Serene Samyesudhas and Dr. Lynn Roy assisted in preparing samples for filming.

1，细胞的卵巢癌细胞株培养和荧光标记<ol><li>制备SKOV3媒体:麦考介质补充有10％胎牛血清（FBS），0.1mM的L-谷氨酰胺，50单位/ ml青霉素和50微克/毫升链霉素。保持OVCA429细胞在最小必需培养基（MEM）中补充有10％FBS，1mM丙酮酸钠，0.1mM的L-谷氨酰胺，50单位/ ml青霉素和50微克/毫升链霉素。 </li><li>在湿润的培养箱中传播的SKOV3和OVCA429细胞系在37℃下用5％的CO 2。细胞生长至40-50％汇合。 </li><li>生成单元，以监测存活在体外和体内表达红色荧光蛋白（RFP）。 <ol><li>以产生SKOV3细胞表达RFP，添加的慢病毒颗粒表达RFP和10微克/毫升的聚凝胺至SKOV3媒体在没有青霉素和链霉素的72小时。 </li><li> 72小时后，选择RFP阳性细胞的荧光激活细胞索尔蒂纳克（FACS），如下所示:Trypsinize RFP和非RFP表达细胞，离心分离机，在125×g离心5分钟，重悬的细胞在含有0.1％BSA的PBS中的10 7个细胞/ ml。在细胞分选仪，使用非RFP的细胞，以确定排序参数。然后收集细胞表达用561 nm激光高的RFP。 </li><li>可选地，如果慢病毒质粒表达的抗生素抗性盒，通过在合适的抗生素培养选择的RFP表达细胞（如10毫克/毫升的杀稻瘟素）。注意:需要使用可以通过分批和由细胞类型而有所不同的慢病毒颗粒的量。合适的慢病毒滴度应为每个小区类型来确定。 </li></ol></li></ol> 2，丰富的肿瘤干细胞<ol><li>治疗SKOV3，SKOV3-RFP或OVCA429细胞系为20μm顺铂72小时。注意:顺铂是有毒的。使用防护服/手套，避免与皮肤，粘膜接触。不要将铂íÑ​​废水。 </li><li>制备CSC媒体:无血清的DMEM / F12培养基补充有5微克/毫升的胰岛素，10毫微克/毫升的人重组表皮生长因子（EGF），10纳克/毫升碱性成纤维细胞生长因子（bFGF），12纳克/毫升白血病抑制因子和0.4％牛血清白蛋白（BSA）。 </li><li> Trypsinize细胞。文化尚存在中信建投的媒体单元。 注:在培养非贴壁球体2天将形成。 </li><li>通过收集介质含有细胞并在125 XG离心5分钟，改变介质每2天。然后重悬在新鲜CSC媒体沉淀。检查细胞活力，每2天计数细胞并进行台盼蓝染色。 </li><li> （137 mM氯化钠，2.7 mM的氯化钾，和11.9 mM磷酸盐缓冲液，pH 7.4的PBS）与然后通过离心129×g离心5分钟，重悬沉淀在含有苯酚胍硫氰酸盐（RNA），磷酸盐缓冲盐水收集的CSCs在进一步的实验0.1％BSA（流式细胞术），或CSC媒体继续培养该细胞。 </li><li>使用光学显微镜在20X和40X的放大倍率监控CSC细胞形态。 </li></ol>通过对干细胞标记基因的表达分析3，中信建投表征<ol><li>收集的CSCs的3制剂通过收集介质含有细胞，在129 XG离心5分钟，重新悬浮沉淀中含有苯酚胍硫氰酸盐的溶液（也可以使用其他方法来提取RNA）。 </li><li>使用TRIzol试剂根据生产商的方案制备的总RNA。利用市售的cDNA反转录试剂盒从0.1-1微克的RNA合成互补DNA（cDNA）。 </li><li>进行定量反转录聚合酶链式反应（QRT-PCR）。 <ol><li>弥补主混合物对于每一组含有QRT-PCR混合物，引物和水至总量每样本8微升引物（引物用不同的荧光团可在同一井被放大）。例如，生成一个主混音的Oct4-FAM，Nanog的六角，和GAPDH-CY5。生成巢和PROM1单独的主混音。加入2μl的样品cDNA模板。 请注意:在本研究中，QRT-PCR使用表1中所列的引物进行。 </li><li>执行所有的荧光定量PCR检测的重复利用实时PCR热循环仪能够检测多种荧光团各样本。包括无模板对照。 </li><li>标准化的干细胞的基因表达，以GAPDH表达为使用ΔΔCT法各样品。 </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0" width="746"><colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="63" rowspan="3" style="height:62px;width:111px;">辛-4-FAM </td><td style="width:636px;">引物1:5&#39;-CCCAAGGAATAGTCTGTAGAAGTG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2:5&#39;-TGCATGAGTCAGTGAACAGG-3&#39;</td></tr><tr height="21"><td height="21" style="height:21px;"> FAM探针:5&#39;-CTTCCAAGC / ZEN / TG​​CCCACCTAACTTCT / 3IABkFQ -3&#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> NANOG-HEX </td><td>引物1:5&#39;-CCTTCTGCGTCACACCATT-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2:5&#39;-AACTCTCCAACATCCTGAACC-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> HEX探针:5&#39;-CTGCCACCT / ZEN.CTTAGATTTCATTCTCTGGT / 3IABkFQ-3&#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> GAPDH-CY5 </td><td>引物1:5&#39;-TGTAGTTGAGGTCAATGAAGGG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2:5&#39;-ACATCGCTCAGACACCATG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> CY5探针:5&#39;-AAGGTCGGAGTCAACGGATTTGGTC / 3IABRQSP-3&#39; </td><html></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;">巢蛋白FAM </td><td>引物1:5&#39;-AGGACCTGAGCGATCTGG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2:5&#39;-CGTTGGAACAGAGGTTGGAG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> FAM探针:5&#39;-AACTTTTCA / ZEN / GTAGCCCGCAGCC / 3IABkFQ -3&#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> CD133-HEX </td><td>引物1:5&#39;-ACTCTCTCCAACAATCCATTCC-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2:5&#39;-AAACAATTCACCAGCAACGAG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> HEX探针:5&#39;-ACAATCACT / ZEN / GAGCACTCTATACCAAAGCG / 3IABkFQ-3 </td></tr></tbody></table> 表1引物用于CSC的表征定量RT-PCR检测。 e_title"&gt; 4。在细胞表面标记CD117和CD133的肿瘤干细胞分析<ol><li>收获的CSCs通过收集介质含有细胞和在125 XG离心5分钟。添加的非蛋白水解细胞剥离溶液来分手单元500微升。离心细胞，在125×g离心5分钟以除去细胞剥离溶液。然后洗涤细胞两次用冷的PBS中。 </li><li>孵育在正常生长培养基中进行1小时标记细胞之前。降速细胞5分钟，在129×g下。重悬的细胞在PBS中的0.1％BSA中与抗CD133-PE和抗-CD117-FITC。 </li><li>在1％多聚甲醛中过夜固定细胞。注意:多聚甲醛是有毒的（疑似致癌物）。请在通风橱中，并在一周内使用。 </li><li>流式细胞仪进行流量分析CD117（用抗-CD117 FITC偶联）和CD133（用抗-CD133的缀合PE）上的流式细胞仪的细胞表面表达。收集细胞在FL1（为FITC）和FL3（体育）频道的数据。产生门，用于细胞同时表达FITC和PE。生成的点积与4个象限（FITC-和PE-，FITC + PE-，FITC-PE +，和FITC +和PE +），并判断在每个象限中的细胞数。 </li></ol> 5，分析肿瘤干细胞的治疗反应<ol><li>板5000细胞每孔（SKOV3，SKOV3-RFP，SKOV3 CSC和SKOV3-RFP的CSCs）在96孔板过夜。 </li><li>治疗与顺铂（0，1，10，100，和1000μM）或紫杉醇（0，0.01，0.1，1，10，和100μM）为96小时。 </li><li>进行MTT（3（4,5二甲基-2 - 基）-2,5 - 二苯基溴化）细胞活性分析。 <ol><li>每孔100μl板5000细胞在96孔板（板的细胞，一式两份，并且包括媒体只对照孔）。 </li><li> 24小时后，加入10微升的10毫克/毫升的MTT。在37℃下进行2-4小时（直至形成沉淀）。 </li><li>加入100微升的MTT溶剂4mM的盐酸，0.1％NP-40的异丙醇。孵育在黑暗中15分钟。 </li><li>阅读板块，在570 nm处对酶标仪中进行eader。 </li></ol></li></ol>

