上皮性卵巢癌侧群细胞分离、鉴定及其在肿瘤形成过程中的作用研究
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摘要
上皮性卵巢癌侧群细胞分离、鉴定及其在肿瘤形成过程中的作用研究
目的：将上皮性卵巢癌细胞以Hoechst 33342染色后进行流式细胞仪检测,探讨卵巢癌侧群(SP)细胞的分离方法、干细胞特性和裸鼠体内致瘤能力,研究卵巢癌SP细胞在肿瘤形成过程中的作用。并通过慢病毒将GFP基因转入卵巢癌细胞中,分选GFP-SP细胞后以GFP作为示踪标记,追踪GFP-SP细胞在裸鼠体内的荧光表达和肿瘤成像检测。
材料和方法：1.选用人卵巢癌细胞株ES-2、HO-8910PM、HO-8910、SK-OV-3和CAOV-3细胞作为研究对象,取对数生长期细胞制成单细胞悬液,以Hoechst 33342染色,以流式细胞仪检测细胞拒染Hoechst 33342的比例。Verapamil作用下的Hoechst 33342拒染率作为对照。2.分选拒染Hoechst 33342的侧群(SP)细胞和NSP细胞,以单克隆抗体检测SP细胞、NSP细胞和未分选细胞表面CD24、CD44、CD45、CD54、CD62L、CDll7、CD133等表面标记的表达。3.收集SP细胞、NSP细胞和未分选细胞,以TRIZOL裂解液分离提取RNA,以RT-PCR法检测Nanog、Oct-4和BCRP等多能基因的表达,并以软琼脂半固体培养法和裸鼠皮下注射体内成瘤比较SP、NSP细胞的集落形成和致瘤能力。4.通过对细胞Transwell跨微孔膜迁移、穿透Matrigel以及贴壁能力的比较,了解SP细胞、NSP细胞在肿瘤侵袭能力方面的差异,并以人类全基因芯片分析SP细胞和NSP细胞在细胞黏附、侵袭方面的基因表达的不同,探究SP细胞和NSP细胞在肿瘤发生、转移中的作用。5.以慢病毒转染GFP至人卵巢癌细胞株ES-2、HO-8910PM,摸索Ubiquitin启动子构建的慢病毒颗粒对该2株细胞的最佳转移MOI。6.以流式细胞仪分选表达GFP的卵巢癌细胞株,获得高纯度的GFP-ES2和GFP-H08910PM,检测GFP表达对Hoechst 33342染色的影响。再以流式细胞仪分选GFP-SP细胞,将分选后GFP-SP细胞注射入裸鼠皮下,观察GFP-SP细胞的的致瘤能力,并在体观测GFP-SP来源肿瘤的荧光成像。
结果：1.卵巢癌细胞株中有一群能将Hoechst 33342泵出细胞外而不被Hoechst33342染色的细胞,Verapamil能降低细胞拒染Hoechst 33342的能力,这群细胞即为侧群细胞。2.SP细胞的比例和细胞BCRP的表达水平有关。ES-2/SP细胞表达Nanog、Oct-4等多能性基因,软琼脂中形成集落的数量比NSP细胞多。3.SP细胞体外贴壁培养时多呈簇状或集落性生长,细胞呈现鹅卵石或圆形；形成的集落细胞排列比较致密,向集落外扩展迁移的能力低。NSP细胞体外贴壁培养时多散在分布,细胞呈梭形或长条形；形成的细胞团比较疏松,向集落外扩展迁移的能力强。4.SP细胞穿过微孔膜的活动度和侵袭力都弱于NSP细胞。5.人类全基因芯片分析发现p-整合素、ICAM-5、CAM-1、各型胶原、层连蛋白、纤连蛋白等多种黏附分子受体/配体在SP细胞中的表达低于NSP细胞,protocadherin 7和CXCR4的表达高于NSP细胞。6.SP细胞在体外培养一定时间后,有部分细胞转为NSP细胞,但细胞拒染Hoechst 33342的比例仍高于NSP细胞培养后的Hoechst 33342拒染率。7.经流式细胞仪分选的HO-8910PM/SP细胞直接植入裸鼠皮下,其成瘤能力强于NSP细胞。8.分选纯化ES-2／SP细胞直接植入裸鼠皮下不能形成肿瘤,但体外培养后却能成瘤,而且致瘤能力仍强于NSP细胞,说明SP细胞在培养过程中细胞的生物学特性发生了变化。9.Ubiquitin启动子构建的慢病毒转染不影响卵巢癌细胞拒染Hoechst33342的能力,分选的GFP-SP细胞仍能在裸鼠体内形成表达GFP的肿瘤,成瘤能力和未转染细胞相比没有显著变化,GFP阳性肿瘤在成像系统中可见荧光表达。
结论：1.卵巢癌细胞株中有拒染Hoechst33342的SP细胞,SP细胞表达多能性基因,具有干细胞特征。2.SP细胞和NSP细胞相比,集落形成能力高于NSP细胞,但黏附能力和侵袭能力比NSP细胞弱,说明在肿瘤生成过程中,SP细胞作为肿瘤干细胞决定了肿瘤的发生,NSP细胞则有助于肿瘤细胞在局部组织中的侵袭,肿瘤干细胞在不断的自我更新和向NSP转化的过程中完成在局部的积聚和浸润,最终导致肿瘤的形成。3.慢病毒转染并不影响卵巢癌SP细胞的成瘤活性,病毒转染和GFP标记可以作为研究某一特性基因在卵巢癌发生、发展中作用的工具以及肿瘤细胞在动物活体内生物学行为的示踪标记。
Side population in epithelial ovarian cancer and its role in tumorigenesis
Objective: After FACs analysis and method modification, we sorted ovarian cancer cells-derived side population (SP) to identify its stem cell feature, including surface markers expression, capability of colony formation and its role in tumorigenesis. GFP-positive ovarian cancer cell lines were generated by lentiviral transduction. Tumorigenicity of GFP-SP cells were compared to that of non-transducing SP cells and fluorescence image of GFP-positive tumor in vivo was taken to testify whether GFP is able to be a marker to trace cancer cell mobility in vivo.
Materials and Methods:1. Human ovarian cancer cell lines, ES-2, HO-8910PM, HO-8910, SK-OV-3 and CAOV-3, were used to perform SP analysis by Hoechst 33342 staining. Cells which were growing well were trypsinized to form single-cell suspension. After stained with Hoechst 33342, Hoechst 33342-negative cells were gated as SP cells. Cells pretreated with Verapamil were used as a control.2. SP cells and non-side population (NSP) cells were sorted respectively. Expression of cell surface markers on SP cells or NSP cells, including CD24, CD44, CD45, CD54, CD62L, CD117 and CD133, were determined by FACs analysis.3. RNA from SP cells and NSP cells was isolated by TRIzol homogenization. Expression of Nanog, Oct-4 and Breast Cancer Resistant Protein (BCRP) were analyzed by RT-PCR. The capability of colony formation and tumorigenesis of SP cells were