Name: in vitro enrichment of ovarian cancer tumor-initiating cells
Uploaded: 2015-02-18T14:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells at JoVE.com

We thank Dr. John Risinger and Memorial Health University Medical Center, Inc. for generously providing the ACI-23 cell line. The authors acknowledge financial support through the Postdoctoral Research Training (PRAT) Award to C. House from The National Institute for General Medical Sciences.

卵巢癌细胞系1.传统文化（贴壁条件） 注:细胞和培养基的所有处理应该在无菌组织培养罩<ol><li>准备传统的细胞培养基。使用1:1的DMEM:F12（+ L-谷氨酰胺，+ 15毫米的HEPES）和补充有10％热灭活的胎牛血清和1％青霉素/链霉素。使用0.45微米真空过滤机的过滤方案。 </li><li>制备传统的聚苯乙烯75厘米2组织培养瓶interest.Remove的细胞系细胞从超低冷冻或液氮小瓶的标记的名称，日期，和通道号。通过放置在37℃水浴5分钟除霜。 </li><li>从小瓶转移悬浮液到装有3毫升的传统的细胞培养介质（上面制备的）15ml离心管中。在4℃离心悬浮液5分钟，于400×g下。同时，加16毫升传统细胞培养基到烧瓶中。 </li><li>吸上清。使用2毫升传统的细胞培养基的细胞吸管上下松动沉淀并转移至悬浮液的烧瓶中。轻轻晃动烧瓶均匀分布cells.Place烧瓶在加湿细胞培养孵化器设置为37℃和5％的CO 2。每天监测细胞，直到80％汇合为止。 </li><li>一旦各信元80％汇合分裂培养1:在细胞培养罩2。吸去上清液，并用温水磷酸盐缓冲盐水（无钙和镁）（PBS）中。添加1.5毫升胰蛋白酶0.25％-EDTA并培育3-5分钟在室温。 </li><li>准备2个新的聚苯乙烯75厘米2烧瓶，并添加16毫升传统细胞培养基到每个烧瓶中。悬浮细胞胰酶消化4毫升传统的细胞培养基和分装等体积每一个新瓶中。 </li><li>在加湿细胞培养孵化器的地方瓶中设定为37℃和5％的CO 2。每天监测细胞，直到80％汇合为止。继续分裂细胞培养物或冷冻并储存在-80或在液氮中。 </li></ol> 2.代多细胞球体从卵巢癌细胞系采用浮动或贴壁细胞注:细胞和培养基的所有处理应该在无菌组织培养罩。培养下，传统的培养方法2株后 - 3通道（第1节）继续进行下面的步骤。 <ol><li>准备干细胞培养基。使用1:1的DMEM:F12（+ L-谷氨酰胺，+ 15毫米的HEPES）中，用1％青霉素/链霉素补充（以100单位的终浓度/ ml青霉素和100μg/ ml链霉素），1％敲除血清替代品，0.4％牛血清白蛋白，和0.1％胰岛素 - 转铁蛋白 - 硒。使用0.45微米真空过滤机的过滤方案。 </li><li>补充干细胞培养基与人重组表皮生长因子（EGF）和人重组碱性成纤维细胞生长因子（FGF）为20毫微克/毫升和10 N的最终浓度克/毫升。 注意:生长因子应在使用前右侧加入新鲜的干细胞媒体。 </li><li>创建浮子TIC文化。除去传统的贴壁培养条件下生长的80％的融合细胞瓶。收集媒体和所有的漂浮细胞（单和聚合），身后留下贴壁的人口，并转移至50ml聚丙烯离心管中。在4℃离心悬浮液5分钟，于400×g下。 注:浮动细胞和聚集是显而易见的24 - 72小时后贴壁细胞附着于传统文化下的烧瓶中。从约1.0 80％汇合板检索- 5.0×10 5个活漂浮细胞可以预计，这取决于细胞系。在这项研究中，从80％汇合的贴壁培养物中收集漂浮细胞的平均数目为5.0×10 5，用于ACI-23，4.8×10 5，用于OVCAR5，3.5×10 5，用于CAOV3，和1.0×10 5，用于TGS- 3。转让时，多节ular球体的形成在传统的和漂浮物TIC培养发生在几天之内，尽管这是多少有些细胞系依赖。例如，ACI-23细胞形成聚集体比更容易CAOV3细胞。 </li><li>同时，准备一个超低附着表面聚苯乙烯75cm 2的组织培养瓶标有名称，日期，和细胞系的传代次数。加入10 mL干补充有生长因子的烧瓶细胞培养基。 </li><li>一旦离心完成后，吸出培养基并转移到粒料低附着烧瓶中加入6毫升干细胞培养基和上下吹打松开粒料。在加湿细胞培养孵化器的地方瓶中设定为37℃和5％的CO 2。监测细胞每隔2 - 3天观察球体形成。 </li><li>打造传统文化的TIC。除去传统的贴壁培养条件下生长的80％的融合细胞瓶。吸媒体和用温水洗净PBS。加入1.5 ml胰蛋白酶的0.25％EDTA，并培育3 - 5分钟RT。 </li><li>制备超低附着表面聚苯乙烯75cm 2的组织培养瓶标有名称，日期，和细胞系的传代次数。加入10 mL的干细胞培养基，补充EGF和FGF，如上所述，到该烧瓶中。 </li><li>添加6毫升干细胞培养基与胰蛋白酶的烧瓶中。吸管的细胞溶液上下并尝试从烧瓶表面松开的细胞。转移细胞的整个体积含有10毫升干细胞介质的超低附着烧瓶中。在加湿细胞培养孵化器代替烧瓶设定为37℃和5％的CO 2。 </li><li>补充TIC培养，每48 - 72小时用另外2毫升，干细胞培养基和新鲜的EGF和FGF为20纳克/毫升的最终浓度和10ng / ml的分别。 </li><li>监测细胞直到60 - 80％汇合为止。分裂培养物通过将等体积的2到新超低附着培养瓶并加入新鲜的干细胞培养基以达到在18 ml的终体积。替代LY，离心机在4℃下，整个悬浮液5分钟，于400×g下。悬浮细胞在干细胞培养基补充EGF和FGF和分发同样进入新的超低附件瓶。