Study On The Features Of Normal Endothelial Cells And Fibroblasts Induced By The Supernatant Of Tumor Cells

The growth and progression of tumors closely depend on the formation of the supporting stroma, including ECM protein, neovascular, and active stromal fibroblasts. Among the supporting cells, endothelial cells and fibroblasts are singled out, along with tumor cells, the interaction among them induce the production of ECM components, growth factors and proteinase for the aggression of tumors.To explore the aggressive phenotypes of endothelial cells and fibroblasts derived from tumors, and to afford valuable cell models for studying their transforming mechanism and developing drugs targeting the tumor stroma in the future. We have induced the normal human umbilical vein endothelial cell ECV304 and mice fibroblast L929 by the supernatant of human ovarian cancer cell SKOV3 and mice hepatocellular carcinoma cell H22 respectively for three and half months, and we acquired two cell lines, ECV304-SKOV3 and L₉₂₉-H₂₂, with stable growth and some biological features of tumor stromal cells. Then the changes of their biological characteristics were determined by light microscopy, electron microscopy, karyotype analysis, MTT assay, flow cytometry etc; the abnormal expression of PCNA, TGF-β1, TN, MMP-9, bcl-2 and tolomerase activity were detected by immunocytochemistry and RT-PCR; the migration, tube formation of endothelial cells and invasive potential induced by fibroblasts were analyzed and compared through co-culture. The results as follows:After three and half months, ECV304-SKOV3 and L₉₂₉-H₂₂ cell lines were thirty passages. The two cell lines can growth stablely and have some biological charateristics of tumor stromal cells:1. The morphological changes of ECV304-SKOV3 and L₉₂₉-H₂₂ cell lines were significant and their chromatosome mode increased slightly.2. As compared with parental cells, the population doubling time of ECV304-SKOV3 and L₉₂₉-H₂₂ cell lines shortened; their mitotic indices increased; their expression of PCNA enhanced; the cell number of ECV304-SKOV3 cells in S-phase increased while in G0/G1-phase decreased significantly, proliferating index hightened.3. ECV304-SKOV3 cells was more sensitive than ECV304 to anticancer drugs with inhibition effect on S-phase or S-phase and M-phase, in accordance with SKOV3 cells; both ECV304-SKOV3 and ECV304 cell lines were P-gp-negative. L₉₂₉-H₂₂ cells was more sensitive than L929 cells to anticancer drugs with inhibition effect on S-phase or S-phase and M-phase or cell skeleton.4. The cellular total protein of ECV304-SKOV3 and L₉₂₉-H₂₂ cell lines were more than ECV304 and L929 cell lines respectively, and the former two expressed PCNA, MMP-9, bcl-2 more, while L₉₂₉-H₂₂ cells expressed TN, TGF-β1 more too.5. Tolomerase activity were enhanced significantly.6. Adhesion potential of L₉₂₉-H₂₂ cells increased, while ECV304-SKOV3 cells decreased.7. The ECV304-SKOV3 and ECV304 cells-derived conditioned medium could induce the growth of SKOV3 cells, but the difference was not significant. On the third day, 25% CM of L₉₂₉-H₂₂ cells stimulate the growth of H22 cells significantly.8. L₉₂₉-H₂₂ and L929 cell lines enhanced the invasiveness of human mammary cancer MDA-MB-231 cells through artificial basement membrane (Mtrigel) 1.21 and 0.48 respectively.9. The migratory capability of ECV304-SKOV3 cells enhanced 2.10.10. Without SKOV3 cells, ECV304-SKOV3 cells developed morphogenesis; while ECV304-SKOV3 cells formed more tubular structures and longer than ECV304 cells did when co-cultured with SKOV3 cells. Fibroblast L₉₂₉-H₂₂ stimulated endothelial cells to form more tubes and longer than L929 did.11. Both ECV304-SKOV3 and L₉₂₉-H₂₂ cell lines induced morphogenesis of ECV304 cells.The above results suggest that ECV304-SKOV3 and L₉₂₉-H₂₂ cell lines had some biological characteristics of tumor stromal cells: Compared with parental cells, ECV304-SKOV3 and L₉₂₉-H₂₂ cell lines proliferate more actively, survive longer and are more functional; express and...