compared to those of NSP cells by agarose colony formation and tumorigenicity in nude mice.4. In terms of capability of transmigration and invasion of SP cells and NSP cells, the sorted-cells were applied onto Transwell upper chamber in the presence or in the absence of Matrigel. After 12 hours,24 hours or 48 hours, the cells having been migrated to lower chamber were fixed and counted. Microarray gene profile assay was used to determine the gene expression related to cell attachment and migration.5. ES-2 cells and HO-8910PM cells were transduced by lentivirus at various MOI. Expression of transgene was clarified by FACs analysis. The best transducing MOI to different cell line under the control of Ubiquitin promoter was determined referring to GFP expression.6. GFP-ES2 and GFP-HO8910PM were collected by FACsorting and passaged under regular culture condition. GFP-SP cells were sorted after stained with Hoechst33342 and injected subcutaneously into nude mice. The local tumorigenicity of GFP-SP cells at injection site was compared to that of non-transduced cells. The fluorescence image of tumor-bearing mouse was taken under a small animal fluorescence imaging system.
Results:1.There was a minor population in ovarian cancer cells which can pump out Hoechst33342 to result in Hoechst33342 staining-negative cells. Taking the difference in Hoechst 33342 staining between this population and major population, it was named as side population.2. The percentage of SP cells was related to gene expression of BCRP. SP cells expressed Nanog, Oct-4 and BCRP by analyses of RT-PCR, and had higher potential of colonigenicity in semisolid agarose gel.3. Sorted-SP cells grew in round-shape, cobblestone-like aggregates in regular cell culture plastic vessels. SP-derived aggregates were more compacted and formed less outgrowth than NSP-derived ones, which were scatter-distributed and spindle-shaped in plastic vessels.4. The differences between SP cells and NSP cells in attachment, transmigration and invasion were determined via Transwell chamber in the presence or in the absence of Matrigel. Cells migrated to the lower chamber were counted after fixation and stained by hematoxylin. The numbers of ES2-SP cells or HO8910PM-SP cells transmigrated through micropore membrane were less than those of NSP cells, either with or without Matrigel.5. Microarray of gene profile showed that expression of adhesion receptors/ligands, including integrin-β, ICAM-5, CAM-1, collagen, laminin and fibronectin, were down-regulated in HO8910PM-SP cells compared to HO8910PM-NSP cells, whereas protocadherin 7 and CXCR4 were up-regulated.6. SP cells can change to NSP phenotype under regular culture condition. However, during two weeks of cultivation, the percentage of Hoechst33342-negative cells in cultured SP population remained higher than that in cultured NSP one.7. Freshly sorted HO8910PM-SP cells were easier to be engrafted at subcutaneous injection site than NSP cells. However, ES2-SP cells showed higher local tumorigenicity only after cultured for a certain period of time when some of SP cells changed to NSP phenotype.8. Lentiviral transduction under the control of Ubiquitin promoter didn't effect on cells capability to pump Hoechst33342 out. GFP-SP cells from HO-8910PM could form GFP-positive tumor in nude mice. There was no difference in tumorigenecity between GFP-SP cells and non-transduced SP cells. Fluorescence in tumor cells at subcutaneous injection site can be detected under a small animal fluorescence imaging system.
