VEGFR1,VEGF-D,bFGF,PDGF-AA.The expression of VEGF in the tumor microenvironment of primary tumors were detected lower than the control group(P<0.05),while in the metastasis the difference was not obvious;The expression of MMP-9 in primary tumors and metastasis were detected lower than the control group(P<0.01,P<0.05).Part seven:The protein phosphorylation signaling pathway chips showed that the down-regulation signaling pathway in tumor under the JPTQ treatment including:Integrin beta-3/FAK,RAc/EMT,MEK1/ERK8,FAK/PI3K/AKT,AMPK/JNK,JAK/STAT1/NF-KB/REL,ATK/c-MYC,etc and down-regulation signaling pathway in MDSC in tumor including JAK/C-JUN/STAT3,JAK,SRC,STAT1,STAT3.The western bolt showed that protein phosphorylation of Stat5a、pStat5a、Stat1、pStatl、Stat3、IKK、NFκB、pNF-κB、pAKT、c-Jun were down-regulation in the tumor tissue with JPTQ treatment,protein phosphorylation of Stat5a and pStatl were down-regulation in MDSC in tumor.Conclusion:Part one:The proliferation and migration ability of 4-T1 was inhibited by JPTQ medicated serum in a dose-dependent manner in vitro,the blocking effect of JPTQ medicated serum occurred in G2 stage.Part two:During the early tumor-burdened period,the tumor growth was delayed by JPTQ,then it was not affected;the pulmonary metastasis was delayed by JPTQ.Part three:the accumulation of MDSC in tumor tissue was decreased by JPTQ;its inhibitory dysfunction related genes ARG-1 and iNOS was reduced by JPTQ.Part four:The amount of CD4+and CD8+T cells in tumor microenvironment were increased by JPTQ,and the Tim3+in T cells with JPTQ tended to decrease.There was no effect in the amount of F4/80+CD11c+and F4/80+CD206+in blood/spleen/tumor by JPTQ.The Treg cells in blood/spleen/tumor in the JPTQ group tended to decrease.Part five:The inflammatory cytokines and metastasis related cytokines from primary tumor microenvironment including MGF-E8,gp130,SDF-1a,IGF-1,CRP,CD40-L,Eotaxin,Eotaxin-2,CCL28,MIP-1b,MMP-10,VEGFR1,VEGF-D,bFGF,PDGF-AA were weakened by JPTQ.The expression of VEGF and MMP-9 in primary tumors and metastasis were down-regulation by JPTQ.Part six:The protein phosphorylation of Stat5a、pStat5a、Stat1、pStat1、Stat3、IKK、NF-κB、pNF-κB、pAKT、c-Jun in primary tumor tissue were weakened by JPTQ.The protein phosphorylation of Stat5a and pStatl were down-regulation in MDSC in tumor by JPTQ. |
