The Role Of Cancer Associated Fibroblasts(CAFs) In Pancreatic Cancer Treated By Qingrehuasi Herbal Medicine

ObjectiveTo study the role of cancer associated fibroblasts (CAFs) in pancreatic cancer treated by Qingrehuasi herbal medicine(Qingyihuaji formula, QYHJ).MethodsPart I:Nude mice with subcutaneous transplanted Capanl tumor models were divided into two groups, including control group(Treated with saline) and QYHJ group(Treated with QYHJ). The effect of QYHJ on subcutaneous transplanted tumor were observed. Nude mice with orthotopic xenograft were treated in the same methods to evaluated the effect of QYHJ in the prevent of pancreatic cancer liver metastasis.Part Ⅱ Herbal medicine serum contained QYHJ and the normal control serum were obtained from rabbits after feed the rabbits with QYHJ or saline for7days. Prepared the inactive QYHJ serum and control serum by heating them in56℃for30min. The effect of these serums in the growth, migratory capacity and invasiveness of pancreatic cancer cells were evaluated by CCK-8assay,migration and invasion assay(Transwell cell chamber).PartⅢ:Establish two pairs of NFs and CAFs from human pancreatic normal tissues and cancer tissues by primary cultured, and identified them with immunofluorescence(ICC) and Western-blot assay. The different expression of CXCL1、2、8among NFs、CAFs and CAFs treated with QYHJ serum were detected by RT-PCR and Western-blot. The proliferation of CAFs treated with QYHJ serum were evaluated by CCK-8in vitro and immunohistochemistry in vivo. The expression of CXCL1、2、8were examined by immunohistochemistry in vivo, and their serum level were detected by Elisa.PartIV:The effect of CXCL1、2、8on enhanced migratory capacity and invasiveness of pancreatic cancer were observed by migration and invasion assay. The effect of CXCL8/CXCR1on the stemness of pancreatic cancer stem cells were accessed with sphere formation assay and the presence of CD44+/CD24cells. Expression of CXCR1, CD24, CD44and CD133were immunohistochemically assessed in pancreatic cancer specimens. The relationships between those markers and clinicopathological features, prognosis were investigated.PartⅤ:Supernatant collected from NFs、CAFs and CAFs pre-treated with QYHJ serum on the enhanced migratory capacity and invasiveness of pancreatic cancer were observed by migration and invasion assay.ResultsPart Ⅰ:The incidence of tumor in different groups were all100%. However, tumors grew slowly in QYHJ group than Control group. There was significantly differences between groups after treated15days, the average tumor weight between Control and QYHJ groups were0.705±0.198809g VS0.435429±0.151751g, the inhibit rate of QYHJ group was38.42%(P<0.05).There was less liver metastases in QYHJ group (P=0.042) and the metastasis rate (P=0.002) in the orthotopic xenograft.Part Ⅱ:The suppression capability of different serums in the proliferation, migratory capacity and invasiveness of pancreatic cancer were:QYHJ serum> normal serum and inactive QYHJ serum> inactive normal serum. The difference between normal serum and inactive normal serum may due to the immune reaction. The difference between inactive QYHJ serum and inactive normal serum may due to the direct effect of its active ingredient. The difference between QYHJ serum and inactive QYHJ serum indicated the exist of indirect effect of QYHJ and that could inhibt in pancreatic cancer.PartⅢ:We successfully established two pairs NFs and CAFs (#1,#2), and identified them with CK, E-cadherin, Vimentin and a-SMA. NFs high expressed the vimentin and the CAFs also high expressed the a-SMA, and neither of them expressed CK or E-cadherin. We further evaluated the expression of CXCL、2、8between them and found out CXCLs were higher in CAFs than NFs, after treated with QYHJ their expression decreased both in vitro and vivo. The proliferation of CAFs was inhibited by QYHJ serum both in vitro and vivo.PartⅣ:The number of pancreatic caner cells passed through chambers was higher in cytokines group(CXCL1、2、8) than control group and inhibitor group. The colonies in the CXCL8group is more than control group(P=0.000) and inhibitor group(P=0.002). The rate of CD44+/CD24+in the CXCL8group is more than control group(P=0.002) and inhibitor group(P=0.03). The analysis of clinical data revealed that CXCR1expressiong was significantly associated with CD44/133and lymph node metastasis. Univariate and multivariate Cox regression analyses of CXCR1expression for OS of patients with PDAC indicated that CXCR1was an independent factor for the prognosis of pancreatic cancer patients.Part V:The number of pancreatic caner cells passed through chambers was higher in supernatant collected from CAPs than that collected from NFs and CAFs pre-treated with QYHJ (P<0.05)ConclusionQYHJ inhibited the proliferation and liver metastases of pancreatic cancer. Their anti-cancer effect included direct effect cuased by active ingredient and its indirect effect, one of the targets would be CAFs.QYHJ inhibited both the CAFs proliferation and the secretion of CXCL1、2、8in CAFs, which enhanced the migratory capacity and invasiveness of pancreatic cancer cells, and CXCL8/CXCR1was associated with the sternness of pancreatic cancer stem cells.