细胞没有被解离成单细胞悬浮液传代。 注意:此时的培养物可以继续分裂，并进行培养，冷冻，或实验操纵进行分析。在60 - 80％汇合进行检索的ACI-23细胞的以下浓度:8.0×10 6个为传统粘附，4.0×10 6，用于传统TIC，和5.0×10 6个为浮TIC培养物。虽然这可能是细胞系相关的，我们已经发现，5 - 10天培养是最适合于TIC富集为CD133 + ALDH +细胞的百分比内的第3天的低，峰值在5 - 10天，再次减小在14天。 </li></ol> 3.代上皮性卵巢癌的细胞株和多细胞球体从化疗耐药患者腹水此协议获得机构审查委员会的批准:注意。细胞和培养基的所有处理应该在无菌组织培养罩。 <ol><li>板腹水液1:1与传统的细胞培养介质。如第1节准备的媒体和加入10毫升的几个传统的聚苯乙烯75cm 2的组织培养瓶。 </li><li>腹水通常在1L无菌真空瓶提供;除去真空盖。小心加入10 mL腹水流体烧瓶含有10ml传统媒体和放置在潮湿的细胞培养箱中设为37℃和5％的CO 2。离开静置72 - 96小时，做一个完整的媒体变化之前。更换介质每2 - 3天，直到细胞达到60％ - 80％汇合</li><li>如在第2节第1节开始TIC文化描述2: - 一旦细胞达到60 80％汇合，拆分1。 注:红细胞出现在ascitES将第一媒体的变化后，因为它们是非粘附除去。存在的任何的成纤维细胞将用胰蛋白酶处理后在很大程度上除去，因为它们是更敏感，将使以最小暴露于胰蛋白酶。卵巢癌细胞的纯度通过流式细胞分析使用抗体对CA-125（MUC16）测定。 </li></ol> 4.染色细胞TIC标记流式细胞仪的确认<ol><li>收集TIC文化。转移烧瓶内容物到50ml聚丙烯离心管中。离心悬浮液5分钟，于400×g下。吸媒体和用温水洗净PBS。加2ml非酶细胞解离溶液，孵育3 - 5分钟，在37℃，并且吸管上下分手团块和离解成单细胞。添加4毫升干细胞培养基。 </li><li>收集贴壁培养。吸媒体和用温水洗净PBS。添加3毫升非酶细胞解离溶液孵育3 - 5分钟，在37℃。加3毫升水传统的细胞培养基和吸管上下反复从瓶中分离细胞，并收集到离心管中。 </li><li>离心机均TIC和贴壁悬浮液5分钟400×克。弃上清，重悬在1ml的PBS含有3％胎牛血清每个粒料。在室温下孵育30分钟。与此同时，计数用台盼蓝活力细胞的数目。观察显微镜下的细胞，以确认球状体解离成单细胞。 </li><li>标签5×1.5毫升Eppendorf管用于每个以下培养条件:1）未染色的（未染色的对照补偿），2）的ALDH（单个染色的阳性对照补偿），3）的CD133（单染色阳性对照补偿），4 ）ALDH + CD133（实验测试样品），和5）DEAB + ALDH（对于背景荧光阴性对照在FL-1和FL-4通道，分别）。 </li><li>分发各约5×10 5个细胞于管＃1 - 4离心悬浮液5分钟，于400×g下。吸supernataNT和颗粒重悬在500微升ALDEFLUOR缓冲液。添加10微升DEAB（ALDH抑制剂）至管＃5，用于实验的阴性对照。 </li><li>添加5微升激活ALDEFLUOR试剂管＃2赔偿阳性对照，一抖混合。添加5微升激活ALDEFLUOR试剂管＃4实验分析。弗里克管混合，并立即从管＃4转移细胞的体积的一半到管＃5（250微升），其中包含了DEAB。 </li><li>弗里克管拌匀。孵育所有试管30 - 45分钟，37℃避光。弗里克管中途潜伏期为细胞将沉淀在底部。离心所有管5分钟，在400×g下。吸上清和悬浮细胞在500微升ALDEFLUOR缓冲液。 </li><li>对于与CD133染色管（＃3和＃4）中，添加CD133-APC 1点11分，直接进入ALDEFLUOR测定缓冲液。在冰上孵育避光15分钟。离心机的CD133管5分钟，在400×g下。用洗一次将500μlALDEFLUOR测定缓冲液。重悬的细胞在500μlALDEFLUOR测定缓冲液，分别</li><li>过滤悬浮成单个聚苯乙烯圆底管与细胞滤网盖。确保整个体积是通过帽过滤。 </li><li>利用流式细胞仪来分析细胞群。用管＃1-3设定补偿控制。使用前向和侧向散射参数，门的细胞，一定要排除细胞碎片。建立ALDH +和CD133 +使用FL-1和FL-4通道，分别为双细胞。 </li><li>使用流式细胞分析程序，确认培养条件增强TIC细胞的TIC丰富文化的百分比应该有这两个标记更高染色。 注意:除了CD133和乙醛脱氢酶，其他markersof卵巢癌国际信托可以用在任何一个样品依赖于流式细胞仪的功能中使用的标记的数量进行分析。 </li></ol> 5. 在体内 </eM&gt;皮下注射，以确认细胞群的致瘤性此协议获得机构审查委员会的批准:注意。分发无胸腺雌性裸鼠6 - 8周龄入实验组中的3笼，并允许前注射适应环境几天。按照下人性化的实验指南按NIH动物护理和使用委员会小鼠;监测体重，身体外观包括体重减轻或获得大于20％，瘦弱的外观，腹泻或皮炎和安乐死的小鼠用CO 2窒息拟合这些标准。 <ol><li>如1毫升的PBS在第2悬浮每个细胞群描述收集文化。使用计数台盼蓝法活细胞。 </li><li>制备15ml离心管中。为一式三份的措施添加稀释在2ml PBS中1.5×10 6个活细胞到每个管（5×10 5个细胞在0.5毫升的体积将被皮下注射到正确f每只小鼠的平直）。准备0.5毫升PBS只为对照组。 </li><li>使用27G的针头，subcutaneouslyinject从传统的贴壁培养物，传统的TIC培养0.5毫升细胞悬浮液，漂浮物TIC文化，或PBS到各每组3只小鼠的右胁。返回小鼠到原来的笼子后喷射。 </li><li>称重小鼠并测量任何可触知的肿瘤的两维两次weeklyby游标卡尺，确立的长度和宽度。 注:肿瘤延迟时间通常为20 - 50天，取决于细胞系。 </li><li>计算肿瘤体积的湿通过标准方法（V =（宽度）2×长度/ 2）。 注意:虽然本研究比较了国际信托对粘附细胞为第一代仅在体内串行相似条件下生长的抽动传代已经由他人11,20完成的致瘤性。 </li></ol>

