Malignant Biological Behavior Of Fibroblasts Derived From Cancer Cells

Background:Lung cancer(LC)and Colorectal cancer(CRC)are the human cancers with the first and third incidence rates in the world,respectively.The rapid growth,infiltration and metastasis of tumors are the important causes of death of patients.Besides tumor cells,tumor matrix also plays an important role in their occurrence and development.For example,the interaction between tumor cells and cancer-associated fibroblasts(CAFs),which expressα-smooth muscle actin(αSMA)and fibroblast activation protein(FAP),runs through the whole process of tumor occurrence and development,but the exact source and function of CAFs are still unclear.CAF is rhombic and with the thicker smooth muscle protein in cytoplasm,which are quite different from normal fibroblasts(NFs).A large number of clinical and basic studies have shown that CAFs only exists in tumor sites,but not in normal tissues.Therefore,we propose to assume that most CAFs come from the tumor in situ,within which the absolute number of non-stem cell-like tumor cells and a small number of tumor stem cells with self-renewal,multidirectional differentiation potential and high tumorigenicity are undoubtedly the primary targets of our investigation.Objective:To determine whether CD133~+tumor cells have the characteristics of tumor stem cells,and to further explore whether CAFs could be derived from tumor cells and the malignant biological behavior of CAFs.Methods:We used the stem cell marker CD133 as screening condition,and divided tumor cells in human lung cancer cell line(A549),lung cancer clinical specimen(PLC),human intestinal cancer cell line(HT-29)and colorectal cancer clinical specimen(PCC)into CD133~+cell group and CD133~-cell group,and then carried out cell identification.We used the fibroblast induction scheme to induce CD133~+and CD133~-cells in vitro and then selected the induced CAFs,and compared them with the CAFs isolated from subcutaneous transplanted tumors in the cell morphology and biology,then explored the key inducing factors.Then,we used transgenic mice(Pvillin-Cre/p CAG-i DTR-EGFP)tumor model to verify the source of CAFs,and studied their role in tumor microenvironment(TME)and their own tumorigenic characteristics.Results:CD133~+cells were found in A549,PLC,HT-29 and PCC,and CD133~+cells sorted by magnetic beads were superior to CD133~-cells in microsphere formation,proliferation,clone formation and tumor formation.After in vitro induction by fibroblast induction program,CD133~+cells and CD133~-cells have successfully induced a certain amount of CAFs,which have similar cell morphology and the expression of cell surface molecules FAP andαSMA with CAFs from xenografts,and TGF-βis the key inducing factor.CAFs isolated from primary intestinal cancer of transgenic mice have the same doubling phenomenon of c-Myc,EGFR and MDM2 oncogenes as tumor cells,and simultaneously express enhanced green fluorescent protein(EGFP)of small intestine.CAFs has the similar invasion and migration ability to tumor cells,which can reconstruct tumor tissue matrix,showing connective tissue hyperplasia accompanied by increased vascular density and increased expression of various tumor-promoting cytokines.In addition,CAFs differentiated from tumor cells can form tumors themselves.Conclusions:CD133~+and CD133~-cells were successfully separated,which confirmed that CD133~+cells have the characteristics of tumor stem cells.In vitro and in vivo experiments have proved that CD133~-non-stem cell-like tumor cells,like tumor stem cells,can be induced into CAFs,of which TGF-βis the most critical inducing factor.In vitro induced CAFs have the same morphology,surface marker expression and amplification signals of c-Myc,EGFR and MDM2 oncogenes as subcutaneous transplanted tumor CAFs.CAFs increases the density of tumor vessels by affecting the expression of HGF,SDF-1,IL-11,TGF-βand VEGF,thus promoting tumor growth.CAFs transformed from tumor cells has the same malignant biological behaviors as tumor cells,such as invasion,migration and tumor formation.