Conclusions: 1.There is a minor population of Hoechst33342-rejecting SP cells in ovarian cancer cells. SP cells had stem cell features with expression of multi-potential genes.2. Being compared to NSP cells, SP cells had higher tumorigenicity and less capability to attachment to and invasion into matrix. It indicated that, during the process of tumor formation, stem-like SP cells undergo self-renewal to accumulate at primary site. Meanwhile, some SP cells changed to NSP phenotype which represents high potential to infiltrate into surrounding tissues. Both fortunate tumorigenesis.3. Lentiviral transduction does not effect on capability of ovarian cancer SP cells-derived tumor formation. Lentiviral transduction can be a useful tool in the study of tumorigenesis and development of ovarian cancer. GFP protein can be a marker to trace cancer cells in vivo.
目的：将上皮性卵巢癌细胞以Hoechst 33342染色后进行流式细胞仪检测,探讨卵巢癌侧群(SP)细胞的分离方法、干细胞特性和裸鼠体内致瘤能力,研究卵巢癌SP细胞在肿瘤形成过程中的作用。并通过慢病毒将GFP基因转入卵巢癌细胞中,分选GFP-SP细胞后以GFP作为示踪标记,追踪GFP-SP细胞在裸鼠体内的荧光表达和肿瘤成像检测。
材料和方法：1.选用人卵巢癌细胞株ES-2、HO-8910PM、HO-8910、SK-OV-3和CAOV-3细胞作为研究对象,取对数生长期细胞制成单细胞悬液,以Hoechst 33342染色,以流式细胞仪检测细胞拒染Hoechst 33342的比例。Verapamil作用下的Hoechst 33342拒染率作为对照。2.分选拒染Hoechst 33342的侧群(SP)细胞和NSP细胞,以单克隆抗体检测SP细胞、NSP细胞和未分选细胞表面CD24、CD44、CD45、CD54、CD62L、CDll7、CD133等表面标记的表达。3.收集SP细胞、NSP细胞和未分选细胞,以TRIZOL裂解液分离提取RNA,以RT-PCR法检测Nanog、Oct-4和BCRP等多能基因的表达,并以软琼脂半固体培养法和裸鼠皮下注射体内成瘤比较SP、NSP细胞的集落形成和致瘤能力。4.通过对细胞Transwell跨微孔膜迁移、穿透Matrigel以及贴壁能力的比较,了解SP细胞、NSP细胞在肿瘤侵袭能力方面的差异,并以人类全基因芯片分析SP细胞和NSP细胞在细胞黏附、侵袭方面的基因表达的不同,探究SP细胞和NSP细胞在肿瘤发生、转移中的作用。5.以慢病毒转染GFP至人卵巢癌细胞株ES-2、HO-8910PM,摸索Ubiquitin启动子构建的慢病毒颗粒对该2株细胞的最佳转移MOI。6.以流式细胞仪分选表达GFP的卵巢癌细胞株,获得高纯度的GFP-ES2和GFP-H08910PM,检测GFP表达对Hoechst 33342染色的影响。再以流式细胞仪分选GFP-SP细胞,将分选后GFP-SP细胞注射入裸鼠皮下,观察GFP-SP细胞的的致瘤能力,并在体观测GFP-SP来源肿瘤的荧光成像。
结果：1.卵巢癌细胞株中有一群能将Hoechst 33342泵出细胞外而不被Hoechst33342染色的细胞,Verapamil能降低细胞拒染Hoechst 33342的能力,这群细胞即为侧群细胞。2.SP细胞的比例和细胞BCRP的表达水平有关。ES-2/SP细胞表达Nanog、Oct-4等多能性基因,软琼脂中形成集落的数量比NSP细胞多。3.SP细胞体外贴壁培养时多呈簇状或集落性生长,细胞呈现鹅卵石或圆形；形成的集落细胞排列比较致密,向集落外扩展迁移的能力低。NSP细胞体外贴壁培养时多散在分布,细胞呈梭形或长条形；形成的细胞团比较疏松,向集落外扩展迁移的能力强。4.SP细胞穿过微孔膜的活动度和侵袭力都弱于NSP细胞。5.人类全基因芯片分析发现p-整合素、ICAM-5、CAM-1、各型胶原、层连蛋白、纤连蛋白等多种黏附分子受体/配体在SP细胞中的表达低于NSP细胞,protocadherin 7和CXCR4的表达高于NSP细胞。6.SP细胞在体外培养一定时间后,有部分细胞转为NSP细胞,但细胞拒染Hoechst 33342的比例仍高于NSP细胞培养后的Hoechst 33342拒染率。7.经流式细胞仪分选的HO-8910PM/SP细胞直接植入裸鼠皮下,其成瘤能力强于NSP细胞。8.分选纯化ES-2／SP细胞直接植入裸鼠皮下不能形成肿瘤,但体外培养后却能成瘤,而且致瘤能力仍强于NSP细胞,说明SP细胞在培养过程中细胞的生物学特性发生了变化。9.Ubiquitin启动子构建的慢病毒转染不影响卵巢癌细胞拒染Hoechst33342的能力,分选的GFP-SP细胞仍能在裸鼠体内形成表达GFP的肿瘤,成瘤能力和未转染细胞相比没有显著变化,GFP阳性肿瘤在成像系统中可见荧光表达。
结论：1.卵巢癌细胞株中有拒染Hoechst33342的SP细胞,SP细胞表达多能性基因,具有干细胞特征。2.SP细胞和NSP细胞相比,集落形成能力高于NSP细胞,但黏附能力和侵袭能力比NSP细胞弱,说明在肿瘤生成过程中,SP细胞作为肿瘤干细胞决定了肿瘤的发生,NSP细胞则有助于肿瘤细胞在局部组织中的侵袭,肿瘤干细胞在不断的自我更新和向NSP转化的过程中完成在局部的积聚和浸润,最终导致肿瘤的形成。3.慢病毒转染并不影响卵巢癌SP细胞的成瘤活性,病毒转染和GFP标记可以作为研究某一特性基因在卵巢癌发生、发展中作用的工具以及肿瘤细胞在动物活体内生物学行为的示踪标记。
