Abstract
BackgroundPancreatic cancer is one of the direct causes of cancer-related death. High level of chemoresistance is one of the major obstacles of clinical treatment. In recent years, cancer stem cells have been widely identified and indicated as the origin of chemoresistance in multi-types of solid tumors. Increasing evidences suggest that cancer stem cells reside in the cells capable of forming holoclones continuously. However, in pancreatic cancer, holoclone-forming cells have not been characterized yet. Therefore, the goal of our present study was to indentify the holoclone-forming pancreatic cancer stem cells and develop an in vitro continuous colony formation system, which will greatly facilitate the study of pancreatic cancer stem cells.Methodology/Principal FindingsPancreatic cancer cell line BxPC3 was submitted to monoclonal cultivation to generate colonies. Based on the morphologies, colonies were classified and analyzed for their capacities of secondary colony formation, long-term survival in vitro, tumor formation in vivo, and drug resistance. Flowcytometry and quantitative RT-PCR were performed to detect the expression level of cancer stem cells associated cell surface markers, regulatory genes and microRNAs in distinct types of colonies. Three types of colonies with distinct morphologies were identified and termed as holo-, mero-, and paraclones, in which only holoclones generated descendant colonies of all three types in further passages. Compared to mero- and paraclones, holoclones possessed higher capacities of long-term survival, tumor initiation, and chemoresistance. The preferential expression of cancer stem cells related marker (CXCR4), regulatory genes (BMI1, GLI1, and GLI2) and microRNAs (miR-214, miR-21, miR-221, miR-222 and miR-155) in holoclones were also highlighted.Conclusions/SignificanceOur results indicate that the pancreatic tumor-initiating cells with high level of chemoresistance were enriched in holoclones derived from BxPC3 cell line. Generation of holoclones can serve as a novel model for studying cancer stem cells, and attribute to developing new anti-cancer drugs.
Highlights
Pancreatic cancer is currently the fourth leading cause of cancer-related mortality
We addressed the heterogeneity in pancreatic cancer cell lines BxPC3 [22] and PC3 [23] based on the morphology of colonies derived from single cancer cells and demonstrated that cancer stem cell properties were enriched in holoclones exclusively
The first aim of our study was to determine whether the diversity of clonal morphologies exists in pancreatic cancer cell population, so monoclonal cultivation was carried out (Fig. 1A) with pancreatic cancer cell line BxPC3
Summary
Pancreatic cancer is currently the fourth leading cause of cancer-related mortality. Less than 5% patients survive for 5 years after diagnosis with the median survival period of 4 to 6 months [1,2]. Chemotherapy is considered as an important option in clinical therapy, but it usually produces poor effects [4,5].it is necessary to decipher the mechanisms underlying the high level chemoresistance of pancreatic cancer cells. Cancer stem cells (or termed as tumor initiating cells) have been identified as an integral part in multi types of solid tumors [6,7,8,9,10,11]. Cancer stem cells have been widely identified and indicated as the origin of chemoresistance in multi-types of solid tumors. The goal of our present study was to indentify the holoclone-forming pancreatic cancer stem cells and develop an in vitro continuous colony formation system, which will greatly facilitate the study of pancreatic cancer stem cells
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