Identification And Characterization Of Cancer Stem Cell-like Side Population Cells In Human Pancreatic Cancer

Pancreatic cancer is one of the most lethal cancers in digestive system, as indicated by a 5year survival rate of 5％-10％even for patients received curative radical excision. It istherefore essential to develop a deeper understanding of the biological characteristics ofthis disease to develop more effective therapies. Emerging evidence showed that onlycancer stem cells, a small subset of cells within a tumor, is able to self-renew and give riseto heterogeneous populations of daughter cells that comprise the tumor. The existence ofcancer stem cells was initially proven in hematological malignancy and subsequentlyverified in breast carcinoma, brain cancers, prostate, lung and colon by specific cell surfacemakers. They were proven to play a decisive role in the pathogenesis of their correspondingtumors. Recently, the cancer stem cell hypothesis has been explored in pancreatic cancer,but the consensus marker of pancreatic cancer stem cells is undetermined. Li et al identifieda CD44+CD24+ESA+ subpopulation as putative pancreatic cancer stem cells, whileHermann et al defined human pancreatic cancer stem cells by means of the surface markerCD133. Moreover, these studies mainly indicate that pancreatic cancer is caused by cancerstem cells, the cell signaling pathways governing the maintenance and development ofpancreatic cancer stem cells, which may become targets to disrupt cancer stem cellactivities, are not well defined. Side population isolated from tumors has been proven to bea generally accepted alternative to study cancer stem cells biology since they are enriched in cancer stem cells. The present study was undertaken to investigate the prevalence of SPcells, study the biological characteristics of them and investigate the role of PI3K/mTORpathway in the survival and proliferation of them.PartⅠIsolation and phenotype analysis of side population cells inhuman pancreatic cancerObjective To identify side population in pancreatic cancer and define its phenotype.Method To detect the persistence of SP cells in 6 pancreatic cancer cell lines and 3 clinicalsamples by Hoechst 33342 dyeing and FACS analysis. To perform phenotype analysis onSP cells in three typical pancreatic cancer cell linesResults All cell lines and clinical samples were found to exhibit verapamil-sensitive SPcells except BXPC-3. The proportions of SP cells in human pancreatic cancer cell linesPANC-1,CAPAN-1,ASPC-1, PC-3 and SW-1990 were 7.84％, 11.22％, 16.88％, 0.43％,and 2.25％, respectively. The proportions of SP cells in clinical pancreatic cancer sampleswere 0.33％, 2.79％, and 0.98％, respectively. Most SP cells in PANC-1, CAPAN-1 andASPC-1 were CD133~+ and CD44~+CD24~-ESA~+Conclusion Side population cells do exist in human pancreatic cancer. The phenotype ofSP cells in pancreatic cancer were CD133~+ and CD44~+CD24~-ESA~+PartⅡThe biological characteristics and mechanism of drugresistance of cancer stem-like side population cells in pancreaticcancerObjective To identify side population cells in pancreatic cancer and investigate the biological characteristics and mechanism of drug resistance of them.Method To detect the presentation of SP cells in pancreatic cancer cell line, PANC-1, byHoechst 33342 dyeing and FACS analysis. To compare the clone formation efficiency andtumorigenicity between SP cells and non- SP cells by clone formation assay andNOD/SCID xenograft transplantation experiment. To Study the differentiation ability of SPcells by flow cytometry reanalysis of SP-derived tumors and cultured SP cells. To evaluatethe self-renew ability of SP cells by sphere formation assay. Transwell invasion assay andMTT assay were conducted to compare the invasiveness and drug resistance between SPcells and non-SP cells. Real-time PCR was used to detect the expression of ABCB1 andABCG2 in both SP cells and non-SP cells. Cell cycle analysis was also performed to thesetwo populations.Results SP cells do exist in PANC-1. SP and NSP cells were collected for subsequentexperiments, the purity was 96.5％for SP cells and 99.11％for NSP cells. PANC-1 SPwere demonstrated to possess greater self-renew ability and tumorigenicity and be capableof generating nontumorigenic non-SP cells through asymmetrical cell division. SP cellswere more invasive and more resistant to chemotherapeutic drugs than NSP cells. ABCB1and ABCG2 were found to express at higher concentrations in SP than NSP cells. And mostSP cells are in G1 cell cycle arrest.Conclusion There is a significant enrichment of stem-like invasive tumor-initiating cells inthe SP of pancreatic cancer. Their high resistance to chemotherapy was related to theincreased expression of ATP-binding cassette transporters and relative quiescence in cellcycle. It may help explain why conventional therapies often fail to improve long-termsurvival of pancreatic cancer and why even patients who respond to primary chemotherapyultimately develop to local recurrence and metastasis. PartⅢPI3K/mTOR pathway is critical for survival andproliferation of pancreatic cancer stem-like side population cellsObjective To characterize side population cells in pancreatic cancer and investigate therole of PI3K/mTOR pathway in the survival and proliferation of them.Method To detect the presentation of side population cells in pancreatic cancer cell lines,PANC-1, by FACS analysis, as well as the SP proportion change in PANC-1 treated withPI3K/mTOR-specific inhibitor LY294002 and rapamycin. MTT assay and clone formationassay were done to assess the inhibition of LY294002 and rapamycin.Results LY294002 and rapamycin decreased the SP fraction in PANC-1. Compared withcontrol PANC-1 cells, which contained 7.60±0.27％SP cells, PANC-1 cells treated withLY294042 and rapamycin had only 1.90±4.22％(P=0.400) and 1.14±0.24％(P=0.000)SP cells, respectively. Both LY294002 and rapamycin preferentially inhibited the SP ratherthan NSP cells. LY294002 and rapamycin inhibited PANC-1 SP cell proliferation by 47.87±3.82％and 57.04±2.78％(1-relative SR) respectively, while inhibited the NSP cellproliferation by 27.64±2.09％and 35.99±3.11％only. In addition to proliferation,LY294042 and rapamycin also preferentially inhibited the colony-formation ability of SPcells by 54±6.56％and 48±3.61％(1-relative CEF), respectively, compared with that ofthe NSP cells by 34.67±3.06％and 30.67±3.21％.Conclusion PI3K/mTOR pathway is critical for Pancreatic SP cells maintenance, whichcould be selectively targeted for inhibiting cancer stem-like cells for improved treatment.