Heterogeneity beyond tumor heterogeneity—SULF2 involvement in Wnt/β-catenin signaling activation in a heterogeneous side population of liver cancer cells

Introduction:Sulfatase 2(SULF2),an endogenous extracellular sulfatase,can remove 6-O-sulfate groups of glucosamine residues from heparan sulfate(HS)chains to modulate the Wnt/β-catenin signaling pathway,which plays an important role in both liver carcinogenesis and embryogenesis.Side population(SP)cells are widely identified as stem-like cancer cells and are closely related to carcinoma metastasis,recurrence,and poor patient prognosis.However,the roles of SULF2 in SP cells of hepatomas are unclear,and the underlying mechanism is undefined.Objectives:This study aimed to compare the heterogeneity between SP cells and non-side population(NSP)cells derived from three different liver cancer cell lines and to elucidate the involvement of the SULF2-Wnt/β-catenin axis in liver cancer stem cells(CSCs)and its impact on the processes of carcinogenesis and invasiveness.Methods:In this work,three different liver cancer SP cells(HepG2,Huh7,and PRC/PRL/5)were sorted by flow cytometry.We also examined the migration and invasion behaviors of SP and NSP cells.To determine if this high tumorigenic potential of SP cells is correlated to SULF2,qPCR,western blotting,and immunofluorescence analysis were conducted.We also performed nude mouse xenograft experiments for in vivo analysis.Results:The results from the in vitro colony formation assay showed that SP cells exhibited a 2-fold higher colony formation efficiency compared to their NSP counterparts.The SP cells exhibited significantly higher potentials in terms of their migratory capacity and invasive ability compared to NSP cells.We found that higher expression of SULF2 in SP cells was associated with greater capabilities for clonogenicity,migration,and invasion.It was also linked to higher activation of the Wnt/β-catenin signaling pathway via stimulation of key downstream factors,particularlyβ-catenin,c-Myc,and cyclin D1.Further,a positive correlation between the upregulated SULF2 expression and tumorigenesis in the in vivo nude mouse xenograft models was demonstrated,highlighting that the potential underlying mechanism was Wnt/β-catenin signaling pathway activation.Conclusion:Our findings show that variable SULF2 expression was associated with differential activation of the Wnt/β-catenin signaling pathway,which could lead to behavioral differences between SP and NSP cells and also among the SP cells of the three liver cancer cell lines assessed.It was reasonably concluded that the SULF2-Wnt/β-catenin axis could play an important role in the tumorigenicity of liver cancer stem cells.

Side Population Cells in Human Gallbladder Cancer Cell Line GBC-SD Regulated by TGF-β-induced Epithelial-mesenchymal Transition

Mounting evidence has shown that side population （SP） cells are enriched for cancer stem cells （CSCs） responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP cells in human gallbladder cancer cell line GBC-SD, and SP cells which had superior potential for proliferation in vitro and tumorigenesis in vivo were identified. Importantly, the abundance of GBC-SD SP cells was increased by a transforming growth factor-β （TGF-β）-induced epithelial-mesenchymal transition （EMT）, and this effect was accompanied with a strong up-regulation of ABCG2 mRNA expression, and a decreased sensitivity to mitoxantrone. SP cells were restored upon the removal of TGF-β and the reversion of the cells to an epithelial phenotype, and smad3-specific siRNA reduced SP abundance in response to TGF-β. In conclusion, TGF-β-induced EMT by smad-dependent signaling pathway promotes cancer development and anti-cancer drug resistant phenotype by augmenting the abundance of GBC-SD SP cells, and a better understanding of mechanisms involved in TGF-β-induced EMT may provide a novel strategy for preventing cancer progression.

Isolation and Identification of Cancer Stem-like Cells from Side Population of Human Prostate Cancer Cells

