This study was supported by ITT (Istituto Toscano Tumori) Grant Proposal 2010.

<ol>
	<li>Reddick, R. L., Michelitch, H. J., Levine, A. M., Triche, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+sarcoma:+a+study+of+the+ultrastructure.">Osteogenic sarcoma: a study of the ultrastructure.</a> Cancer. 45 (1), 64-71 (1980).</li><li>Gatta, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+cancer+survival+trends+in+Europe:+a+EUROCARE+Working+Group+study.">Childhood cancer survival trends in Europe: a EUROCARE Working Group study.</a> J. Clin. Oncol. 23 (16), 3742-3751 (2005).</li><li>Olstad, O. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+heterogeneity+in+human+osteosarcoma+demonstrated+by+enriched+mRNAs+isolated+by+directional+tag+PCR+subtraction+cloning.">Molecular heterogeneity in human osteosarcoma demonstrated by enriched mRNAs isolated by directional tag PCR subtraction cloning.</a> Anticancer. Res. 23 (3B), 2201-2216 (2003).</li><li>Geller, D. S., Gorlick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma:+a+review+of+diagnosis,+management,+and+treatment+strategies.">Osteosarcoma: a review of diagnosis, management, and treatment strategies.</a> Clin Adv Hematol Oncol. 8 (10), 705-718 (2010).</li><li>Tang, N., Song, W. X., Luo, J., Haydon, R. C., He, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma+development+and+stem+cell+differentiation.">Osteosarcoma development and stem cell differentiation.</a> Clin. Orthop. Relat. Res. 466 (9), 2114-2130 (2008).</li><li>Kempf-Bielack, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma+relapse+after+combined+modality+therapy:+an+analysis+of+unselected+patients+in+the+Cooperative+Osteosarcoma+Study+Group+(COSS).">Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS).</a> J. Clin. Oncol. 23 (3), 559-568 (2005).</li><li>Meyers, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma:+a+randomized,+prospective+trial+of+the+addition+of+ifosfamide+and/or+muramyl+tripeptide+to+cisplatin,+doxorubicin,+and+high-dose+methotrexate.">Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate.</a> J. Clin. Oncol. 23 (9), 2004-2011 (2005).</li><li>Gorlick, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+childhood+osteogenic+sarcoma+and+potential+targets+for+therapeutic+development:+meeting+summary.">Biology of childhood osteogenic sarcoma and potential targets for therapeutic development: meeting summary.</a> Clin. Cancer Res. 9 (15), 5442-5453 (2003).</li><li>Hayden, J. B., Hoang, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma:+basic+science+and+clinical+implications.">Osteosarcoma: basic science and clinical implications.</a> Orthop. Clin. North Am. 37 (1), 1-7 (2006).</li><li>Thomas, D., Kansara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+modifications+in+osteogenic+differentiation+and+transformation.">Epigenetic modifications in osteogenic differentiation and transformation.</a> J. Cell. Biochem. 98 (4), 757-769 (2006).</li><li>Araki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+retinoblastoma+gene+in+primary+osteosarcomas+and+other+bone+and+soft-tissue+tumors.">Involvement of the retinoblastoma gene in primary osteosarcomas and other bone and soft-tissue tumors.</a> Clin. Orthop. Relat. Res. 270 (270), 271-277 (1991).</li><li>Chou, A. J., Gorlick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+resistance+in+osteosarcoma:+current+challenges+and+future+directions.">Chemotherapy resistance in osteosarcoma: current challenges and future directions.</a> Expert. Rev. Anticancer Ther. 6 (7), 1075-1085 (2006).</li><li>Arndt, C. A. S., Crist, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+musculoskeletal+tumorsof+childhood+and+adolescence.">Common musculoskeletal tumorsof childhood and adolescence.</a> N. Engl. J. Med. 341 (5), 342-352 (1999).</li><li>Longhi, A., Errani, C., De Paolis, M., Mercuri, M., Bacci, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+bone+osteosarcoma+in+the+pediatric+age:+state+of+the+art.">Primary bone osteosarcoma in the pediatric age: state of the art.