<ol>
	<li>Coleman, R. L., Monk, B. J., Sood, A. K., Herzog, 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=Latest+research+and+treatment+of+advanced-stage+epithelial+ovarian+cancer.">Latest research and treatment of advanced-stage epithelial ovarian cancer.</a> Nat Rev Clin Oncol. 10, 211-224 (2013).</li><li>Ushijima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+for+recurrent+ovarian+cancer-at+first+relapse.">Treatment for recurrent ovarian cancer-at first relapse.</a> J Oncol. 2010, 497429 (2010).</li><li>Erickson, B. K., Conner, M. G., Landen, C. N. <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+the+fallopian+tube+in+the+origin+of+ovarian+cancer.">The role of the fallopian tube in the origin of ovarian cancer.</a> Am J Obstet Gynecol. 209, 409-414 (2013).</li><li>King, S. 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=The+impact+of+ovulation+on+fallopian+tube+epithelial+cells:+evaluating+three+hypotheses+connecting+ovulation+and+serous+ovarian+cancer.">The impact of ovulation on fallopian tube epithelial cells: evaluating three hypotheses connecting ovulation and serous ovarian cancer.</a> Endocr Relat Cancer. 18, 627-642 (2011).</li><li>Flesken-Nikitin, 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=Ovarian+surface+epithelium+at+the+junction+area+contains+a+cancer-prone+stem+cell+niche.">Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche.</a> Nature. 495, 241-245 (2013).</li><li>Paik, D. 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=Stem-like+epithelial+cells+are+concentrated+in+the+distal+end+of+the+fallopian+tube:+a+site+for+injury+and+serous+cancer+initiation.">Stem-like epithelial cells are concentrated in the distal end of the fallopian tube: a site for injury and serous cancer initiation.</a> Stem Cells. 30, 2487-2497 (2012).</li><li>Connor, M. 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=Cancer+stem+cells:+A+contentious+hypothesis+now+moving+forward.">Cancer stem cells: A contentious hypothesis now moving forward.</a> Cancer Lett. 344, 180-187 (2014).</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=Tumour+stem+cells+and+drug+resistance.">Tumour stem cells and drug resistance.</a> Nat Rev Cancer. 5, 275-284 (2005).</li><li>Bapat, S. A., Mali, A. M., Koppikar, C. B., Kurrey, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+progenitor-like+cells+contribute+to+the+aggressive+behavior+of+human+epithelial+ovarian+cancer.">Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer.</a> Cancer Res. 65, 3025-3029 (2005).</li><li>Szotek, P. 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=Ovarian+cancer+side+population+defines+cells+with+stem+cell-like+characteristics+and+Mullerian+Inhibiting+Substance+responsiveness.">Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.</a> Proc Natl Acad Sci U S A. 103, 11154-11159 (2006).</li><li>Zhang, 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=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68, 4311-4320 (2008).</li><li>Wang, L., Mezencev, R., Bowen, N. J., Matyunina, L. V., McDonald, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+stem-like+cells+from+a+human+ovarian+cancer+cell+line.">Isolation and characterization of stem-like cells from a human ovarian cancer cell line.</a> Mol Cell Biochem. 363, 257-268 (2012).</li><li>Kryczek, I., 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=Expression+of+aldehyde+dehydrogenase+and+CD133+defines+ovarian+cancer+stem+cells.">Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells.</a> Int J Cancer. 130, 29-39 (2012).</li><li>Meng, E., 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=CD44+/CD24-+ovarian+cancer+cells+demonstrate+cancer+stem+cell+properties+and+correlate+to+survival.">CD44+/CD24- ovarian cancer cells demonstrate cancer stem cell properties and correlate to survival.</a> Clin Exp Metastasis. 29, 939-948 (2012).</li><li>Alvero, A. 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=Molecular+phenotyping+of+human+ovarian+cancer+stem+cells+unravels+the+mechanisms+for+repair+and+chemoresistance.">Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance.</a> Cell Cycle. 8, 158-166 (2009).</li><li>Chen, J., 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=Evaluation+of+characteristics+of+CD44+CD117++ovarian+cancer+stem+cells+in+three+dimensional+basement+membrane+extract+scaffold+versus+two+dimensional+monocultures.">Evaluation of characteristics of CD44+CD117+ ovarian cancer stem cells in three dimensional basement membrane extract scaffold versus two dimensional monocultures.</a> BMC Cell Biol. 14, 7 (2013).</li><li>Guo, R., Wu, Q., Liu, F., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+the+CD133++subpopulation+of+the+human+ovarian+cancer+cell+line+OVCAR3.">Description of the CD133+ subpopulation of the human ovarian cancer cell line OVCAR3.</a> Oncol Rep. 25, 141-146 (2011).</li><li>Curley, M. 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=CD133+expression+defines+a+tumor+initiating+cell+population+in+primary+human+ovarian+cancer.">CD133 expression defines a tumor initiating cell population in primary human ovarian cancer.