It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues.However,due to lack of specific stem cell surface markers,CSCs are very difficult to be separated from some cancer cells,which becomes the key barrier of functional studies of CSCs.The sorting method by side population cells (SP) lays a solid foundation for in-depth and comprehensive study of CSCs.To identify the existence of SP in prostate cancer cell lines,we applied flow cytometry sorting by SP to cultures of prostate cancer cell lines (TSU,LnCap,and PC-3),and the cancer stem-like characteristics of SP were verified through experiments in vitro and in vivo.The proportion of SP in TSU cells was calculated to be 1.60%±0.40% (±s),and that in PC-3 and LnCap cells was calculated to be 0.80%±0.05% and 0.60%±0.20%,respectively.The colony formation assay demonstrated that the colony formation rate of SP to non-SP sorted from TSU via flow cytometry was 0.495±0.038 to 0.177±0.029 in 500 cells,0.505±0.026 to 0.169±0.024 in 250 cells,and 0.088±0.016 to 0.043±0.012 in 125 cells respectively.In the in vivo experiments,tumors were observed in all the mice on the 10th day after injecting 50 000 cells subcutaneously in SP group,whereas when 5×106 cells were injected in non-SP group,tumors were developed in only 4 out of 8 mice until the 3rd week before the end of the experiment.Our results revealed that prostate cancer cells contain a small subset of cells,called SP,possessing much greater capacity of colony formation and tumorigenic potential than non-SP.These suggest that SP in prostate cancer cells may play a key role in the self-renewal and proliferation,and have the characteristics of cancer stem-like cells.Dissecting these features will provide a new understanding of the function of prostate CSCs in tumorigenicity and transformation.

Isolation of Side Population Cells and Detection of ABCG2 from SW480

Objective：Side population cells （SP cells） are a new type of stem cells. They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342. Many studies showed that side population cells were able of self-renewal, differentiation and carcinogenesis in cancers. Our investigation aimed at isolation of side population cells and ABCG2 positive subpopulation.from colon cancer cell line SW480 and identification of their characteristics of cancer stem cells. Methods： side population cells and non-side population cells of colon cancer cell line SW480 were isolated with DNA dye Hoechst33342 and their cell cycles were measured by flow cytometry. Expression of ABCG2 of SW480 was measured by immunohistochemistry and immunofluorescence, and its proportion was measured by flow cytometry. Results： SW480 contained 2.29% side population ceils. The fraction of side population ceils decreased greatly to 0.40% by treatment with verapamil. The fraction of side population cells in S-G2M cell cycle was 16.14%, which was much lower than the fraction （34.05%） of non-side population cells in S-G2M. In SW480, ABCG2 positive cells, which proportion was 9.66%, were small, circular or oval, lack of psuedopods, similar to poor differentiation. On the contrary, the ABCG2 negative cells were large, polygonal, with many psuedopods, similar to high differentiation. ConclusJon： our assay identified that side population cells did exist in SW480 and had a quiescence characteristic of stem cells. ABCG2 positive subpopulation occupied about 9.66% of SW480 and may have the ability to promote cell self-renewal and inhibit cell differentiation. Therefore, to isolate ABCG2 positive subpopulation from side population cells may be an alternative to study colorectal cancer stem cells.

Identification and Characterization of Side Population Cells in Human Lung Adenocarcinoma SPC-A1 Cells

Objective：There has been an increasing interest in recent years in the role of stem cells.With an extensive understanding of their biology,a major role for stem cells in the malignant process has been proposed and the existence of cancer stem cells（CSCs） has been confirmed in hematopoietic malignancies and solid organ malignancies including brain cancer,breast,prostate,colon,and pancreatic cancer.Lung cancer is the leading cause of cancer mortality in most large cities of China.It is possible that lung cancer contains cancer stem cells responsible for its malignancy.The aim of this study is to identify,characterize and enrich the CSC population that drives and maintains lung adenocarcinoma growth and metastasis.Methods：Side population（SP） cell analysis and sorting were applied on human lung adenocarcinoma cell line and an attempt to further enrich them by preliminary serum-free culture before fluorescence activated cell sorting（FACS） was done.Stem cell properties of SP cells were evaluated by their proliferative index,colony-forming efficiency,tumorigenic potential,bi-differentiation capacity and the expression of common stem cell surface markers.Results：Lung cancer cells could grow in a serum-free Medium（SFM） as non-adherent spheres similar to neurospheres or mammospheres.The proportion of SP cells in cell spheres was significantly higher than that in cells grown as monolayers.SP cells had a greater proliferative index,a higher colony-forming efficiency and a greater ability to form tumor in vivo.SP cells were both CCA positive and SP-C positive while non-SP cells were only SP-C positive.Flow cytometric analysis of cell phenotype showed that SP cells expressed CD133 and CD44,the common cell surface markers of cancer stem cells,while non-SP cells only expressed CD44.Conclusion：SP cells existed in human lung adenocarcinoma cell lines and they could be further enriched by preliminary serum-free culture before FACS sorting.SP cells possessed the properties of cancer stem cells.