</a> Cancer. Treat. Rev. 32 (6), 423-436 (2006).</li><li>Bacci, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+outcome+for+patients+with+nonmetastatic+osteosarcoma+of+the+extremity+treated+at+the+istituto+ortopedico+rizzoli+according+to+the+istituto+ortopedico+rizzoli/osteosarcoma-2+protocol:+an+updated+report.">Long-term outcome for patients with nonmetastatic osteosarcoma of the extremity treated at the istituto ortopedico rizzoli according to the istituto ortopedico rizzoli/osteosarcoma-2 protocol: an updated report.</a> J. Clin. Oncol. 18 (24), 4016-4027 (2000).</li><li>Clarke, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells-perspectives+on+current+status+and+future+directions:+AACR+workshop+on+cancer+stem+cells.">Cancer stem cells-perspectives on current status and future directions: AACR workshop on cancer stem cells.</a> Cancer. Res. 66 (19), 9339-9344 (2006).</li><li>Lapidot, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+initiating+human+acute+myeloid+leukaemia+after+transplantation+into+SCID+mice.">A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.</a> Nature. 367 (6464), 645-648 (1994).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nat. Med. 3 (7), 730-737 (1997).</li><li>Visvader, J. E., Lindeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+solid+tumors:+accumulating+evidence+and+unresolved+questions.">Cancer stem cells in solid tumors: accumulating evidence and unresolved questions.</a> Nat. Rev. Cancer. 8 (10), 755-768 (2008).</li><li>Bapat, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+cancer+stem+cells.">Evolution of cancer stem cells.</a> Semin. Cancer Biol. 17 (3), 204-213 (2007).</li><li>Rubio, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+human+adult+stem+cell+transformation.">Spontaneous human adult stem cell transformation.</a> Cancer. Res. 65 (8), 3035-3039 (2005).</li><li>Burns, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenic+heterogeneity+in+cancer+stem+cells+evolved+from+long-term+cultures+of+telomerase-immortalized+human+mesenchymal+stem+cells.">Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells.</a> Cancer. Res. 65 (8), 3126-3135 (2005).</li><li>Zhang, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+etiology+and+treatment+of+cancer.">Stem cells in the etiology and treatment of cancer.</a> Curr. Opin. Genet. Dev. 16 (1), 60-64 (2006).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+transgenes+encoding+components+of+the+Wnt+signaling+pathway+preferentially+induce+mammary+cancers+from+progenitor+cells.">Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells.</a> Proc. Natl Acad. Sci. USA. 100 (26), 15853-15858 (2003).</li><li>Sell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+origin+of+cancer:+dedifferentiation+or+stem+cell+maturation+arrest?.">Cellular origin of cancer: dedifferentiation or stem cell maturation arrest?.</a> Environ Health Perspect. 101 (Suppl 5), 15-26 (1993).</li><li>Wernig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448 (7151), 318-324 (2007).</li><li>Niwa, H., Miyazaki, J., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+expression+of+oct-3/4+defines+differentiation,+dedifferentiation+or+self-renewal+of+ES+cells.">Quantitative expression of oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.</a> Nat. Genet. 24 (4), 372-376 (2000).</li><li>Hochedlinger, K., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+reprogramming+and+pluripotency.">Nuclear reprogramming and pluripotency.</a> Nature. 441 (7097), 1061-1067 (2006).</li><li>Dean, M., Fojo, T., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stem+cells+and+drug+resistance.">Tumor stem cells and drug resistance.</a> Nat. Rev. Cancer. 5 (4), 275-284 (2005).</li><li>Ma, S., Lee, T. K., Zheng, B. J., Chan, K. W., Guan, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++HCC+cancer+stem+cells+confer+chemoresistance+by+preferential+expression+of+the+Akt/PKB+survival+pathway.">CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway.</a> Oncogene. 27 (12), 1749-1758 (2008).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+cells+isolated+from+mesenchymal+neoplasms+have+tumor+initiating+potential.">Side population cells isolated from mesenchymal neoplasms have tumor initiating potential.</a> Cancer. Res. 67 (17), 8216-8222 (2007).</li><li>Ma, I., Allan, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+human+aldehyde+dehydrogenase+in+normal+and+cancer+stem+cells.">The role of human aldehyde dehydrogenase in normal and cancer stem cells.</a> Stem. Cell. Rev. 7 (2), 292-306 (2011).</li><li>Awad, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+ALDH+activity+identifies+chemotherapy-resistant+Ewing's+sarcoma+stem+cells+that+retain+sensitivity+to+EWS-FLI1+inhibition.">High ALDH activity identifies chemotherapy-resistant Ewing's sarcoma stem cells that retain sensitivity to EWS-FLI1 inhibition.</a> PLoS One. 5 (11), e13943 (2010).</li><li>Fujii, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphere-forming+stem-like+cell+populations+with+drug+resistance+in+human+sarcoma+cell+lines.">Sphere-forming stem-like cell populations with drug resistance in human sarcoma cell lines.</a> Int. J. Oncol. 34 (5), 1381-1386 (2009).</li><li>Di Fiore, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+molecular+characterization+of+the+human+osteosarcoma+3AB-OS+cancer+stem+cell+line:+a+possible+model+for+studying+osteosarcoma+origin+and+stemness.">Genetic and molecular characterization of the human osteosarcoma 3AB-OS cancer stem cell line: a possible model for studying osteosarcoma origin and stemness.</a> J. Cell. Physiol. 228 (6), 1189-1201 (2013).</li><li>Reynolds, B. A., Tetzlaff, W., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multipotent+EGF-responsive+striatal+embryonicprogenitor+cell+produces+neurons+and+astrocytes.">A multipotent EGF-responsive striatal embryonicprogenitor cell produces neurons and astrocytes.</a> J. Neurosci. 12 (11), 4565-4574 (1992).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255 (5052), 1707-1710 (1992).</li><li>Gibbs, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-like+cells+in+bone+sarcomas:+implications+for+tumorigenesis.">Stem-like cells in bone sarcomas: implications for tumorigenesis.</a> Neoplasia. 7 (11), 967-976 (2005).</li><li>Beccari, N., Mazzi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manuale di tecnica microscopic. Casa Editrice Dr. Francesco Vallardi Societ&#224; Editrice Libraria. , 99-100 (1966).</li><li>Majumdar, M. K., Thiede, M. A., Mosca, J. D., Moorman, M., Gerson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+comparison+of+cultures+of+marrow-derived+mesenchymal+stem+cells+(MSCs)+and+stromal+cells.">Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells.</a> J. Cell. Physiol. 176 (1), 57-66 (1998).</li><li>Tirino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+bone+sarcomas+contain+CD133++cancer+stem+cells+displaying+high+tumorigenicity+in+vivo.">Human primary bone sarcomas contain CD133+ cancer stem cells displaying high tumorigenicity in vivo.</a> FASEB J. 25 (6), 2022-2030 (2011).</li><li>Tirino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+solid+tumors:+an+overview+and+new+approaches+for+their+isolation+and+characterization.">Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization.</a> FASEB J. 27 (1), 13-24 (2013).</li><li>Martins-Neves, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+implications+of+an+enriched+cancer+stem-like+cell+population+in+a+human+osteosarcoma+cell+line.">Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line.</a> BMC Cancer. 12 (1), 139 (2012).</li><li>Tang, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+osteosarcoma+stem+cells+by+chemotherapy.">Enrichment of osteosarcoma stem cells by chemotherapy.</a> Chin. J. Cancer. 30 (6), 426-432 (2011).</li></ol>