</a> Stem Cells. 27, 2875-2883 (2009).</li><li>Baba, 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=Epigenetic+regulation+of+CD133+and+tumorigenicity+of+CD133++ovarian+cancer+cells.">Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells.</a> Oncogene. 28, 209-218 (2009).</li><li>Liao, J., 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=Ovarian+cancer+spheroid+cells+with+stem+cell-like+properties+contribute+to+tumor+generation,+metastasis+and+chemotherapy+resistance+through+hypoxia-resistant+metabolism.">Ovarian cancer spheroid cells with stem cell-like properties contribute to tumor generation, metastasis and chemotherapy resistance through hypoxia-resistant metabolism.</a> PLoS One. 9, e84941 (2014).</li><li>Silva, I. 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=Aldehyde+dehydrogenase+in+combination+with+CD133+defines+angiogenic+ovarian+cancer+stem+cells+that+portend+poor+patient+survival.">Aldehyde dehydrogenase in combination with CD133 defines angiogenic ovarian cancer stem cells that portend poor patient survival.</a> Cancer Res. 71, 3991-4001 (2011).</li><li>Jaggupilli, A., Elkord, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+CD44+and+CD24+as+cancer+stem+cell+markers:+an+enduring+ambiguity.">Significance of CD44 and CD24 as cancer stem cell markers: an enduring ambiguity.</a> Clin Dev Immunol. 2012, 708036 (2012).</li><li>Irollo, E., Pirozzi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133:+to+be+or+not+to+be,+is+this+the+real+question.">CD133: to be or not to be, is this the real question.</a> Am J Transl Res. 5, 563-581 (2013).</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, 292-306 (2011).</li><li>McLean, 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=Human+ovarian+carcinoma&#8211;associated+mesenchymal+stem+cells+regulate+cancer+stem+cells+and+tumorigenesis+via+altered+BMP+production.">Human ovarian carcinoma&#8211;associated mesenchymal stem cells regulate cancer stem cells and tumorigenesis via altered BMP production.</a> J Clin Invest. 121, 3206-3219 (2011).</li><li>Amara, I., Touati, W., Beaune, P., de Waziers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+cellular+vehicles+for+prodrug+gene+therapy+against+tumors.">Mesenchymal stem cells as cellular vehicles for prodrug gene therapy against tumors.</a> Biochimie. , (2014).</li><li>Brabletz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMT+and+MET+in+metastasis:+where+are+the+cancer+stem+cells.">EMT and MET in metastasis: where are the cancer stem cells.</a> Cancer Cell. 22, 699-701 (2012).</li><li>Latifi, 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=Isolation+and+characterization+of+tumor+cells+from+the+ascites+of+ovarian+cancer+patients:+molecular+phenotype+of+chemoresistant+ovarian+tumors.">Isolation and characterization of tumor cells from the ascites of ovarian cancer patients: molecular phenotype of chemoresistant ovarian tumors.</a> PLoS One. 7, e46858 (2012).</li><li>Liu, 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=Breast+Cancer+Stem+Cells+Transition+between+Epithelial+and+Mesenchymal+States+Reflective+of+their+Normal+Counterparts.">Breast Cancer Stem Cells Transition between Epithelial and Mesenchymal States Reflective of their Normal Counterparts.</a> Stem Cell Reports. 2, 78-91 (2014).</li><li>Malecki, 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=TRA-1-60(+),+SSEA-4(+),+Oct4A(+),+Nanog(+)+Clones+of+Pluripotent+Stem+Cells+in+the+Embryonal+Carcinomas+of+the+Ovaries.">TRA-1-60(+), SSEA-4(+), Oct4A(+), Nanog(+) Clones of Pluripotent Stem Cells in the Embryonal Carcinomas of the Ovaries.</a> J Stem Cell Res Ther. 2, (2012).</li><li>Malecki, M., Tombokan, X., Anderson, M., Malecki, R., Beauchaine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> TRA-1-60(+), SSEA-4(+), POU5F1(+), SOX2(+), NANOG(+) Clones of Pluripotent Stem Cells in the Embryonal Carcinomas of the Testes. J Stem Cell Res Ther. 3, 10-4172 (2013).</li><li>Rajasekhar, V. K., Studer, L., Gerald, W., Socci, N. D., Scher, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-initiating+stem-like+cells+in+human+prostate+cancer+exhibit+increased+NF-&#954;B+signalling.">Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-&#954;B signalling.</a> Nat Commun. 2, 162 (2011).</li><li>Loh, Y. 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=The+Oct4+and+Nanog+transcription+network+regulates+pluripotency+in+mouse+embryonic+stem+cells.">The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.</a> Nat Genet. 38, 431-440 (2006).</li><li>Tay, Y., Zhang, J., Thomson, A. M., Lim, B., Rigoutsos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+to+Nanog,+Oct4+and+Sox2+coding+regions+modulate+embryonic+stem+cell+differentiation.">MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation.</a> Nature. 455, 1124-1128 (2008).</li><li>Lee, 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=Prognostic+impact+of+the+cancer+stem+cell-related+marker+NANOG+in+ovarian+serous+carcinoma.">Prognostic impact of the cancer stem cell-related marker NANOG in ovarian serous carcinoma.</a> Int J Gynecol Cancer. 22, 1489-1496 (2012).</li></ol>