Targeting BCRP/ABCG2 by RNA Interference Enhances the Chemotherapy Sensitivity of Human Colon Cancer Side Population Cells

Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics.This study isolated side population（SP） cells from a colon cancer cell line（Colo-320） and examined their self-renewal and differentiation abilities.Compared to non-SP（NSP） cells,SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies.Additionally,more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2.We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like,small interfering RNA（si RNA） eukaryotic expression plasmid targeting BCRP/ABCG2.

Side population cells from HXO-Rb44 retinoblastoma cell line have cancer-initiating property

AIM: To ascertain whether side population (SP) cells in HXO-Rb44 retinoblastoma cell line have cancer stem cell-like property in vitro and in vivo. METHODS: We analyzed and sorted SP from HXO-Rb44 retinoblastoma cell line by Hoechst 33342 staining on flow cytometry. SP and NSP cells were determined their ability of proliferation and self-renewal by SP reanalysis, soft agar assay and tumor sphere assay in vitro. Clone formation was detected by seeding HXO-Rb44 and HXO-Rb44 -RFP cells into soft agar. The expression of ABCGZ MDRI Bmi-1 and Oct-4 was determined by RT-PCR between SP and non-SP (NSP) cells. Moreover, they were injected into nude mice to determine their tumorigency in vivo. RESULTS: SP from HXO-Rb44 retinoblastoma cell line could grow clonally in soft agar assays and form tumor spheres from single cells in conditioned media. The expressions of ABCG2, MDRI Bmi-1 and Oct-4 were significantly higher in SP than NSP cells. As few as SP cells resulted in tumor formation in 6 of 12 injected sites, however, the injection of NSP cells failed to form new tumor. CONCLUSION: SP cells isolated by Hoechst 33342 from the HXO-Rb44 retinoblastoma cell line had property of high tumorigency in vivo and in vitro Therefore, SP might be a target while developing retinoblastoma therapies.

Distinct Side Population Cell Subtypes Have Different Stemness Levels in Human Ovarian Cancer Cells

The stemness of different side population(SP)cell subtypes in ovarian cancer cells was studied,and the heterogeneity of ovarian cancer stem cells was analyzed.The cisplatin-resistant human serous ovarian cancer cell line C13 was stained with the bisbenzimide Hoechst 33342.A flow cytometry-based fluorescence-activated sorting method was used to obtain lower-SP(LSP)cells,upper-SP(USP)cells,and non-SP cells(NSP)based on their sensitivity to the staining time and Hoechst dye concentration.The sphere-forming capability,expression levels of stem cell markers,resistance to high concentrations of cisplatin,and subcutaneous tumorigenicity in NOD/SCID mice of the different cell subtypes were evaluated.The C13 cells contained SP cells with stemness characteristics,and the LSP cell subtype expressed higher levels of stem cell markers,had higher in vitro sphere-forming capability,higher cisplatin resistance and higher in vivo subcutaneous tumorigenesis than USP cells(P<0.05).NSP cells had no stemness.In conclusion,different subtypes of ovarian cancer SP cells have different stemness levels,and ovarian cancer stem cells may be heterogeneous.

miR-888 in MCF-7 Side Population Sphere Cells Directly Targets E-cadherin

Side population （SP） cells are a small subset of cells isolated from a cultured cancer cell line that exhibit characteristics similar to those of cancer stem cells （CSCs）, such as high metastatic and tumorigenic potential. The molecular mechanisms that give rise to the malignant properties of SP cells are not clear. We isolated SP cells from the MCF-7 breast cancer cell line and profiled microRNA （miRNA） expression patterns between SP cell-derived spheroids and non-SP cells. SP spheroids were found to possess 42 up-regulated miRNAs and 27 down-regulated ones （above 5-fold changes）. One of the up-regulated miRNAs, miR-888 computationally predicted to participate in the adherens junction （AJ） pathway, was investigated. Over-expression of miR-888 in MCF-7 cells reduced the mRNA levels of all four AJ pathway genes （E-cadherin, ACTG1, PTPRTand CDC42） that were selected for testing, whereas knocking down miR-888 reversed the trends. Western blot and flow cytometric quantitation of the membrane E-cadherin levels showed the same trend of change under these treatments. Luciferase reporter assay showed E-cadherin is a direct target of miR-888. As a potential role in intercellular adhesiveness and maintenance of malignant tissue architecture, the results indicate that miR-888 is a repressor of the AJ pathway in MCF-7 cells and that up-regulation of miR-888 contributes to aggressiveness in MCF-7 SP cells.