The authors declare that they have no competing financial interest.

CSCが腫瘍バルク中のこの特定の細胞サブセットの同定を可能にするいくつかのプロパティを持っています。そのようなATP結合カセットの多剤排出トランスポーターの過剰発現28細胞毒性化学療法剤に対する獲得耐性のようなこれらの特性に基づいて、32、33、またはそのような発現のためのALDH 32として解毒酵素の発現のアップレギュレーションのためにCSCを単離するためのこのようなCD133、CD44、CD34、CD90、および他の30、34、35、41、いくつかの異なる方法として、特定の表面マーカーの42-44が開発されてきました。これらの技術の一つは、非付着条件下で増殖するためのCSCの能力に基づいて球体形成アッセイです。 球体を形成するための組織幹細胞とのCSCの能力は、最初のレイノルズらによる神経幹細胞の同定の研究に記載された。37。その後、ギブスら38たちこれらの研究edは骨肉腫から、特に、固形腫瘍からのCSCを単離することを開始します。私たちは、ギブスらによって示されている球形成アッセイ法を使用することを決定した。従来のOSの生検から得られたOSA細胞株からのCSCを隔離します。我々は、このアッセイの結果を向上させ、他の癌細胞株のための再現性を容易にするための独自の方法を適合しました。球形成アッセイの確立を参照すると、我々は、40,000細胞/ウェルをプレーティングすることが確認され、アッセイの開始時に分離して細胞を維持するための良い方法です。このトリックは、球状コロニーが細胞凝集からではなく、非付着条件下で増殖し、球状のコロニーを形成するための単一のCSCの特定と排他的な能力に由来する可能性を回避するために非常に重要です。この能力は、このアッセイの特に重要な点です。 また、球形成の良好な速度を得るためにそれを認定します、元の方法に記載のように増殖因子のアリコートごとに3次元ではなく、毎日更新するのに十分です。本研究では、我々はまた、確立され、広範囲に非接着条件下で培養したときに形成される球状のコロニーを単離するための良い方法を説明しました。それらを損傷することなく、各ウェル内に形成されている可能な限り球のような多くを隔離しようとすることは非常に重要であるため、この手順は、このアッセイにおいて重要です。唯一の球ではなく、アッセイの期間中、懸濁液中に残っている可能性があり、単一の細胞を単離することも重要です。これらの重要な点を克服するために、我々は、CSCの単離のための良好な結果が得られ、上記のように、特定の分離法を開発しました。明らかに、そこに形成された全ての球を回収することができない可能性があるが、損失率は非常に低いです。実際に、我々はまた、（フォーム大きくなった後に球を単離するために40μmの孔を有するフィルターを使用する可能性を有していますEDは約100から200細胞）。 この分離は、球形成を停止しますが、単一細胞、メチルセルロース残基の一部、および最小の球をフィルタリングすることができます。プロトコールに記載されるように、この除去は、完全な濾過により行われます。 また、最小の球状コロニーの必然的な損失と40μmのメッシュを介して最大の球状コロニーの選択は1つが球状コロニーを形成する最大容量で、より大きな幹細胞性とのCSCを選択することができます。これらの修飾の全ては、アッセイを改善し、理解し、球体形成アッセイの元の方法の最も重要なステップを再現するためのCSCを研究する研究者を支援するために行きました。 CSCを単離するためのin vitro法に関する研究の中で、これは球形成アッセイを適応方法を示すことを目的としたこの研究からのCSCを単離するための良い方法かもしれませんOSAの細胞株。オリジナルの方法と詳細な分離技術への適応は、その有効性を改善します。短時間でのCSCのかなりの数を得ることができ、いくつかの実験のために使用されます。したがって、迅速にOS-のCSCを特徴付ける二幹様の表現型を研究するために、特に、幹様の表現型を確認し、することが可能です。したがって、この修正されたアッセイは、CSCをを単離し、それらの生物学を研究するための優れた手法である可能性があります。将来的には、この方法は、追加の適応でも希少な固形腫瘍の生検により得られた他の有限の癌細胞株からのCSCを単離するために使用されてもよいです。 OSなど希少な固形腫瘍からのCSCを単離する可能性は、この特定の癌に関する研究の改善を可能にするだけでなく、それらの単離のためのより良い方法を開発する異なるタイプの癌の研究にとの生物学の今後の研究にも及ぶだけでなく、この重要な細胞サブセット。したがって、我々として本研究で行っている、おそらくのために責任があるこの特定の細胞サブセットに対して向けの非常に特定の抗癌治療を、分子標的を発見し、開発の最終目標で、CSC生物学の研究を通じてCSC分離の方法を向上させることが重要です原発腫瘍の維持、再発の開発、およびいくつかの器官における転移の起源。 CSC生物学の研究でも、このようなネオアジュバント治療後の生存率は非常に貧しいままであるためにOS、などの癌の治療法に鋭いかもしれない治療法を見出すために重要です。

