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...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.展开更多
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 ...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.展开更多
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 G...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.展开更多
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 th...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.展开更多
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 a...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.展开更多
背景与目的目前研究认为侧群细胞(side population cell,SP cell)富集了肿瘤干细胞,是肿瘤生长和发展的根源;并且SP细胞对放化疗高度耐受,成为肿瘤复发转移的重要因素。为此,我们探讨了肺癌SP细胞与非SP细胞(non-SP)miRNA分子表达谱差异...背景与目的目前研究认为侧群细胞(side population cell,SP cell)富集了肿瘤干细胞,是肿瘤生长和发展的根源;并且SP细胞对放化疗高度耐受,成为肿瘤复发转移的重要因素。为此,我们探讨了肺癌SP细胞与非SP细胞(non-SP)miRNA分子表达谱差异,旨在为进一步研究miRNA在肺癌干细胞中的作用机制打下基础。方法利用Hoechst33342染料法,通过流式细胞仪紫外激光分选功能分选出A549细胞中的SP细胞和non-SP细胞,分别采用Trizol一步法提取两者的总RNA,利用miRNA芯片检测系统进行miRNA表达谱差异分析。结果肺癌SP细胞和non-SP细胞miRNA表达谱存在明显差异。与non-SP细胞相比,在SP细胞中上调2倍以上的miRNA有9条,下调2倍以上的miRNA有25条。结论差异表达的miRNA可能参与肺癌干细胞的发生发展过程,为进一步揭示肺癌干细胞发生的分子机制提供了依据。展开更多
目的:从人肝癌组织中分离培养肝癌干细胞样细胞,为进一步研究肝癌干细胞靶向治疗奠定基础。方法:采用酶消化法和以原代培养结合短暂传代培养的方法从人肝癌组织中分离出含人肝癌干细胞样细胞(human liver cancer stem-like cells,hLCSL...目的:从人肝癌组织中分离培养肝癌干细胞样细胞,为进一步研究肝癌干细胞靶向治疗奠定基础。方法:采用酶消化法和以原代培养结合短暂传代培养的方法从人肝癌组织中分离出含人肝癌干细胞样细胞(human liver cancer stem-like cells,hLCSLCs)的连续传代细胞,分别加入肝素、白蛋白、氢化可的松以筛选适用于hLCSLCs的无血清悬浮成球培养基。以流式细胞术检测hLCSLCs和由无血清悬浮成球培养所获得的成球细胞中旁群(side population,SP)细胞、CD133及CD90等的表达,裸鼠成瘤实验检测这两种细胞的致瘤能力。结果:成功地从人肝癌组织分离获得hLCSLCs,其SP、CD133+和CD90+细胞含量分别为0.9%、0.8%和12.7%,裸鼠皮下接种2000个SP细胞的成瘤率为100%。含有肝素的无血清培养基更适于hLCSLCs的成球培养,所获成球细胞中SP、CD133+和CD90+细胞含量分别为4.6%、9.7%和48%,接种10000个成球细胞即可全部成瘤。结论:成功建立了从人肝癌组织中分离肿瘤干细胞样细胞的方法,添加肝素的无血清悬浮成球培养法可有效富集肝癌干细胞样细胞。展开更多
基金supported by Beijing Natural Science Foundation (No.7102047)
文摘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.
基金the Natural Science Foundation of Shanghai,No. 08ZR 1413800
文摘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.
基金supported in part by the Foundation of State Key Laboratory of Reproductive Medicine,the project funded by the Priority Academic Program Development(PAPD) of Jiangsu Higher Education Institutionsthe National Natural Science Foundation of China(No. 30930080 and 81161120537)
文摘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.
文摘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.
文摘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.
文摘背景与目的目前研究认为侧群细胞(side population cell,SP cell)富集了肿瘤干细胞,是肿瘤生长和发展的根源;并且SP细胞对放化疗高度耐受,成为肿瘤复发转移的重要因素。为此,我们探讨了肺癌SP细胞与非SP细胞(non-SP)miRNA分子表达谱差异,旨在为进一步研究miRNA在肺癌干细胞中的作用机制打下基础。方法利用Hoechst33342染料法,通过流式细胞仪紫外激光分选功能分选出A549细胞中的SP细胞和non-SP细胞,分别采用Trizol一步法提取两者的总RNA,利用miRNA芯片检测系统进行miRNA表达谱差异分析。结果肺癌SP细胞和non-SP细胞miRNA表达谱存在明显差异。与non-SP细胞相比,在SP细胞中上调2倍以上的miRNA有9条,下调2倍以上的miRNA有25条。结论差异表达的miRNA可能参与肺癌干细胞的发生发展过程,为进一步揭示肺癌干细胞发生的分子机制提供了依据。