Identification and analysis of a minority fraction in a U87 cell line Do side-population cells represent bona fide stem cell-like cancer cells?

BACKGROUND： Overwhelming evidence suggests that tumor bulks are comprised of differentiated tumor cells and cancer stem cells （CSCs）. The stem cell-like side-population （SP） cells account for a minor fraction of the total tumor cells, yet are apparently the cells capable of tumor initiation, growth, maintenance, and recurrence. OBJECTIVE： To identify potential stem cell-like cancer cells in a U87 human brain glioma cell line on the basis of dye efflux, clone formation, and multi-drug resistance capacity. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Laboratory of Shanghai Institute of Hematology and Laboratory of Shanghai Institute of Endocrinology in Ruijin Hospital; in vivo contrast observational animal trial was performed at Experimental Animal Center, School of Medicine, Shanghai Jiao Tong University from June 2007 to May 2008. MATERIALS： The U87 cell line was provided by the Shanghai Institute of Cancer Research, Chinese Academy of Science; DMEM/F12 （1 ： 1） and fetal bovine serum were purchased from Gibco Invitrogen, USA; human recombinant basic fibroblast growth factors were purchased from BD Bioscience, USA; Hoechst 33342, Verapamil, and methyl thiazolyl tetrazolium were purchased from Sigma, USA; phycoerythrin-labeled anti-human-CD133 was purchased from Milteny Biotec, Germany; SYBR PrimeScriptTM RT-PCR kit was purchased from TaKaRa Biotechnology, Dalian, China. METHODS： Monolayer cultured cells were harvested by 0.25% Trypsin-EDTA and suspended at a 1 ×10^6/mL dilution in PBS containing 2% FBS, and were stained with Hoechst 33342 dye, either alone or in combination with Verapamil. Following fluorescence-activated cell sorting, SP and non-SP subsets were cultivated with serum-containing （DMEM plus 10% fetal bovine serum） or serum-free culture medium [DMEM/F12 （1： 1） ＋ 1× B27 supplement ＋ 10 ng/mL basic fibroblast growth factors ＋ 1× L-glutamine] to determine growth characteristics in vitro. Finally, single free U87 cells and subsets （SP or non-SP cells） were subcutaneously injected into the backs of 5-week-old nude mice for in vivo tumorigenicity. MAIN OUTCOME MEASURES： Cell morphology and clonogenicity were observed under inverted microscope; SP phenotype and fluorescent antibody labeling were analyzed by MoFIoTM flow cytometry; ABC transporter mRNA expression was evaluated by semi-quantitative real-time RT-PCR; efflux capacity for anti-neoplastic drugs from the U87 cell line and subsets was measured with the MTT assay, then detected by enzyme-linked immunosorbent assay at a wavelength of 490 nm; in vivo tumorigenicity in immunodeficient nude mice was evaluated by diameter size. RESULTS： During in vitro passages, human U87 cells maintained a stable SP fraction profile and exhibited the ability to form neurosphere-like clones. SP cell proliferation decreased compared with non-treated U87 cells. CD133 expression was reduced in the SP and non-SP cells. Freshly sorted SP fractions expressed higher levels of ABC drug transporter genes, and exhibited increased potential for cytotoxic drug resistance. The in vivo malignancy of U87 cells was largely dependent on non-SP cells in nude mice, and tumors that formed from the non-SP fraction developed faster and larger compared with tumors from the SP fraction. CONCLUSION： The SP cell component was a key factor that influenced mRNA expression and cytotoxic drug resistance. In particular, cancer stem cells or tumor-initiating cells were not exclusively enriched in the SP subset of the U87 cell line, and non-SP cells were even more tumorigenic.

Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer

Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.

Endometrial stem/progenitor cells: Properties, origins, and functions

The endometrium is the inner mucosal lining of the uterus that undergoes extensive cyclic growth, regeneration, differentiation, and shedding throughout the menstrual cycle in response to steroid hormones. It repeatedly undergoes approximately 450 cycles of degeneration and regeneration in a woman's lifetime. Endometrial abnormalities can be associated with repeated embryo implantation failure, recurrent spontaneous abortion, and other physiological features responsible for female infertility. This significant regenerative capacity may occur as a result of tissue-resident stem cell populations within the endometrium. Indeed, the existence of endometrial stem cells was only observed in humans and rodents through several isolation and characterization methods in the last few years. Although endometrial stem cells share various biological characteristics with other types of mesenchymal stem cells, they also show some differences in phenotype, self-renewal, and multilineage differentiation potential. Extensive studies over many years on endometrial stem cells will provide new insights into the physiology and mechanisms underlying various gynaecological diseases related to endometrial abnormalities such as female infertility, endometriosis, and endometrial cancer. Here we summarized recent studies about cellular origins and biological characteristics of endometrial stem cells. We also reviewed various recent studies to improve our understanding of their physiological roles. Many preclinical studies on their potential therapeutic applications to various endometrial diseases that could lead to reproductive dysfunction were also reviewed.