The authors have nothing to disclose.

此处所描述的协议提供了一个有效的和一致的富集培养物为细胞从既定卵巢癌细胞系干细胞的特性的方法，并且适用于初级患者样品。这种方法成功地富集用于在各种细胞系国际信托。它可以及时识别，丰富的TIC和/或致瘤表型的条件下，没有考虑时间从内人口非TICS资本流动的TICS资本流动物理排序。以这种方式，不同的信号传导途径的相对水平可以评估其通过比较传统粘附培养到TIC使用各种功能测定培养物的TIC表型贡献。如果纯TIC人口是期望的，隔离可以容易地通过将在流式细胞协议的流程的荧光辅助或磁分选步骤实现的。 它，要记住，但是这是非常重要的TIC标志物的表达S，如CD133，CD44，或ALDH，可能其中的细胞系或患者样本甚至相同肿瘤类型的不同而不同。例如，我们发现，OVCAR-5细胞具有CD44的相对于CD133的高表达，而逆为真ACI-23。因此，有针对性依靠小鼠肿瘤发生和流式细胞仪分析，最终的TIC存在。按照此协议，高ALDH活性物一致时观察到卵巢癌细胞生长在干细胞的条件。有趣的是，CD133的表达是在传统文化的TIC条件下生长的ACI-23细胞持续走高，而ALDH最高的是浮子TIC条件。相比之下，TGS-3主要腹水细胞显示CD133阳性小幅增加浮子TIC条件下，但在ALDH活性传统TIC条件下显着增加。这些发现强调卵巢癌细胞的异质性，而且经常抽动关联的可塑性。为了进一步支持劳委会即时通讯，这些方法提高抽搐，也观察到TRA-1-60阳性细胞富集。 TRA-1-60是一种多能性标记物，并已被用于识别卵巢胚胎癌和睾丸以及前列腺癌TICS 30-32。虽然我们的方法已被成功地与众多的细胞系，这是可能的，标准的TIC条件可能更适合于一些卵巢癌细胞，而修改后的浮动条件更好的为在其他卵巢癌的细胞群国际信托充实。这再次强调了两个病人衍生的和建立的卵巢癌细胞系中的预期的异质性。 除了 ​​细胞表面标记有已被证明以表征卵巢癌国际信托包括Nanog的，SOX2，Oct-4的11多种转录因子，尽管这些标记是不特定于卵巢癌抽动和相当代表因子为胚胎干细胞多能性的重要和DIFferentiation 33,34。我们研究了改变这些因素的蛋白水平，发现Nanog的水平在TIC培养条件增加，在与他人13,35以及CD133，TRA-1-60，和CD117的协议。一个人类干细胞标记基因芯片进一步表明相对于传统的附着条件增加CD133，Nanog的，梅尔克和PODXL基因在TIC条件。重要的是，应该指出的是，作为与细胞表面标记物，与卵巢国际信托相关的转录因子可之间的细胞系和患者样品而异。 我们已观察到，细胞可以是更致瘤性和表达更高水平的干细胞标记物，如果他们是固有非粘附在体外 （ 即漂浮细胞不容易附着到粘附片）。在培养细胞系如ACI-23通常具有下传统文化康迪特生长垂直于堆叠方式许多可行的漂浮细胞和/或细胞离子。虽然从漂浮细胞和聚集抽动成功富集可能适用于相对少的细胞系，包括那些在本研究和OVCAR-3 12，我们的研究结果表明，这可能代表了一种更致瘤的人口。因此，收获培养在标准组织培养板和介质可作为TIC富集的替代方法，对于细胞适应不良商业干细胞培养基的细胞的"浮动"人口。 使用本稿件提出不同的培养方法将使TIC人群的快速致富，并更好地了解是什么因素支持这种表型在不同的细胞。这种方法的当前应用包括表征该信号通路是至关重要，以支持细胞的这种独特的人口的繁殖。这些研究结果将有助于澄清肿瘤进展和复发的机制。