肉腫は胚の中胚葉1から主に由来するまれな悪性の結合組織腫瘍の異種群です。異なるタイプの骨肉腫および軟部組織肉腫を含みます。骨肉腫は、比較的まれ原発腫瘍のグループは、骨肉腫（OS）を含むいくつかのサブタイプ、で構成されています。 OS、骨の最も一般的な原発腫瘍の一つは、大規模な臨床、組織学的、および分子不均質2、3を示す間葉系悪性腫瘍である。残念ながら、OSは5、子供におよび若年成人4で主に発生し、60％を占めます小児期における骨肉腫の一般的な組織学的サブタイプは6、7。OSは通常影響を与える急速な骨の成長によって特徴付けられる骨格領域、（ 例えば 、長骨の骨幹）。 OS、従来のOSの組織学的に異なるサブタイプの中でも、また髄または中央OSと呼ばれる、悪性度の高いグレードとクォータ社を持っています80％8の再。この80％、60％、従来の骨芽細胞のOS、10％軟骨芽OS、および10％の線維芽OS 6、8-10から構成されています。他のOSのサブタイプは、未分化、毛細血管拡張、巨細胞が豊富で小細胞OSが含まれています。 OS管理で組み合わせ、手術と化学療法の進歩にもかかわらず、結果は転移11、12のない患者では65から70パーセントの長期生存率は、貧しいまま。遠い再発が頻繁に転移として、それほど頻繁に、肺転移として発生したり、遠くの骨や地元の再発13へ。転移は、多くの場合、従来の治療に抵抗性です。この抵抗は、10年の無病生存率は診断14、15に転移性疾患を有する患者では約30％である理由です。 正常組織と同様に、癌組織は、細胞型の異種コレクションから構成されています。腫瘍内の細胞は、発生の異なる段階に対応するように見えます。任意のn個以内ormal組織は、このように組織の恒常性のための前駆細胞および成熟細胞を提供し、selfrenewする能力を有する細胞の亜集団を常駐します。同様に、癌が増殖および腫瘍形成能の程度が異なると、発生の異なる段階における細胞の同様の不均一な集団で構成されています。これらの癌細胞のサブセットは、がん幹細胞（CSCを）と呼ばれる、このようにして腫瘍塊16を構成する種々の細胞系統を生成する、腫瘍の悪性度をselfrenewし維持する排他的な能力を有する細胞をselfsustainingのリザーバを構成しています。 1990年代には、急性骨髄性白血病の研究は、CSCの亜集団17、18の存在のための最初の説得力のある証拠を提供した。CSCがあるため、それによって癌研究の中で最も研究テーマの一つになって、固形腫瘍19の多数から単離されています。 CSCが実際に正常を作る遺伝子の変異により正常な幹細胞から生じる可能性があります20-23癌幹細胞。微小環境を持つ複数の転換の突然変異と相互作用ものCSCを代表するselfrenewal容量と不死を取得し、健康な前駆細胞および成熟細胞に寄与し得ます。この変換に関するいくつかの仮説があります。健康な前駆細胞、健康な成熟細胞、癌細胞は、selfrenewal関連遺伝子24-28を活性化することにより幹様表現型を得る、幹細胞に脱分化することができます。いくつかの最近の研究にもかかわらず、CSCをの起源はまだ発見されなければなりません。 CSCの特定の特性は、その容量は異なる薬剤との併用手術と化学療法で構成されたマルチ治療アプローチを、抵抗することです。最近の研究でのCSCはまた、細胞毒性化学療法剤に対する耐性を獲得することができることを示しています。この抵抗のための可能な説明は、ATP結合カセット（ABC）多剤輸送体（ すなわち、の過剰発現を含みますMDR1およびBCRP1）、アルデヒドデヒドロゲナーゼ1（ALDH1）、および/または細胞周期動態30-33の変化として化学療法代謝酵素の過剰発現。これまでに記載されているすべてのこれらの概念の直接的な結果は、CSCの亜集団が完全に排除された場合にのみ、1つでもCSCが生き残った場合は局所再発や遠隔転移が発生する可能性がありながら、癌治療は、効率的であるということです。 それは多くの治療が最初に有効であると思われる理由として可能性のある説明が、患者後に再発していますので、人間の肉腫34、特にOS 35で、または任意の他の骨と軟部組織のがんでのCSCの発見は、大きな臨床的重要性を持っています。そのため、従来のOSに対する今後の戦いのための希望は、このサブの分子特性にOS-のCSCのおかげに向けた革新的な医薬品の開発に基づいて、新しいと特定の標的療法を見つけることです人口とCSC生物学の研究に。 1992年には、幹細胞のサブセットは、成人哺乳類の脳に存在したかどうかを調査したレイノルズらは、幹細胞様細胞36、37であることが疑われる細胞を分離する方法を開発した。この方法は、特定の能力に基づいています非付着条件下で増殖するとき、これらの細胞は球状のコロニーを形成します。同様の技術は、骨肉腫38中の幹細胞様細胞の亜集団を研究するために2005年にギブスらによって採用されました。従来のOSの異なるタイプの初代細胞培養物から、OSのCSCを単離し、特徴づけるために、我々は、OS細胞株は、この技術を適応することを決定しました。 ここでは、球体形成アッセイのこの適合方法を説​​明し、従来のOSのヒト生検に由来する有限の初代細胞株からOS-のCSCを単離することができる「sarcosphereアッセイ」と呼ばれます。我々はまた、すべての技術のuを記述しますこのアッセイにより単離された細胞株の幹様CSC表現型を検証するためのsed：多能性胚性幹細胞（ESC）を特徴づける遺伝子とのCSCのマーカーであるCD133遺伝子の発現の1）の評価を、 2）コロニー形成単位（CFU）アッセイ。 3）適切な分化条件下での骨芽細胞と脂肪細胞に分化するために、これらの細胞の能力を評価します。間葉系幹細胞（MSC）の表面マーカーの4）研究（ すなわち、CD44、CD105及びSTRO-1）の免疫蛍光染色により、フローサイトメトリー分析によって、 5）これらの細胞のALDH活性を評価します。