Comparative study on the stem cell phenotypes of C6 cells under different culture conditions

Background Glioma stem cell （GSC） hypothesis posits that a subpopulation of cells within gliomas have true clonogenic and tumorigenic potential. Significantly, a more controversial correlate to GSC is that cells in different culture conditions might display distinct stem cell properties. Considering these possibilities, we applied an approach comparing stem cell characteristics of C6 glioma cells under different culture conditions.Methods C6 cells were cultured under three different growth conditions, i.e., adherent growth in conventional 10% serum medium, non-adherent spheres growth in serum-free medium, as well as adherent growth on laminin-coated flask in serum-free medium. Growth characteristics were detected contrastively through neurosphere formation assay and cell cycle analysis. Markers were determined by immunofluorescence, relative-quantitative reverse transcription （RT）-PCR,Western blotting and flow cytometry. Side population cells were analyzed via flow cytometry. Tumor models were detected by magnetic resonance imaging and hematoxylin ＆ eosin staining. Data analyses were performed with SPSS software （17.0）.Results C6 cells （C6-Adh, C6-SC-Sph and C6-SC-Adh） showed distinctive growth patterns and proliferation capacity.Compared to suspending C6-SC-Sph, adherent C6-Adh and C6-SC-Adh displayed higher growth ratio. C6-SC-Sph and C6-SC-Adh showed enhanced capability of neurosphere formation and self-renewal. High side population ratio was detected in C6-SC-Sph and C6-SC-Adh. CD133 was not detected in all three kinds of cells. Conversely, Nestin and β-Ⅲ-tubulin were demonstrated positive, nonetheless with no statistical significance （P 〉0.05）. Interestingly, lower expression of glial fibrillary acidic protein was demonstrated in C6-SC-Sph and C6-SC-Adh. C6-Adh, C6-SC-Sph and C6-SC-Adh were all displayed in situ oncogenicity, while statistical difference of survival time was not confirmed.Conclusions C6 glioma cell line is endowed with some GSC phenotypes that can be moderately enriched in vitro when transferred into stem cell culture condition. The resultant tumor-spheres may be not a prerequisite or sound source of GSCs and adherent culture in stem cell medium is not a growth condition in favor of GSCs expanding in vivo.

Preliminary analysis of stem cell-like cells in human neuroblastoma

Background:Neuroblastoma is an embryonic neoplasm originating from the neural crest with cellular heterogeneity as one of its oncobiological characteristics.This study was undertaken to determine whether human neuroblastoma contains stem ell-like cells.Methods:Twenty patients with neuroblastoma who have been treated in our hospital since January 2005 were divided into pre-operative chemotherapy(10 patients)and non-chemotherapy(10)groups.Tumor specimens of the patients were taken and paraffin sections were made.The expressions of stem cell markers CD133,ABCG2,CD117 and nestin were immunohistochemically detected in the specimens.Neuroblastoma cells were stained with Hoechst 33342 and PI.The side population(SP)cells were analyzed by the fluorescence activated cell sorter.The disparity drug resistance to cisplatin(DDP)of SP and non-SP cells was measured with MTT colorimetric assay.The oncogenicity of SP and non SP cells was identified in nude mice.Results:There was no significant difference in the expression intensity of CD117 and nestin between the two groups of specimens(P>0.05).There was a significant difference between the two groups in terms of the expression intensity of CD133 and ABCG2(P<0.05).The SP cells accounted for 0.2%-1.3%of the total human neuroblastoma cells and were decreased to 0.1%-0.5%after verapamil treatment.The SP and non-SP cells showed disparity in cell growth experiment and drug resistance to DDP.Oncogenicity experiment revealed that nude mice could erupt tumor by an injection of 1×10^(6)SH-SY5Y and WIV SP cells.However,the nude mice could not form tumor by an injection of 1×10^(6)non-SP cells.Conclusion:Neuroblastoma might contain cancer stem cell-like cells.