Side population in epithelial ovarian cancer and its role in tumorigenesis
Objective: After FACs analysis and method modification, we sorted ovarian cancer cells-derived side population (SP) to identify its stem cell feature, including surface markers expression, capability of colony formation and its role in tumorigenesis. GFP-positive ovarian cancer cell lines were generated by lentiviral transduction. Tumorigenicity of GFP-SP cells were compared to that of non-transducing SP cells and fluorescence image of GFP-positive tumor in vivo was taken to testify whether GFP is able to be a marker to trace cancer cell mobility in vivo.
Materials and Methods:1. Human ovarian cancer cell lines, ES-2, HO-8910PM, HO-8910, SK-OV-3 and CAOV-3, were used to perform SP analysis by Hoechst 33342 staining. Cells which were growing well were trypsinized to form single-cell suspension. After stained with Hoechst 33342, Hoechst 33342-negative cells were gated as SP cells. Cells pretreated with Verapamil were used as a control.2. SP cells and non-side population (NSP) cells were sorted respectively. Expression of cell surface markers on SP cells or NSP cells, including CD24, CD44, CD45, CD54, CD62L, CD117 and CD133, were determined by FACs analysis.3. RNA from SP cells and NSP cells was isolated by TRIzol homogenization. Expression of Nanog, Oct-4 and Breast Cancer Resistant Protein (BCRP) were analyzed by RT-PCR. The capability of colony formation and tumorigenesis of SP cells were compared to those of NSP cells by agarose colony formation and tumorigenicity in nude mice.4. In terms of capability of transmigration and invasion of SP cells and NSP cells, the sorted-cells were applied onto Transwell upper chamber in the presence or in the absence of Matrigel. After 12 hours,24 hours or 48 hours, the cells having been migrated to lower chamber were fixed and counted. Microarray gene profile assay was used to determine the gene expression related to cell attachment and migration.5. ES-2 cells and HO-8910PM cells were transduced by lentivirus at various MOI. Expression of transgene was clarified by FACs analysis. The best transducing MOI to different cell line under the control of Ubiquitin promoter was determined referring to GFP expression.6. GFP-ES2 and GFP-HO8910PM were collected by FACsorting and passaged under regular culture condition. GFP-SP cells were sorted after stained with Hoechst33342 and injected subcutaneously into nude mice. The local tumorigenicity of GFP-SP cells at injection site was compared to that of non-transduced cells. The fluorescence image of tumor-bearing mouse was taken under a small animal fluorescence imaging system.