卵巢癌是最致命的妇科恶性肿瘤在美国的发病率和死亡率是本病的高复发，化疗耐药特点的主要原因。初级处理通常包括最大减灭术和随后的基于铂的治疗，虽然有一些证据表明新辅助治疗可能是有益的在某些情况下，1。广大患者积极响应基层处理，但遗憾的是上半年将在18个月2内复发。 大多数卵巢恶性肿瘤是上皮癌，可能起源于卵巢或输卵管3,4的表面上皮。几项研究支持体干细胞在雌性生殖系统中大概有助于所需要以下排卵5,6组织修复的存在。在两个过程中ovul卵巢和输卵管的高增殖活性ATION可能是在卵巢癌的发展的一个重要因素（Gharwan 等人提交的手稿）。肿瘤形成细胞的推导是不明的，但它们可以通过使它们无法调节除法或命运突变源自正常干细胞，祖细胞，或分化细胞。这些细胞也被称为"肿瘤干细胞"或"肿瘤起始细胞"，并能长成低附着条件下致瘤，多细胞球状体。尽管TIC发展的层次模型可以是动态的，信托之间有很多共同的相同的功能，正常干细胞，包括静止，化疗耐药，长期的自我更新和分化成各种细胞谱系7,8能力。 几个研究支持抽动存在于卵巢癌和当前正在努力以澄清机制（多个），以使这些细胞支持肿瘤9-11。几种标记物已被提出，以确定卵巢国际信托具有增强的致瘤性，包括CD133，ALDH1A1，CD117，CD44，和MyD88的，虽然每个标志物的确切贡献是不清楚，可能是特定11-16细胞类型。而通用的标记物或一组标志物还没有得到明确证实卵巢抽搐不同群体已通过选择CD44 +，CD133 +和/或细胞以高醛脱氢酶（ALDH）活性13,17-21分离卵巢国际信托更常见。 CD44是一种跨膜糖蛋白，其充当受体透明质酸和调节对肿瘤进展，包括粘附，增殖，迁移，血管发生和分化22重要的几个过程。 CD133是一种跨膜糖蛋白，其功能目前还不清楚，但研究表明，它组织细胞膜拓扑23。 ALDH，其催化醛氧化的胞内酶，可能是最UNIVERS国际信托作为高活性的人标记已被确定在干细胞从多种组织和多个角色的分离已被归因于ALDH在支持正常干细胞和国际信托24。截至目前，CD133和ALDH1似乎是卵巢TICS资本流动13,21的重现性最好的标记。 除了要了解TICS资本流动的特点，也有大的努力，以确定药物，专门针对这一亚群。与卵巢癌相关的高复发率，可能是由于目前的化疗未能成功地根除国际信托。虽然大部分肿瘤易受现有疗法，信托被认为是耐在不可检测通过标准方法的密度。治疗性肿瘤复发阐明机制，以提高反应和卵巢癌患者生存率的关键。 在这里，养殖技术介绍日在充实，从建立和原发性卵巢癌细胞系TICS资本流动。本文所述的培养条件下已经使用几组来诱导国际信托或球体的细胞与干细胞质量11,12,14,16,20的传播。虽然有几种干细胞培养媒体和补充通常用于富集抽搐/球体，我们使用用EGF和FGF一个无血清培养基配方，但不加B27或N-2补充剂。这些补充剂，通常用于神经元细胞培养和富集干细胞，已显示出促进间质表型25,26和仍然在该领域有一些不确定性，以具有间充质或上皮表型国际信托是否更致瘤性27-29 。为了最大限度地减少不确定性和变数，我们选择使用最常见的公式，因为我们正在处理的上皮卵巢癌细胞。 维持细胞在无血清培养基中的低附着烧瓶便于球体形成并支持细胞与CD133的表达和高ALDH活性的繁殖。我们的工作已进一步表明，细胞正常粘附的条件下浮动也可以代表一个更致瘤的TIC的人口。注射在这些条件下进无胸腺裸鼠中生长的细胞的引出相比生长在所附的条件在血清的存在下的细胞更高的致瘤潜力。关于抽动在卵巢癌起始，进展，治疗反应和疾病复发的作用多的信息可以通过使用这些技术来获得。

