Roles Of Pancreatic Adenocarcinoma Cells With Tumor Stem Cell Property In Invasion And Metastasis

Part I Sphere-formation capacity and expression pattern of CD44, CD24, and ESA in human pancreatic adenocarcinoma cell linesObjective:To investigate the capacity of sphere-formation and the expression pattern of CD44, CD24, and ESA in different pancreatic adenocarcinoma cell linesMethod:The capacity of sphere-formation in specific serum free medium(SFM) of four human pancreatic adenocarcinoma cell lines (PANC-1, PC-2, MIA-Paca-2, and BxPC-3) was assessed. In addition, flow cytometry was used to analyze the expression pattern of CD44, CD24, and ESA in these four cell lines. Then, the same assays were performed when tumor cell spheres were developed. The influence of this kind of culture condition on the expression pattern of CD44, CD24, and ESA was analyzed.Results:Only CD44 expression was detected in PC-2 and MIA-Paca-2. In PANC-1, CD44 and CD24 were detected. While, in BxPC-3, all CD44, CD24, and ESA were detected. Tumor cell spheres were developed in PANC-1 and MIA-Paca-2 in SFM. This was accompanied by an increase in CD24 expression (13.32%±6.15%v. 1.22%±0.57%, P<0.01) and a decrease in CD44 expression (85.21%±8.74%v. 98.88%±2.38%, P<0.01). Meanwhile, CD24+CD44+ cells increased to 6.6%-21% (12.60%±5.10% v.1.43%±0.59%, P<0.01). Statistically significant change in CD44 expression percentage was not detected in MIA-Paca-2. Conclusion:PANC-1 and MIA-Paca-2 could develop tumor cell spheres in SFM. The expression patterns of pancreatic cancer stem cell surface markers CD44, CD24, and ESA were obviously different and could varied with SFM in different pancreatic adenocarcinoma cell lines.PartⅡThe biological characteristics and expression of SHH in PANC-1 tumor cell spheresObjective:To identify the biological characteristics of PANC-1 tumor cell spheres and investigate the expression of SHH in themMethod:To identify the differentiation ability of PANC-1 cell spheres, cells from PANC-1 tumor cell spheres were cultured in serum containing medium(SCM). Then, flow cytometry was applied to analyze expression pattern change of CD44, CD24, and ESA. BALB/c-scid mice xenograft transplantation experiment was used to assess the tumorigenicity capacity of PANC-1 tumor cell spheres. The role of sonic hedgehog (SHH) in cells from PANC-1 tumor cell spheres was also explored by RT-PCR and Western blot.Results:The expressoin patterns of CD44 and CD24 changed when the culture condition was shifted to SCM, with a decrease in CD24 expression (1.57%±0.68%v. 13.32%±6.15%, P<0.01) and an increase in CD44 expression (94.80±4.77%v. 85.21±8.74%, P<0.01). Meanwhile, CD24+CD44+cells decreased to 0.7%-2.4% (1.55%±0.75% v.12.60%±5.10%, P<0.01). Besides, the expression patterns of CD44, CD24, and ESA returned to the same level as in PANC-1 cells which they originate from; In PANC-1 cells group, the minimum cell amount to develop tumor was 5×104(1/3), while, the minimum cell amount was 1×104 in PANC-1 tumor cell spheres group(3/3); Comparing with PANC-1 cells, the expression of SHH in PANC-1 tumor cell spheres was up-regulated, either at mRNA or at protein level, accouting for (4.23±0.85 v.1.52±0.60, P<0.01) and (0.374±0.012 v.0.274±0.023,P<0.05), respectively.Conclusion:PANC-1 tumor cell spheres have the properties of tumor stem cells and the SHH signal pathway is up-regulated in themPartⅢRoles of pancreatic adenocarcinoma cells with tumor stem cell property in invasion and metastasisObjective:To investigate the role of pancreatic adenocarcinoma cells with tumor stem cell property in invasion,metastasis and explore the relationship between this role and epithelial-mesenchymal transitions(EMT).Method:Transwell chamber assays were conducted to compare the mobile and invasive capacity in vitro between PANC-1 tumor cell spheres and PANC-1 cells. BALB/c-scid mice xenograft splenic transplantation liver metastasis experiment was applied to assess metastasic abiliy. Western blot was used to detect the expression of E-cadherin and Vimentin in both PANC-1 tumor cell spheres and PANC-1 cells.Results:Comparing with PANC-1 cells, PANC-1 tumor cell spheres possess stronger migration and invasion abiliy in vitro; In PANC-1 cells group, when transplanted with 5×105 cells, liver metastasis was found (2/12), while, in PANC-1 tumor cell spheres group, at least with 1×105 cells, liver metastasis was found, besides, in 5×105, 1×106 and,5×106 cells group, more liver metastasis was verified(p<0.05); The protein expression of E-cadherin in PANC-1 tumor cell spheres was 0.415±0.005, being significantly lower than that in PANC-1 cells(0.415±0.005 v.0.504±0.006, p< 0.01). Meanwhile, the protein expression of Vimentin in PANC-1 tumor cell spheres was 0.504±0.005, being significantly higher than that in PANC-1 cells, which was 0.450±0.009(p<0.01).Conclusion The pancreatic adenocarcinoma cells with tumor stem cell property play a key role in invasion and metastasis. Furthermore, epithelial-mesenchymal transitions may be involved in this procedure.