<ol>
	<li>Pennington, K., Pulaski, H., Pennington, M., Liu, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+Much+of+a+Good+Thing:+Suicide+Prevention+Promotes+Chemoresistance+in+Ovarian+Carcinoma.">Too Much of a Good Thing: Suicide Prevention Promotes Chemoresistance in Ovarian Carcinoma.</a> Curr Cancer Drug Targets. 10, 575-583 (2010).</li><li>Thigpen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rational+approach+to+the+management+of+recurrent+or+persistent+ovarian+carcinoma.">A rational approach to the management of recurrent or persistent ovarian carcinoma.</a> Clin Obstet Gynecol. 55, 114-130 (2012).</li><li>Burgos-Ojeda, D., Rueda, B. R., Buckanovich, R. 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The authors declare they have no competing financial interest.

肿瘤干细胞有抗药性的治疗可能是治疗原发肿瘤后交代复发。肿瘤干细胞的特性可能导致更好的治疗卵巢癌。在建立使用上述协议化疗耐药的CSCs的临界参数的时序治疗与化疗的长度和化疗的浓度。当使用在Ma 等人的协议。人们发现，在7天后用顺铂和紫杉醇治疗的，无活细胞保持4。通过还原处理，以3天（以20μM的顺铂而不是40μM的顺铂和10μM紫杉醇），可行化疗耐药细胞和肿瘤干细?...

Resistance to chemotherapy is a major impediment to the treatment and cure of cancer. Ovarian cancer is the 5th leading cause of cancer-related deaths among women in the United States (American Cancer Society Facts and Figures 2013). Patients initially respond well to chemotherapy, but most patients relapse1. After relapse patients are treated with a variety of additional chemotherapy agents with very little benefit2. General properties of CSCs include unlimited proliferative capabilities, retention of an undifferentiated state, resistance to drug treatment, efficient DNA repair, and ability to generate malignant tumor cells with different phenotypes3. CSCs frequently exhibit expression of stem cell genes such as Nanog, Oct4, Sox2, Nestin, CD133, and CD117. CSCs often express elevated levels of ABCG2, and ALDH genes that may contribute to drug efflux and metabolism3,4. 
The first definitive evidence for CSCs was demonstrated by isolating acute myeloid leukemia-initiating cells that were capable of self-renewal and tumor generation5. These leukemic stem cells expressed surface CD34 and generated leukemia in NOD/SCID (immunocompromised) mice5. Since then CSCs have been identified in many cancer types including leukemias/lymphomas, breast, bladder, colorectal, endometrial, sarcomas, hepatocellular carcinoma, melanomas, gliomas, ovarian, pancreatic, prostate, squamous cell carcinoma, and lung6. Therefore, being able to study this subtype of cancer cell is advantageous.
The goal of this study is to create a protocol for the selection and isolation of chemoresistant CSCs. Several methods have been reported for the isolation of CSCs from ovarian cancer cell lines. Non-adherent spheroids isolated from OVCAR-3, SKOV3, or HO8910 cultures demonstrate stem-like properties7,8. Isolation of CD133+ cells from OVCAR-3 cultures also yields CSCs. CSCs have also been selected in culture by treatment with chemotherapeutic agents. Treating tumor cell lines (OVCA433, Hey, and SKOV3 cells) with cisplatin and paclitaxel allows for the expansion and isolation of CSCs4,9. While culture of some cell types in CSC media leads to isolation of CSCs, SKOV3 cells did not survive culture in serum-free media or form sphere cells4. Therefore, treatment of cells with cisplatin and paclitaxel aided the expansion or isolation of this population4.
Using a modification of the procedure presented by Ma and colleagues4 we developed a method to isolate CSCs from the ovarian cancer cell lines. Our protocol is advantageous as it yields more viable cells while using less toxic chemotherapeutic agents. Cells are treated with cisplatin and subsequently grown in serum-free media supplemented with growth factors (stem cell media). We isolate the resulting non-adherent sphere cells and assay them for their expression of stem cell markers. This model enables the study of CSC properties and response to drug therapy.