Results:1.There was a minor population in ovarian cancer cells which can pump out Hoechst33342 to result in Hoechst33342 staining-negative cells. Taking the difference in Hoechst 33342 staining between this population and major population, it was named as side population.2. The percentage of SP cells was related to gene expression of BCRP. SP cells expressed Nanog, Oct-4 and BCRP by analyses of RT-PCR, and had higher potential of colonigenicity in semisolid agarose gel.3. Sorted-SP cells grew in round-shape, cobblestone-like aggregates in regular cell culture plastic vessels. SP-derived aggregates were more compacted and formed less outgrowth than NSP-derived ones, which were scatter-distributed and spindle-shaped in plastic vessels.4. The differences between SP cells and NSP cells in attachment, transmigration and invasion were determined via Transwell chamber in the presence or in the absence of Matrigel. Cells migrated to the lower chamber were counted after fixation and stained by hematoxylin. The numbers of ES2-SP cells or HO8910PM-SP cells transmigrated through micropore membrane were less than those of NSP cells, either with or without Matrigel.5. Microarray of gene profile showed that expression of adhesion receptors/ligands, including integrin-β, ICAM-5, CAM-1, collagen, laminin and fibronectin, were down-regulated in HO8910PM-SP cells compared to HO8910PM-NSP cells, whereas protocadherin 7 and CXCR4 were up-regulated.6. SP cells can change to NSP phenotype under regular culture condition. However, during two weeks of cultivation, the percentage of Hoechst33342-negative cells in cultured SP population remained higher than that in cultured NSP one.7. Freshly sorted HO8910PM-SP cells were easier to be engrafted at subcutaneous injection site than NSP cells. However, ES2-SP cells showed higher local tumorigenicity only after cultured for a certain period of time when some of SP cells changed to NSP phenotype.8. Lentiviral transduction under the control of Ubiquitin promoter didn't effect on cells capability to pump Hoechst33342 out. GFP-SP cells from HO-8910PM could form GFP-positive tumor in nude mice. There was no difference in tumorigenecity between GFP-SP cells and non-transduced SP cells. Fluorescence in tumor cells at subcutaneous injection site can be detected under a small animal fluorescence imaging system.
Conclusions: 1.There is a minor population of Hoechst33342-rejecting SP cells in ovarian cancer cells. SP cells had stem cell features with expression of multi-potential genes.2. Being compared to NSP cells, SP cells had higher tumorigenicity and less capability to attachment to and invasion into matrix. It indicated that, during the process of tumor formation, stem-like SP cells undergo self-renewal to accumulate at primary site. Meanwhile, some SP cells changed to NSP phenotype which represents high potential to infiltrate into surrounding tissues. Both fortunate tumorigenesis.3. Lentiviral transduction does not effect on capability of ovarian cancer SP cells-derived tumor formation. Lentiviral transduction can be a useful tool in the study of tumorigenesis and development of ovarian cancer. GFP protein can be a marker to trace cancer cells in vivo.
引文
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18. Bruce WR, Van Der Gaag H. A Quantitative Assay for the Number of Murine Lymphoma Cells Capable of Proliferation in Vivo. Nature.1963;199:79-80.
19. Janzen V, Scadden DT. Stem cells:good, bad and reformable. Nature. 2006;441:418-419.
20. Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology. 2006;44:240-251.
21. Clarke RB, Spence K, Anderson E, Howell A, Okano H, Potten CS. A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol.2005;277:443-456.
22. Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, et al. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci U S A.2006; 103:11154-11159.
23. Haraguchi N, Utsunomiya T, Inoue H, et al. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells.2006;24:506-513.
24. Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A. 2004;101:781-786.
25. Bapat SA, Mali AM, Koppikar CB, Kurrey NK. Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Res. 2005;65:3025-3029.
26. Virchow R. Cellular Pathology. Arch Pathol Anat Physiol Klin Med.1855;8:3-39.
27. Cohnheim J. Ueber entzundung und eiterung. Path Anat Physiol Klin Med. 1867;40:1-79.
28. Makino S. The role of tumor stem-cells in regrowth of the tumor following drastic applications. Acta Unio Int Contra Cancrum.1959;15(Suppl 1):196-198.
29. McKay R. Stem cells-hype and hope. Nature.2000;406:361-364.
30. Austin TW, Salimi S, Veres G, et al. Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells. Blood.2000;95:829-836.
