| Objective:In recent years, with the technical advances especially the molecular biologyprogression, the researches on tumorigenesis and development of malignancy progressed,and some research results have been applied to clinical use. e.g. The clinical effective rateof imatinib mesylate for the gastrointestinal stromal tumors exceeds 80%. However, littleprogress has been made in prognosis of pancreatic cancer. The 5-year survival rate is onlyabout 3% and the median survival is lessthan 6 months. For the patients who undergopotentially curative resection, the 5-year survival is less than 24% by reason of localrecurrence and metastasis.The cancer stem cell hypothesis offers new hope for the treatment of highlymalignancy. Cancer stem cells were considered to be the origin of malignancy, and playkey roles in cocarcinogenesis, progression, invasion, metastasis, recurrence of malignancy.Very recently, cancer stem cells have been isolated from some solid malignancies includingbreast, brain, prostate and lung cancers. Prof. Wang proposed that there were also cancerstem cells in pancreatic cancer, and worked at the isolation, identification and originationof pancreatic cancer stem cells. These works were supportted by the National NaturalScience Foundation of China (No.30571817) and the PhD Programs Foundation ofMinistry of Education of China (No. 20050487077).The aim of this study was to confirm the exist of cancer stem cells in pancreatic cancer,and study the possible origin of these pancreatic cancer stem cells.Methods:PANC-1 cells were cultured in medium DMEM-F12 supplemented with EGF, bFGF,insulin, transferrin, selenium and BSA at a density of 1,000 cells/mL. In this condition,cells with properties of stem cells could propagate to spheres. Clone formation assay and tumor formation assay of cells were used to identify the ability of propagation in vitro andin vivo. Spheres were also stained by Hoechst 33342 dye to evalueate their capacity ofexcluding Hoechst dye. Real time PCR was used to detect expressions of LY6E, c-Met,TACSTD1, CD34 and'CD44 mRNA.Immunofluorescence stain was used to detect the expression of CD24 in pancreaticductal cells. Density gradient centrifugation was used to isolate ductal cells from collagendigested pancreatic cells, and fluorescence activated cell sorting was used to separatepancreatic ductal cells into two subpopulation of CD24+ and CD24- cells. Flow cytometryand Immunofluorescence stain were used to detect the expression of CK19 and Insulin,Growth curve was used for evaluate the ability of propagation, and special culture conditionwith basic fibroblast growth factor was used to differentiate pancreatic ductal cells intoother types of cells.Mouse bone marrow derived mesenchymal stem cells were obtained by culture ofwhole bone marrow obtained from the long bones. Theexpression of special surfacemarkers were detected by flow cytometry. Growth curve was used for evaluate the ability ofpropagation. Bone marrow derived mesenchymai stem cells were differentiated intoadipocytes, osteocytes and chondrocytes by culture in different induction medium.Polyethylene glycol was used to fuse the pancreatic ductal cells fromC57BL/6J-Tg(pPGKneobpA)3Ems mouse and bone marrow derived mesenchymal stemcells transfected with green fluorescent protein. G418 selection and fuorescence activatedcell sorting were used for purification of fusion cells. Growth curve was used for evaluatethe ability of propagation, colony forming efficiency in soft agar for anchorage-independentgrowth, and Transwell assay for migration and invasion. Seek spontaneous transformedcells from long-term cultured fusion cells. Growth curve was used for evaluate the ability ofpropagation of transformed cells, colony forming efficiency in soft agar foranchorage-independent growth, karyotpe analysis for aneuploid cells, Transwell assay formigration and invasion; and xenografls in NOD/SCID mice for tumorigenic ability. Results:A subpopulation of 9.83 %±2.61% cells in PANC-1 cells could porpagte to formspheres in specific culture condition. These cells had the hallmark of excluding Hoechst33342 dye. Propagation capacity of these cells was higher than that of normal PANC-1cells both in vitro and in vivo. i.p. injection of 5×105 cells into nude mice could formmacroscopical tumors. Sca-1, ESA and CD44 mRNA were over expressed in these cells,while c-met mRNA was not over expressed.Part of pancreatic ductal cells express CD24. CD24 expression was mainly observedin the ductal epithelium of the interlobular duct and the intralobular duct. In contrast, theductal epithelial cells of the major duct and intercalated duct were mainly CD24-/low.CD24-/low pancreatic ductal cells can differentiate into insulin-secreting cells in vitro whenstimulated with exogenous bFGFPurified fusion cells could be obtained with the usage of G418 selection andfluorescence activated cell sorting. After fused with bone marrow derived mesenchymalstem cells, pancreatic ductal cells obtained capacity of anchorage-independent growth.After long-term culture, aneuploid cells could be detected in these cells, and part of cellstransformed into malignant cells. These cells could form tumors in NOD/SCID mouse.Conclusion:1. A subpopulation of cells with properties of cancer stem cells exist in pancreaticcancer cells. These cells were of capacity of excluding Hoechst 33342 and formingtumors in nude mice.2. Sca-1, ESA and CD44 mRNA were over expressed in pancreatic cancer stem cells,while c-met mRNA was not over expressed.3. CD24- pancreatic ductal cells of mouse can differentiate into insulin-secretingcells in vitro when stimulated with exogenous bFGF. Therefore, CD24- pancreaticductal cells have the potential to be pancreatic progenitor cells.4. Purified fusion cells could be obtained with the usage of G418 selection and fluorescence activated cell sorting.5. After fused with bone marrow derived mesenchymal stem cells, pancreatic ductalcells obtained capacity of anchorage-independent growth.6. Fusion cells of pancreatic ductal cells and bone marrow derived mesenchymalstem cells trend to transform spontaneously after long-term culture. Transformedcells were similar with pancreatic cancer stem cells.Innovations of this study:1. Verify the exist of cancer stem cells in pancreatic cancer by culture pancreaticcancer cells in selective culture condition that only cancer stem cells can propagate,and bring forward that CD44, Sca-1 and ESA were candidate specific surfacemarkers of pancreatic cancer stem cells.2. Verify the CD24- pancreatic ductal cells of mouse can differentiate intoinsulin-secreting cells, and bFGF plays an important role in this process. Bringforward that failor of differentiate pancreatic ductal cells into insulin-secretingcells in other studies should impute to in-appropriate culture conditions.3. Modify the cell fusion technic, use G418 selection and fluorescence activated cellsorting to purify fusion cells.4. Bring forward that pancreatic stem cells may originate from fusion cells ofpancreatic ductal cells and bone marrow derived mesenchymal stem cells. Verifypancreatic ductal cells trend to transform into malignant cells after fused with bonemarrow derived mesenchymal stem cells, and transformed cells are similar withpancreatic cancer stem cell. |
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