establishment of cancer stem cell cultures from human conventional osteosarcoma

骨肉腫（OS）に対する治療における現在の改善は、癌患者の生命を延長しているが、転移が発生した場合に5年の生存率は悪いまま。癌幹細胞（CSC）理論は、腫瘍を維持し、多剤化学療法に抵抗する能力を含む幹様特性を有する腫瘍内の腫瘍細胞のサブセットが存在することを保持します。したがって、OS生物学および病因論のより良い理解は、この特定のサブセットを根絶し、患者の間で罹患率および死亡率を減少させるために標的治療の開発を進めるために必要とされます。 、のCSCの単離のCSCの細胞培養を確立し、それらの生物学を研究すると、OSの生物学および病因の我々の理解を改善するための重要なステップです。 OSの生検からのヒト由来のOS-のCSCの確立がnonadhere下3次元幹細胞培養物を作成する能力を含むいくつかの方法を用いて可能になりましたNT条件。これらの条件下では、CSCを娘幹細胞によって形成された球状の浮遊コロニーを生成することができます。これらのコロニーは、「細胞球」と呼ばれます。ここでは、OSの生検から得られた従来のOSの初代細胞培養物からのCSCの培養物を確立するための方法を記載しています。我々は明らかにのCSCを分離し、特徴付けるために必要ないくつかの通路を説明します。

Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

骨肉腫における癌幹細胞（CSCを）の存在は、最近、その病因に関連しています。本稿では、非接着性条件下で増殖するのCSCの能力を用いて、従来の骨肉腫（OS）の人間の生検から得られた初代細胞培養物からのCSCの単離を提示します。

がんの確立は、ヒト従来の骨肉腫からの幹細胞培養物

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

establishment-cancer-stem-cell-cultures-from-human-conventional

Research

JoVE Journal

Cancer Research

18.9K ビュー.  University of Florence. 骨肉腫における癌幹細胞（CSCを）の存在は、最近、その病因に関連しています。本稿では、非接着性条件下で増殖するのCSCの能力を用いて、従来の骨肉腫（OS）の人間の生検から得られた初代細胞培養物からのCSCの単離を提示します。 

ビデオ: Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

腫瘍と間葉系幹細胞の間細胞小胞を介した通信を定義する骨肉腫のマウスの前臨床モデル

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

がん研究

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

李 Fraumeni 症候群患者由来の誘導多能性幹細胞を用いた骨肉腫をモデリング

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

後根神経節軸軸索-オリゴデンドロサイト共培養におけるヒトグリオーマ細胞移動のリアルタイムモニタリング

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

胃癌患者由来異種移植片モデルおよび原発細胞株の確立

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

婦人科腫瘍および乳がん腫瘍から癌幹細胞球体を取得する

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

3次元スフェロイド培養からの幹様細胞の分離

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

アデノ関連ウイルス-Cas9システムを用いた遺伝子操作マウス頭頸部癌細胞株のインビトロ確立

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

大腸癌細胞の3次元スフェロイド培養におけるプロバイオティクスの細胞遊離上清を用いた細胞死の評価

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

アペレットヒト産物からのガンマデルタ(γδ)T細胞の増殖と濃縮

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...