The Landscape Of Tumor Immune Micro-Environment(TIME) And Gemcitabine Chemotherapy Of Pancreatic Cancer

BackgroundMore than 80%of pancreatic cancer is pancreatic ductal adenocarcinoma(PDAC),it has dismal prognosis.The 5-year overall survival of the stage I-stage IIA of PD AC is only 30%,therefore,surgical resection alone is not enough for these patients.According to the NCCN guideline,chemotherapy should be recommended for any stage of PD AC patients.Nowadays,gemcitabine(GEM)is the first-line drug for adjuvant treatment,however,the response rate is less than 20%,therefore,to improve the efficacy of GEM is of great clinical significance.PDAC has special tumor immune micro-environment(TIME).In the TIME,there are massive infiltrations of immune cells,however,only 10%-30%of the cells are cancer cells.Previous studies showed that targeting TIME could improve the efficacy of chemotherapy,and now it has become to be a very hot topic.However,the landscape of the TIME of PDAC has not been available,therefore,we are still lacking of a systematical and deep recognition of the TIME of PDAC.Objective(1)To depict the landscape of the TIME of PDAC;(2)To explore the roles of the TIME and the traditional clinical-pathological characters to predict the survival of the patients;(3)To explore the inner relationship between the TIME and the traditional clinical-pathological characters;(4)To explore the roles of GEM to remodel the TIME of PDAC;(5)To screen and demonstrate the items of TIME as optimal synergistic targets to improve the fficacy of GEM.Methods(1)We collected 17 clinical-paholtogicl items and obtained tumor tissue and para-tumoral tissue from 97 cases of PDAC patients undergoing radical pancreaticoduodenectomy and adjuvant GEM treatment.We constructed the tumor tissue microarray(TMA)with the representative tissue,and then performed the immunohistochemical staining(IHC)to mark and semi-quantitatively evaluate 15 immune cell populations,1 maker for angiogenesis,1 marker for lymphangiogenesis,11 immune regulatory factors and 2 markers for pancreatic cancer stem cells(PCSCs).For the first time,by integrated analysis of these 30 factors,we depicted the landscape of the TIME of PDAC;(2)We adopted X-tile program to determine the optimal cutoff value of each factor.After uni-variate and multi-variate analysis,we found the independent risk factors to predict poor survival.We also adopted ROC curve(receiver operating characteristic curve)analysis to show the efficacy of these items to predict the death of the patients;(3)By Spearman rank correlation coefficient test,we analyzed the inner relationship of the traditional clinical-pathological factors and the items of TIME which had roles to predict the survival of the patients;(4)We treated human and murine PDAC cell lines by GEM,and then detected the changes of 13 inflammatory regulatory factors and cancer stem cell markers of cancer cells by real time RT-PCR,Western blot and FCM.And then the roles of PDAC cells after GEM treatment to regulate the morphology,phenotypes and functions of macrophages were studied;(5)After integrated analysis of the previous results,we found that the optimal targets in the TIME to improve the efficacy of GEM.And then,we established subcutaneous bearing PDAC in immunocompetent mice,and demonstrated the synergistic roles of the targets by combinational intra-tumoral blockage of these targets.Results(1)For the first time,we depicted the landscape of TIME at a semi-quantitative histo-pathological level,and TGF-?1 and GM-CSF were strong positive expression in the tumor tissue of the most patients;(2)Among all of these 17 clinico-pathological items and 30 items of TIME,we found 22 risk factors,and 8 of them were indepenedent risk factors,including lymph node metastasis,poor differentiation,elevated serum CA19-9,CD163(+)M2 polarized tumor associated macrophages(TAM),IgG4(+)plasma cells,intra-tumoral strong positive expression of GM-CSF and TGF-? 1,and higher density of lymphangiogenesis.In the meanwhile,accroding to the analysis of ROC curve,TGF-? 1 and CD163(+)M2 polarized TAM were top two factors to predict death of the patients;(3)After Spearman rank correlation analysis,we uncovered the inn relationship of these 22 risk factors and found that GM-CSF and TGF-? 1 could promote the malignant biological behavior by different mechanisms;(4)After treatment of different concentrations of GEM,three human and one murine PDAC cell lines produced higher level of GM-CSF and TGF-? 1,and as well the cancer cells showed stronger roles to induce M2 polarized TAM.(5)Based on the previous results:1):GM-CSF and TGF-? 1 were strong positive expression in most tumor tissues;2):GM-CSF and TGF-? 1 were independent risk factors to predict poor survival of the patients;3):GM-CSF and TGF-?1 could promote the malignant biological behaviors of pancreatic cancer by differernt mechanisms;4):GEM could induce the expression GM-CSF and TGF ? 1,we proposed that GM-CSF and TGF-? 1 as the potential synergistic targets.And then the combinational intra-tumoral blockage of GM-CSF and TGF-?1 significantly improved the efficacy of GEM.ConclusionsFor the first time,this study semi-quantitatively depicted the landscape of the TIME of human PD AC,and GM-CSF and TGF-? 1 were strong positive expression in most tumor tissues.We adopted X-TILE programme to define the cutoff value of each factor,after analysis of these 47 items,we found 22 risk factors and 8 of them were independent risk factors to predict poor survival of the patients,including GM-CSF and TGF-? 1.On this base,we uncoved the inner relationship of these 22 factors,and the results showed that GM-CSF and TGF-?1 could promote the malignant biological behvaiors by different mechanisms.Elevated expression of of GM-CSF and TGF-? 1 of cancer cells induced by GEM could lead to the accumulations of M2 polarized TAM which could contribute to the secondary drug resistance.Finally,we proposed and demonstrated that GM-CSF,TGF-? 1 were the optimal synergistic targets and the combinational intra-tumoral blockages of GM-CSF and TGF-? 1 could significantly improve the efficacy of GEM,which presented a new strategy for the integrated treatment of PDAC and had promising future of potential clinical applications.