| Tumor microenvironment is complex cellular ecological system arrounding tumor cells and supports the proliferation, migration, metastasis and drug resistance of tumor cells. The significant pathological characteristic of pancreatic ductal adenocarcinoma is desmoplasia. The molecular biological basis of this phenomenon is that fibroblast cells are activated, grow and produce huge extracellular matrix in tumor microenvironment. As potent supporters of tumor cells, such fibroblast cells are called cancer-associated fibroblast cells. Studies about the origins, activation status and supportive effect of CAFs to tumor cells are of vital importance to the knowledge of tumor microenvironment.There are two parts about the pro-carcinogenesis function of CAFs.1. Mesenchymal stem cells like CAFs (MSC-like CAFs) promoted sternness and migration of tumor cells. MSCs is one of origins of CAFs and is induced by chemokines secreted by tumor cells or produced in inflammation or hypoxic background in tumor microenvironment. Then they are recruited through circulation to tumor stroma and differentiated into CAFs. Some CAFs still have some mesenchymal sternness. It is reported that "normal" MSCs came from bone marrow or adipose supported tumor growth in many ways. While, the function of MSCs-like CAFs in tumor microenvironment is not clear yet.We cultured CAF of human pancreatic ductal adenocarcinoma by outgrowth method. Then the contamination of epithelial cells and immunocytes were excluded by immunohistochemistry stain of CK19and CD45. However, given the thoughts that there might be epithelial cells lost their epithelial antigen during the process of epithelial-mesenchymal transforms, the negative stain of CK19alone couldn’t exclude the possibility of tumor parenchymal contamination. So we tested the Kras status in CAF and corresponding tumor tissues to make sure there was no Kras mutation in CAF cell population. Among CAFs, we sorted the cell population CD90+/CD73+/CD44+/CD49a+by flow cytometry, which had the potential to form colony and differentiate into bone, cartilage and adipose.The function of MSCs-CAFs to pancreatic cancer cells:1.1increase the sternness of tumor cells In groups of direct and indirect co-culture of tumor cells and CAFs, compared with the non-MSCs-CAFs, the MSCs-CAFs had a greater potential to increase the sphere forming number of tumor cells and the ratio of CD44+/c-Met+in tumor cell population. And the direct co-culture group had an even greater effect.1.2promote migration of tumor cellsIn groups of direct and indirect co-culture of tumor cells and CAFs, compared with the non-MSCs-CAFs, the MSCs-CAFs had a greater potential to promote migration of tumor cells in a3D collagen co-culture model. Tumor cells in the direct co-culture group showed discrete growth pattern, and had tumor foci in1case of directly co-culture group.1.3the distinct cytokines secreted by MSCs-CAFsWe tested36common cytokines reported secreted by CAFs, among which there were some cytokines positively stained in our sample. MIF and Serpin E1secreted by MSCs-CAFs and non-MSCs-CAFs. CXCL-1, IL-6, IL-8were exclusively or predominantly expressed in MSCs-CAFs rather than non-MSCs-CAFs. And the GM-CSF was expressed only in MSCs-CAFs groups.From the above study, we found that MSCs-CAFs isolated from pancreatic cancer stroma was a cell population showed higher ability to drive stemness and migration of tumor cells. They exerted their function not only through direct contact with tumor cells, but also involved the roles of soluble cytokines they secreted. Those distinct cytokines might be the molecular basis of their special function to tumor cells.2. Inhibitory effect of retinoic acid to CAFs. Some researchers found that normal fibroblast cells in pancreas stroma were activated by stimulations such as inflammation, hypoxia and cytokines secreted by tumor cells. When they are activated,Vitamin A fat droplets in the plasma are lost, which is related with the activation status of CAFs. Activated CAFs supported tumor initiation and development in many ways. Our research utilized retinoic acid-small molecular lipophilic derivatives to inactivate CAFs, by which to investigate the function of CAFs to migration of tumor cells and the indirect inhibition effect of all-trans retinoic acid (ATRA) and9-cis-RA to pancreatic cancer cells. We found that2.1The effect of RA to different pancreatic cancer cells varied. There were inhibition effect of ATRA and9-cis-RA to proliferation of Aspc-1. And RAs treatment maintained Aspc-1in Gl phase. But there was no proapoptosis effect to Aspc-1. While, to Panc-1, high concentration (20μM) group of RAs promoted early phase of apoptosis, although there was no significant difference of late phase of apoptosis rate between experiment groups and control group. And the effect of9-cis-RA was more evident than that of ATRA to Panc-1. However, RAs didn’t show proliferation inhibition of Panc-1.2.2RA could inactivate CAF. With the treatment of RAs, there were more fat droplets showed in CAFs plasma. And the expression level of CAF activation-related protein α-SMA and FAP were lower than control group, as so to ECM production gene, such as collagen Ⅰ, fibronectin and laminin, and cytokines as IL6/8. However, the inhibition effect of RAs was not through the promotion of apoptosis of CAFs.2.3RA could inhibit the soluble cytokines of CAFs, which promoted epithelial-mesenchymal transform (EMT) of tumor cells. By such, RA inhibited the migration and EMT of tumor cells indirectly. We found that incubation with the CAFs conditioned media previously treated with RAs, the migration distance and numbers of tumor cells crossing the borderline were less than control groups. And the EMT of tumor cells induced by3D collagen was inhibited, but RAs did no effect on migration and EMT of tumor cells directly. So the cytokines inhibited by RAs treatment in CAFs conditioned media was responsible for the migration of tumor cells.In this part, we found that RAs could inactivate CAFs, which were related with low level of activation-associated protein, ECM production, cell surface receptor and cytokines. Static CAFs showed weaker support to migration and EMT of tumor cells. Cytokines IL-6secreted by CAFs were involved in the process.In conclusion, the CAFs in pancreatic cancer microenvironment have a characteristics of heterogeneity, among which, MSCs-like CAFs have more potent ability to drive sternness and migration of tumor cells and secrete special cytokines. While through the study about inhibitory effect of retinoic acid, we found CAFs promote migration of tumor cells by secreting soluble cytokines. And retinoic acid can inhibit tumor migration and EMT by inactivation of CAFs. |
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