Dynamic Changes Of Myeloid Derived Suppressor Cells In The Development Of Mouse Pancreatic Cancer Aud Induced Differentiation Of Tumor Associated Macrophages In Vitro

Background and objectiveRecently pancreatic cancer immunotherapy is becoming a research focus,and it is most important for in vivo immunotherapy research to construct appropriate pancreatic cancer models with immunocompetent animals.So animal models can similarly recapitulate the pancreatic cancer microenvironment in human body.Myeloid derived suppressor cells(MDSC) and tumor associated macrophages(TAM) are both critical tumor immunomodulatory cells which can inhibit functions of multiple immune effector cells, and both have close relationships with tumorigenesis and development of pancreatic cancer.In our experiments we attempt to construct subcutaneous allograft pancreatic cancer models using mouse pancreatic ductal adenocarcinoma Pane02cell line.Furthermore,we observe dynamic changes of MDSC in the development of PDAC.Meanwhile,we in vitro attempt to use conditioned mediums of Pane02cells to induce differentiation of mouse macrophages cell line RAW264.7towards tumor associated macrophages. MethodsCultivate Panc02cells in vitro.When in logarithmic phase, Panc02cells are collected and1x106/100μl Panc02cells are inoculated under left anterior axillary skin of C57BL/6J mouse.Observe growth of subcutaneous allograft and take a biopsy of allograft tissue for H&E staining in time. Neoplasm staging includes x,1,2,3,4five stages according to the size of tumor tissue.It is defined as Tx (suspected tumorigenesis without obvious solid tumor formation),T1(0<tumor volume≦500mm3),T2(500mm3<tumor volume≦1750mm3),T3(1750mm3<tumor volume≦4000mm3),T4(4000mm3<tumor volume).Aquire peripheral blood and spleen samples from tumor-bearing mice and normal control respectively and undertake flow cytometric analysis.Then analyze dynamic changes of CD11b+Gr-1+MDSC and its subsets (CD11b+Ly6C+MDSC, CD11b+Ly6G+MDSC) in peripheral blood and spleen samples in the development of PDAC.Use conditioned mediums collected from Panc02cells culture to induce differentiation of mouse macrophages cell line RAW264.7.After induction for72h cells are collected and undertake flow cytometric analysis to examine differentiation of RAW264.7.ResultsThe average tumorigenic time for mice is12days,with emerging rate of tumor100%.After inoculation,the neoplasm accelerate growing,and comes into rapid growth period in the3-4th week. Tumor texture is hard,with ulceration bleeding appearance in advanced tumor. We can observe disarrangement of tissue cells,loss of tissue polarities and frequently meet cells in mitotic phase with obvious atypia in H&E stained pathological sections of subcutaneous allograft.Besides atypical glandular lumens and cancer nests are observed.All observations are in line with manifestation of malignant ductal epithelial carcinoma.The ratio of CD11b+Gr-1+cells in peripheral blood from normal control is7.3%,and the ratios of CDllb+Gr-l+cells in peripheral blood from Tx、T1、T2、T3、T4are3.7%、4.9%、11.6%、16.7%、25%in order; the ratio of CD11b+Ly6C+cells in peripheral blood from normal control is6.2%,and the ratios of CD11b+Ly6C+cells in peripheral blood from Tx、T1、T2、T3、T4are4.9%、12.6%、8.5%、18%、24.7%in order; the ratio of CD11b+Ly6G+cells in peripheral blood from normal control is4.8%,and the ratios of CD11b+Ly6G+cells in peripheral blood from Tx、 T1、T2、T3、T4are3.8%、9.5%、6.6%、19.9%、24.9%in order. The ratio of CD11b+Gr-1+cells in spleen from normal control is3.4%,comparing with20.4%in T4tumor-bearing mouse; the ratio of CD11b+Ly6C+cells in spleen from normal control is3.9%,comparing with15.5%in T4tumor-bearing mouse; the ratio of CD11b+Ly6G+cells in spleen from normal control is3.6%,comparing with13.9%in T4tumor-bearing mouse. The ratios of M1(F4/80+CD16/32+) and M2(F4/80+CD206+)TAM cells in conditioned mediums stimulating group are obviously higher than those in control group(44.2%vs28.5%,18.5%vs9.4%).ConclusionThe immunocompetent subcutaneous allograft pancreatic cancer mouse model is successfully constructed with short tumorigenic time and high emerging rate of tumor,and it can provide an useful tool for pancreatic cancer immunity reaearch.The ratios of MDSC and its subsets in peripheral blood from mice are in positive correlation with the increasing pancreatic tumor burden.The ratios of MDSC and its subsets in spleen from late stage tumor-bearing mouse are much higher than those in normal mouse.The differentiation induction of macrophages towards tumor associated macrophages(including Ml、 M2) with conditioned mediums collected from mouse pancreatic cancer Panc02cell line culture is successful.