31. Wolf NS, Kone A, Priestley GV, Bartelmez SH. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp Hematol. 1993;21:614-622.
32. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med.1997;3:1337-1345.
33. Gussoni E, Soneoka Y, Strickland CD, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature.1999;401:390-394.
34. Hirschmann-Jax C, Foster AE, Wulf GG, et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A. 2004;101:14228-14233.
35. Matsui W, Wang Q, Barber JP, et al. Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Res.2008;68:190-197.
36. Murase M, Kano M, Tsukahara T, et al. Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas. Br J Cancer.2009;101:1425-1432.
37. Zen Y, Fujii T, Yoshikawa S, et al. Histological and culture studies with respect to ABCG2 expression support the existence of a cancer cell hierarchy in human hepatocellular carcinoma. Am J Pathol.2007; 170:1750-1762.
38. Kato K, Takao T, Kuboyama A, et al. Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage. Am J Pathol.2010;176:381-392.
39. Pecorelli S, Favalli G, Zigliani L, Odicino F. Cancer in women. Int J Gynaecol Obstet.2003;82:369-379.
40. Zhang S, Balch C, Chan MW, et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res.2008;68:4311-4320.
41. Sales-Pardo I, Avendano A, Martinez-Munoz V, et al. Flow cytometry of the Side Population: tips & tricks. Cell Oncol.2006;28:37-53.
42. Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A.1998;95:15665-15670.
43. Moserle L, Indraccolo S, Ghisi M, et al. The side population of ovarian cancer cells is a primary target of IFN-alpha antitumor effects. Cancer Res. 2008;68:5658-5668.
44. Condeelis J, Segall JE. Intravital imaging of cell movement in tumours. Nat Rev Cancer.2003;3:921-930.
45. Williams DA, Cancelas JA. Leukaemia: niche retreats for stem cells. Nature. 2006;444:827-828.
46. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res. 2006;66:7833-7836.
47. Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol.2004;5:816-826.
48. Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol.1962;59:223-239.
49. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem.1985;260:3440-3450.
50. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science.1994;263:802-805.
51. Tsien RY. The green fluorescent protein. Annu Rev Biochem.1998;67:509-544.
1. Virchow R. Cellular Pathology. Arch Pathol Anat Physiol Klin Med. 1855;8:3-39.
2. Cohnheim J. Ueber entzundung und eiterung. Path Anat Physiol Klin Med. 1867;40:1-79.
3. Sell S. Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol.2004;51:1-28.
4. McKay R. Stem cells-hype and hope. Nature.2000;406:361-364.
5. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature.2001;414:105-111.
6. Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature.2006;444:756-760.
7. Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133+ cancer stem cells in glioblastoma. Mol Cancer.2006;5:67.
8. Huang EH, Heidt DG, Li CW, Simeone DM. Cancer stem cells:a new paradigm for understanding tumor progression and therapeutic resistance. Surgery. 2007;141:415-419.
9. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science.1988;241:58-62.
10. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A. 1992;89:2804-2808.
11. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med.1997;3:730-737.
12. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A.2003;100:3983-3988.
13. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature.2004;432:396-401.
14. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med.1996;183:1797-1806.
15. Challen GA, Little MH. A side order of stem cells:the SP phenotype. Stem Cells. 2006;24:3-12.
16. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A.2002;99:12339-12344.
17. Wulf GG, Luo KL, Jackson KA, Brenner MK, Goodell MA. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica.2003;88:368-378.
18. Iwatani H, Ito T, Imai E, et al. Hematopoietic and nonhematopoietic potentials of Hoechst(low)/side population cells isolated from adult rat kidney. Kidney Int. 2004;65:1604-1614.
19. Imai N, Hishikawa K, Marumo T, et al. Inhibition of histone deacetylase activates side population cells in kidney and partially reverses chronic renal injury. Stem Cells. 2007;25:2469-2475.
20. Montanaro F, Liadaki K, Volinski J, Flint A, Kunkel LM. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc Natl Acad Sci U S A.2003;100:9336-9341.
21. Welm B, Behbod F, Goodell MA, Rosen JM. Isolation and characterization of functional mammary gland stem cells. Cell Prolif.2003;36 Suppl 1:17-32.
22. Liu BY, McDermott SP, Khwaja SS, Alexander CM. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci U S A.2004; 101:4158-4163.
23. Falciatori I, Borsellino G, Haliassos N, et al. Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis. Faseb J.2004;18:376-378.
24. Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology. 2006;44:240-251.
25. Clarke RB, Spence K, Anderson E, Howell A, Okano H, Potten CS. A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol.2005;277:443-456.
26. Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, et al. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci U S A.2006; 103:11154-11159.
27. Haraguchi N, Utsunomiya T, Inoue H, et al. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells.2006;24:506-513.
28. Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A. 2004;101:781-786.
29. Hemmings C. The elaboration of a critical framework for understanding cancer: the cancer stem cell hypothesis. Pathology;42:105-112.
30. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K. Autocrine TGF-beta signaling maintains tumorigenicity of glioma-initiating cells through Sry-related HMG-box factors. Cell Stem Cell.2009;5:504-514.
31. Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell.2008;133:704-715.
32. Chen D, McKearin D. Dpp signaling silences bam transcription directly to establish asymmetric divisions of germline stem cells. Curr Biol.2003;13:1786-1791.
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45. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res. 2006;66:7833-7836.
46. Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol.2004;5:816-826.
47. Calabrese C, Poppleton H, Kocak M, et al. A perivascular niche for brain tumor stem cells. Cancer Cell.2007;11:69-82.
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2. Hemmings C. The elaboration of a critical framework for understanding cancer: the cancer stem cell hypothesis. Pathology.2010;42:105-112.
3. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science.1988;241:58-62.
4. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A. 1992;89:2804-2808.
5. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature.2001;414:105-111.
6. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med.1996;183:1797-1806.
7. Challen GA, Little MH. A side order of stem cells:the SP phenotype. Stem Cells. 2006;24:3-12.
8. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A.2002;99:12339-12344.
9. Wulf GG, Luo KL, Jackson KA, Brenner MK, Goodell MA. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica.2003;88:368-378.
10. Iwatani H, Ito T, Imai E, et al. Hematopoietic and nonhematopoietic potentials of Hoechst(low)/side population cells isolated from adult rat kidney. Kidney Int. 2004;65:1604-1614.
11. Imai N, Hishikawa K, Marumo T, et al. Inhibition of histone deacetylase activates side population cells in kidney and partially reverses chronic renal injury. Stem Cells. 2007;25:2469-2475.
12. Montanaro F, Liadaki K, Volinski J, Flint A, Kunkel LM. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc Natl Acad Sci U S A.2003;100:9336-9341.
13. Welm B, Behbod F, Goodell MA, Rosen JM. Isolation and characterization of functional mammary gland stem cells. Cell Prolif.2003;36 Suppl 1:17-32.
14. Liu BY, McDermott SP, Khwaja SS, Alexander CM. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci U S A.2004; 101:4158-4163.
15. Falciatori I, Borsellino G, Haliassos N, et al. Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis. Faseb J.2004;18:376-378.
16. Fidler IJ, Hart IR. Biological diversity in metastatic neoplasms:origins and implications. Science.1982;217:998-1003.
17. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med.1997;3:730-737.
18. Bruce WR, Van Der Gaag H. A Quantitative Assay for the Number of Murine Lymphoma Cells Capable of Proliferation in Vivo. Nature.1963;199:79-80.
19. Janzen V, Scadden DT. Stem cells:good, bad and reformable. Nature. 2006;441:418-419.
20. Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology. 2006;44:240-251.
21. Clarke RB, Spence K, Anderson E, Howell A, Okano H, Potten CS. A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol.2005;277:443-456.
22. Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, et al. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci U S A.2006; 103:11154-11159.
23. Haraguchi N, Utsunomiya T, Inoue H, et al. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells.2006;24:506-513.
24. Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A. 2004;101:781-786.
25. Bapat SA, Mali AM, Koppikar CB, Kurrey NK. Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Res. 2005;65:3025-3029.
26. Virchow R. Cellular Pathology. Arch Pathol Anat Physiol Klin Med.1855;8:3-39.
27. Cohnheim J. Ueber entzundung und eiterung. Path Anat Physiol Klin Med. 1867;40:1-79.
28. Makino S. The role of tumor stem-cells in regrowth of the tumor following drastic applications. Acta Unio Int Contra Cancrum.1959;15(Suppl 1):196-198.
29. McKay R. Stem cells-hype and hope. Nature.2000;406:361-364.
30. Austin TW, Salimi S, Veres G, et al. Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells. Blood.2000;95:829-836.
31. Wolf NS, Kone A, Priestley GV, Bartelmez SH. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp Hematol. 1993;21:614-622.
32. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med.1997;3:1337-1345.
33. Gussoni E, Soneoka Y, Strickland CD, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature.1999;401:390-394.
34. Hirschmann-Jax C, Foster AE, Wulf GG, et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A. 2004;101:14228-14233.
35. Matsui W, Wang Q, Barber JP, et al. Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Res.2008;68:190-197.
36. Murase M, Kano M, Tsukahara T, et al. Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas. Br J Cancer.2009;101:1425-1432.