<table><tbody><tr><td>McCoy</td><td>Life Technologies</td><td>16600-108</td><td>Warm to 37 &amp;#176;C prior to use</td></tr><tr><td>DMEM / F12 serum free</td><td>Life Technologies&amp;nbsp;</td><td>11320-033</td><td>Warm to 37 &amp;#176;C prior to use</td></tr><tr><td>Minimal Essential Media</td><td>Life Technologies&amp;nbsp;</td><td>42360032</td><td>Warm to 37 &amp;#176;C prior to use</td></tr><tr><td>Sodium pyruvate</td><td>Life Technologies&amp;nbsp;</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/11360070&quot;&gt;11360070&lt;/a&gt;</td><td></td></tr><tr><td>Polybrene</td><td>Millipore</td><td>TR-1003-G</td><td></td></tr><tr><td>Blasticidin</td><td>Life Technologies&amp;nbsp;</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/R21001&quot;&gt;R21001&lt;/a&gt;</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Atlas Biologicals</td><td>F-0500-A</td><td></td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Cisplatin</td><td>Sigma-Aldrich</td><td>T7402-5MG</td><td>Caution: Toxic. Use precautions</td></tr><tr><td>pLenti-suCMV-Rsv</td><td>Gentarget</td><td>LVP023</td><td>BSL2 approval needed</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I-1882</td><td></td></tr><tr><td>Human Recombinant EGF&amp;nbsp;</td><td>Cell Signaling Technology</td><td>8916LC</td><td></td></tr><tr><td>bFGF</td><td>BD biosciences</td><td>354060</td><td></td></tr><tr><td>LIF</td><td>Santa Cruz</td><td>sc-4988A</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Roche</td><td>03 116 956 001</td><td></td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>15596-018</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit&amp;nbsp;&amp;nbsp;</td><td>Applied Biosystems</td><td>4368813</td><td></td></tr><tr><td>IQ Multiplex Powermix</td><td>BioRad</td><td>1725849</td><td></td></tr><tr><td>Accumax</td><td>Millipore</td><td></td><td></td></tr><tr><td>Primers</td><td>Integrated DNA Technology</td><td>individually designed and ordered (see protocol for sequnces)</td><td></td></tr><tr><td>Anti-CD133 PE</td><td>Milenyl</td><td>130-098-826</td><td>Primer/probe sets are light sensitive</td></tr><tr><td>CD117-Biotin</td><td>Miltenly</td><td>130-098-570</td><td></td></tr><tr><td>AntiBiotin-FITC</td><td>Miltenly</td><td>130-098-796</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148-1KG</td><td>Caution: Toxic. Always prepare in hood and make fresh.</td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/25300062&quot;&gt;25300062&lt;/a&gt;</td><td></td></tr><tr><td>MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>25200-072</td><td></td></tr><tr><td>EVOS Fl Epifluorescence and Transmitted Light Microscope</td><td>Advanced Microscopy Group</td><td></td><td></td></tr><tr><td>Biorad CFX96 C1000 System</td><td>Biorad</td><td></td><td></td></tr><tr><td>Beckman Coulter FC500 Flow Cytometer&amp;nbsp;</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Spectramax 340PC384&amp;nbsp;</td><td>Molecular Devices</td><td></td><td></td></tr></tbody></table>