<table><tbody><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Ultra - Low Attachment Flask</td><td>Corning</td><td>3814</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Tissue Culture Flask</td><td>Sarstedt</td><td>83.1813.002</td><td></td></tr><tr><td>Aldefluor Kit</td><td>Stemcell Technologies</td><td>1700</td><td></td></tr><tr><td>CD133-APC</td><td>Miltenyi Biotec Inc.</td><td>130-090-826</td><td></td></tr><tr><td>Fibroblast Growth Factor - Basic human recombinant</td><td>Sigma-Aldrich</td><td>F0291</td><td>Prepared according to manufacturer&#039;s instructions</td></tr><tr><td>Epidermal Growth Factor - human recombinant</td><td>Sigma-Aldrich</td><td>E9644</td><td>Prepared by Dissolving 0.2 mg with 1 ml sterile PBS</td></tr><tr><td>DMEM:F12 (+L-glutamine, +5 mM HEPES)</td><td>Gibco</td><td>11330-032</td><td></td></tr><tr><td>1x Dulbecco&#039;s Phosphate-Buffered Saline (without calcium and magnesium)</td><td>Corning cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Cellstripper Non-enzymatic Cell Dissociation Solution</td><td>Corning Cellgro</td><td>25-056-CI</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418-10G</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Gibco</td><td>51500-056</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>10828010</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Fetal Bovine Serum (heat inactivated)</td><td>Gemini</td><td>100-106</td><td></td></tr><tr><td>Rapid-Flow Sterile Dsiposable Filter Units with CN Membrane (0.45 &amp;mu;m)</td><td>Nalgene</td><td>450-0045</td><td></td></tr><tr><td>15 ml Sterile Polypropylene Centrifuge Tube</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>50 ml Sterile Polypropylene Conical Tube</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Trypan Blue 0.4% solution in 0.85% NaCl</td><td>Lonza</td><td>17-942E</td><td></td></tr></tbody></table>

in vitro enrichment of ovarian cancer tumor-initiating cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

