Establishment Of A Cell Line With Highly Lymphatic Metastatic Potential And Further Investigation Of Its Stem Cell-like Properties

Pancreatic cancer is one of the highest malignant solid tumors. Morbidity is close to mortality and the incidence rate of this disease is increasing year by year. It is serious to lives and property of people. Currently a radical surgery is still the only possible means to cure it for pancreatic cancer patients. But even for the patients with a radical operation,5-year survival rate remains below 20%. Studies have shown that lymph node metastasis is an important independent poor prognostic factor for patients after radical surgery. And the proportion of patients after surgery verified pathologically to be with lymph node metastasis is up to 60%. Hence, the aspect of research on lymph node metastasis characteristics and relevant treatment of pancreatic cancer has become a focus. Lack of an ideal cell line with high lymphatic metastasis at home and abroad has been a choke point for further insight into the mechanism of lymphatic metastasis. Therefore, the establishment of a stable cell line with high lymphatic metastasis of pancreatic cancer will be of great significance.Part I. Establishment of human pancreatic cancer BxPC-3 cell lines (BxPC-3LN) with a highly lymph-metastasis potentialObjective:To establish a cell line with highly lymphatic metastatic potential employing an in vivo highly continuous selection methodMethods:An in vivo highly continuous selection method was employed to establish a cell line with highly lymphatic metastatic potential. To research the difference between BxPC-3 and BxPC-3LN was tested by optical microscopy, electron microscopy, flow cytometry, cell proliferation assay and transwell as well as cDNA microarray.Results:The BxPC-3-LN subline, derived from the BxPC-3 human pancreatic cancer cell line, was established through serial passages in nude mice via footpad injections. The subline was able to develop notable lymphatic metastases in 100% of the recipient mice 8 weeks after tumor cell implantation. Compared with the parental BxPC-3 cells, BxPC-3-LN cells were more aggressive, displaying invasive ultrastructure, increased migration and invasion ability, and chemoresistance. Metastasis-related gene alteration including upregulation of MMP14, MMP24, MIF and ADRM1, and downregulation of TGFB2 and ROBO1 were also observed in BxPC-3-LN cells by cDNA microarrays.Part Ⅱ:Preliminary study of stem cell-like properties of the established cell line BxPC-3LNObjective:To further explore the stem cell-like properties and related regulatory mechanisms of the established cell line BxPC-3LN.Methods:Evaluation of the stem cell-like properties of the highly lymphatic metastatic pancreatic cancer cells BxPC-3-LN, including high lymphatic metastasis potential, self-renewal ability and chemoresistance by orthotopically implanted tumors in nude mice, flow cytometry, the ability of sphere formation and clone formation, immunofluorescence, RT-PCR and miRNAs PCR array.Results:Compared with the parental BxPC-3 cells, the BxPC-3-LN cells showed stem cell-like properties, including high lymphatic metastasis potential, self-renewal ability and chemoresistance. In addition, the BxPC-3-LN cells also expressed higher levels of sonic hedgehog and migrating cancer stem cell surface markers (CD 133 and CXCR4) compared to the parental BxPC-3 cells. The growth of BxPC-3-LN cells was significantly inhibited by gemcitabine combined with the sonic hedgehog inhibitor cyclopamine. The BxPC-3-LN cells expressed lower levels of let-7, miR-34, miR-107, miR-125, miR-128, miR-130, miR-132 and miR-141 than the parental BxPC-3 cells detected by microRNA PCR array, which were reported to have close relation to stem cell factors.Conclusions:(1)The new established cell line can provide an accessible model for research on the mechanisms of lymphatic metastasis of pancreatic cancer and screening anticancer drugs. And it has a very important scientific significance and application prospects. (2) Cancer stem cells were considered to be involved in the process of lymph node metastasis of pancreatic cancer. Some miRNAs may affect the process of invasion and metastasis of pancreatic cancer by modulation of the formation and maintenance of cancer stem cells.