enrichment for chemoresistant ovarian cancer stem cells from human cell lines

癌症干细胞（CSCs的）被定义为慢骑自行车和未分化细胞的不对称分裂，产生高度增殖，浸润和化疗耐药的肿瘤细胞的一个子集。因此，肿瘤干细胞是细胞的一个有吸引力的人群为目标的治疗。 CSCs的被预测为向多种类型的恶性肿瘤，包括那些在血液，脑，肺，胃肠道，前列腺和卵巢的。分离和富集肿瘤细胞群的肿瘤干细胞将使研究人员能够研究性质，遗传学和肿瘤干细胞治疗的反应。我们产生的一种协议，它可再现丰富的卵巢癌细胞株（SKOV3和OVCA429）卵巢癌肿瘤干细胞。细胞系是用20μM的顺铂处理3天。存活细胞的分离及培养在含细胞因子和生长因子的无血清干细胞培养基。我们通过分析分离出的细胞对于k证明这些肿瘤干细胞纯化的浓缩nown干细胞标记物的Oct4，Nanog和PROM1（CD133）和CD177和CD133的细胞表面表达。在社区体育会显示增加的耐药。这种方法对于肿瘤干细胞的分离，是研究耐药和肿瘤复发的肿瘤干细胞的作用，一个有用的工具。

表明我们用顺铂治疗中分离的CSC从卵巢上皮癌的细胞系，我们首先在治疗之前和选择后获得的细胞系中的图像。我们使用光学显微镜拍摄附着的（未处理的）的SKOV3和OVCA429细胞和SKOV3和OVCA429的CSCs（ 图1）的图像。 CSCs的出现圆形和未附着到组织培养板中（ 图1和2）。我们还表明，这表明该细胞是可行的（ 图2B）中的CSCs的分离后SKOV3细胞转导的RFP保...

Watch this Scientific Journal Video about Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines at JoVE.com

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

肿瘤干细胞（肿瘤干细胞）有牵连的肿瘤复发，由于化疗。我们已经进行了优化的协议选择和扩展，从卵巢癌细胞系的CSCs的。通过处理细胞与化疗顺铂和在干细胞培养促进我们丰富的非粘附CSC培养介质。

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, ...

enrichment-for-chemoresistant-ovarian-cancer-stem-cells-from-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

13.4K 次观看.  Indiana University School of Medicine. 肿瘤干细胞（肿瘤干细胞）有牵连的肿瘤复发，由于化疗。我们已经进行了优化的协议选择和扩展，从卵巢癌细胞系的CSCs的。通过处理细胞与化疗顺铂和在干细胞培养促进我们丰富的非粘附CSC培养介质。 

视频: Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

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

科研

癌症研究

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

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.

从妇科和乳腺癌肿瘤获得癌症干细胞球

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

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

<meta charset="utf8"></head><body>使用人基质促进转移的卵巢癌原位小鼠模型

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

使用人基质促进转移的卵巢癌原位小鼠模型

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

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 ...

医学

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 ...

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 ...

丰富的人力细胞系化疗耐药性卵巢癌干细胞

摘要

探索更多视频

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

从妇科和乳腺癌肿瘤获得癌症干细胞球

使用人基质促进转移的卵巢癌原位小鼠模型

使用人基质促进转移的卵巢癌原位小鼠模型

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

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