建立卵巢癌细胞系和在传统贴壁培养条件下的血清显示附着，鹅卵石形态存在下生长的初级腹水细胞系，而在TIC培养条件下生长的相同细胞显示浮动球体的形态，在TIC群体普遍。在图1中所显示的明场图像清楚地表明，在培养条件下的形态差异。 图1B示出replating的ACI-23和TGS-3 TIC培养在传统的贴壁条件后所取得的分化形态。播种早在附着条件后仅24小时，大部分的多细胞球体的分解，并附着在表面，类似于典型的贴壁细胞。 要验证TIC条件富集细胞的干细胞标记物流式细胞术采用卵巢抽动两种常见的标志物进行分析 - CD133 expression和乙醛脱氢酶的活性。在图2所示点图的高亮CD133 +细胞的低附着，无血清条件下增加的百分比。同样地，所述ALDH酶的活性是在这些条件下相比传统贴壁培养条件下更高。虽然传统的贴壁培养物包含CD133 +细胞，即提高球体形成的条件下的百分比增加。一个多能性标志物分析，TRA-1-60，借给进一步支持旅游业议会条件富集TICS资本流动， 图2 B。定量图2 C表明，双阳性（CD133 + ALDH +）ACI-23细胞主要限于TIC提高的条件下生长的文化。还审议了变化，发生在后的TIC条件5天文化的ACI-23细胞不同的转录因子的蛋白质水平。在每个标记的累进加价s被从传统的可见相比，相对较低的水平见于贴壁培养， 图2个D到漂浮物TIC条件。有趣的是，在各种条件下生长的人腹水细胞株显示了传统的TIC条件下很少的最高标记表型没有出现在浮子TIC条件下染色， 图2 E。 的TIC培养条件的致瘤性，通过从所有的条件到无胸腺裸鼠的队列获得的活细胞的数目相等的皮下注射进行了验证。 图3表明，ACI-23（A，B）和OVCAR-5（℃）从细胞中传统的贴壁培养均较从TIC文化少致瘤。需要注意的是浮子TIC培养在更短的时间周期比传统粘附或传统TIC培养产生较大的肿瘤。这些ðATA表明，即使有适度增加卵巢TIC标志，有一个戏剧性的表型改变。但是与其它细胞系（未示出）的传统的TIC培养物比传统的贴壁和漂浮物TIC培养细胞更致瘤性。 <img alt="图1" src="/files/ftp_upload/52446/52446fig1highres.jpg" /> 成立图1.球体发展在不同的培养条件。（A）的卵巢癌细胞系（ACI-23，OVCAR-5和CAOV3）和生长在低附着瓶中用无血清主患者来源腹水细胞（TGS-3）干细胞媒体容易形成多细胞球体。图像显示，传统的贴壁培养条件下维持上皮鹅卵石形态ACI-23，OVCAR-5，CAOV3和TGS-3细胞，而在TIC条件，在96小时形成球体相同的细胞。比例尺为100μm。 （B）在24小时曝光传统的贴壁培养条件下，TIC文化形态显示有区别的表型相似的原贴形态。比例尺100微米。 <a href="https://www.jove.com/files/ftp_upload/52446/52446fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52446/52446fig2highres.jpg" /> 图2.低附着，无血清条件富集细胞与TIC标志物:（A）流式细胞仪使用的APC缀合的CD133抗体ACI-23细胞的分析表明CD133 +细胞中的低附着烧瓶增加的百分比与无血清干细胞介质。流式细胞仪使用ALDEFLUOR测定节目分析在细胞中的低附着瓶中用无血清干细胞培养基中培养增加ALDH活性。 CD133阴性对照不包含抗体和ALDH负CON控制是激活的衬底中，所述ALDH抑制剂（DEAB）的存在。 （B）分析使用FITC共轭TRA-1-60抗体展示TRA-1-60 +细胞在ACI-23细胞浮子TIC情况比例最高。阴性对照不包含抗体。 （C）CD133 + ALDH +细胞是最传统的TIC和漂浮物TIC条件的ACI-23细胞系。误差棒:SEM，N = 6，* P &lt;0.05。 （D）的 Western印迹分析示出增加的干细胞中的ACI-23细胞在5天TIC条件下培养的转录标记物蛋白质的水平。全细胞裂解液（50微克）用GAPDH作为加样对照进行分析。 （E）代表散点图分析，作为一个 TGS-3主要腹水细胞。 <a href="https://www.jove.com/files/ftp_upload/52446/52446fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.低附着，无血清条件富集细胞具有增加的致瘤性。（A）的 5×10 5活的ACI-23细胞皮下注射到无胸腺裸小鼠中，一式三份。小鼠称重和肿瘤通过测径器每周两次测量。 A =传统的贴壁培养，F =浮动框TIC文化，S =传统TIC文化。 * P &lt;0.05。 （B）的后使用不同的培养条件下生长的细胞25天从ACI-23形成的肿瘤的代表性图像。 （C）5×10 5可行的OVCAR-5细胞皮下注射到无胸腺裸鼠一式三份和监控作为一个 。 <a href="https://www.jove.com/files/ftp_upload/52446/52446fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells at JoVE.com

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

in-vitro-enrichment-of-ovarian-cancer-tumor-initiating-cells

Research

JoVE Journal

Medicine

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

13.7K 次观看.  National Cancer Institute. Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

视频: In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

科研

医学

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

癌症研究

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

用于临床前药物测试的卵巢癌患者来源的类器官模型

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

标准化卵巢癌类器官生物样本库

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

作者聚焦：推进卵巢癌的个性化医疗 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

在卵巢癌肿瘤起始细胞体外富集

摘要

探索更多视频

此视频中的章节

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

异型三维

高级别浆液性卵巢癌患者来源类器官的生成和培养

为获取原发性卵巢癌细胞固体样品的方法

小鼠模型的非侵入性成像检测和监测卵巢癌复发

丰富的人力细胞系化疗耐药性卵巢癌干细胞

用于临床前药物测试的卵巢癌患者来源的类器官模型

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性