37. Zen Y, Fujii T, Yoshikawa S, et al. Histological and culture studies with respect to ABCG2 expression support the existence of a cancer cell hierarchy in human hepatocellular carcinoma. Am J Pathol.2007; 170:1750-1762.
38. Kato K, Takao T, Kuboyama A, et al. Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage. Am J Pathol.2010;176:381-392.
39. Pecorelli S, Favalli G, Zigliani L, Odicino F. Cancer in women. Int J Gynaecol Obstet.2003;82:369-379.
40. Zhang S, Balch C, Chan MW, et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res.2008;68:4311-4320.
41. Sales-Pardo I, Avendano A, Martinez-Munoz V, et al. Flow cytometry of the Side Population: tips & tricks. Cell Oncol.2006;28:37-53.
42. Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A.1998;95:15665-15670.
43. Moserle L, Indraccolo S, Ghisi M, et al. The side population of ovarian cancer cells is a primary target of IFN-alpha antitumor effects. Cancer Res. 2008;68:5658-5668.
44. Condeelis J, Segall JE. Intravital imaging of cell movement in tumours. Nat Rev Cancer.2003;3:921-930.
45. Williams DA, Cancelas JA. Leukaemia: niche retreats for stem cells. Nature. 2006;444:827-828.
46. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res. 2006;66:7833-7836.
47. Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol.2004;5:816-826.
48. Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol.1962;59:223-239.
49. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem.1985;260:3440-3450.
50. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science.1994;263:802-805.
51. Tsien RY. The green fluorescent protein. Annu Rev Biochem.1998;67:509-544.
1. Virchow R. Cellular Pathology. Arch Pathol Anat Physiol Klin Med. 1855;8:3-39.
2. Cohnheim J. Ueber entzundung und eiterung. Path Anat Physiol Klin Med. 1867;40:1-79.
3. Sell S. Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol.2004;51:1-28.
4. McKay R. Stem cells-hype and hope. Nature.2000;406:361-364.
5. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature.2001;414:105-111.
6. Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature.2006;444:756-760.
7. Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133+ cancer stem cells in glioblastoma. Mol Cancer.2006;5:67.
8. Huang EH, Heidt DG, Li CW, Simeone DM. Cancer stem cells:a new paradigm for understanding tumor progression and therapeutic resistance. Surgery. 2007;141:415-419.
9. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science.1988;241:58-62.
10. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A. 1992;89:2804-2808.
11. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med.1997;3:730-737.
12. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A.2003;100:3983-3988.
13. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature.2004;432:396-401.
14. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med.1996;183:1797-1806.
15. Challen GA, Little MH. A side order of stem cells:the SP phenotype. Stem Cells. 2006;24:3-12.
16. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A.2002;99:12339-12344.
17. Wulf GG, Luo KL, Jackson KA, Brenner MK, Goodell MA. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica.2003;88:368-378.
18. Iwatani H, Ito T, Imai E, et al. Hematopoietic and nonhematopoietic potentials of Hoechst(low)/side population cells isolated from adult rat kidney. Kidney Int. 2004;65:1604-1614.
19. Imai N, Hishikawa K, Marumo T, et al. Inhibition of histone deacetylase activates side population cells in kidney and partially reverses chronic renal injury. Stem Cells. 2007;25:2469-2475.
20. Montanaro F, Liadaki K, Volinski J, Flint A, Kunkel LM. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc Natl Acad Sci U S A.2003;100:9336-9341.
21. Welm B, Behbod F, Goodell MA, Rosen JM. Isolation and characterization of functional mammary gland stem cells. Cell Prolif.2003;36 Suppl 1:17-32.
22. Liu BY, McDermott SP, Khwaja SS, Alexander CM. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci U S A.2004; 101:4158-4163.
23. Falciatori I, Borsellino G, Haliassos N, et al. Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis. Faseb J.2004;18:376-378.
24. Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology. 2006;44:240-251.
25. Clarke RB, Spence K, Anderson E, Howell A, Okano H, Potten CS. A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol.2005;277:443-456.
26. Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, et al. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci U S A.2006; 103:11154-11159.
27. Haraguchi N, Utsunomiya T, Inoue H, et al. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells.2006;24:506-513.
28. Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A. 2004;101:781-786.
29. Hemmings C. The elaboration of a critical framework for understanding cancer: the cancer stem cell hypothesis. Pathology;42:105-112.
30. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K. Autocrine TGF-beta signaling maintains tumorigenicity of glioma-initiating cells through Sry-related HMG-box factors. Cell Stem Cell.2009;5:504-514.
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