作者感谢达雷尔Kotton博士在波士顿大学提供噬菌体EF1aL - 红色荧光蛋白-UBC-GFP-W在这些研究中使用高滴度的慢病毒载体的生产和协议。这项工作是由国防部BCRP W81XWH-11-1-0101（DMC），ACS IRG＃57-001-53（DMC），NCI K22CA181250（DMC）和R01 CA140985（CAS）资助.NCI P30CA046934中心授予支持活体成像和组织培养内核，AMC科罗拉多大学。

所有需要在这个协议中使用动物的步骤如下科罗拉多动物实验伦理委员会的大学（IACUC）的指导方针。 1，仪器，文化传媒等试剂的制备<ol><li>准备含100毫升微球体含贝科的改良Eagle培养基和汉族的F-12培养基（DMEM / F12）媒体（1:1），碱性成纤维细胞生长因子（bFGF，20纳克/毫升），表皮生长因子（EGF，10毫微克/毫升），肝素（4微克/毫升），1×B27，青霉素（100单位/毫升），链霉素（100微克/毫升）。使在无菌条件媒体和储存在4℃下进行长达3个月。 </li><li>制备含有PBS pH 7.2的，0.5％牛血清白蛋白（BSA），2mM EDTA的上皮富集缓冲液（EEB）。过滤消毒和储存在4℃下长达6个月。 </li><li>使在-20℃，5毫升分装在无菌的15毫升锥形管中并储存消化缓冲液几个月。肿瘤消化，解冻消化缓冲液的前一天晚上（5毫升，每500毫克肿瘤）在冰上，在4℃下。在使用之前加入1X抗生素 - 抗真菌剂的组合。 </li><li>高压灭菌（121℃，30分钟）至少两个镊子，解剖刀和剪刀PDXs解剖。 </li><li>使含DMEM500毫升洗涤缓冲液:F12和5％胎牛血清（FBS）。存放在4℃数月。分装10毫升每个肿瘤的肿瘤切除的一天。 </li><li>在4微克/微升用无菌水准备一个凝聚胺的股票。过滤器和分装成含有各100微升，储存在-20°C的1.5 ml离心管中。 </li></ol> 2.高滴度的慢病毒颗粒的生成携带标记可溯源<ol><li>购买或携带生成一个报告基因的高滴度的慢病毒颗粒（ 即 ，GFP和吕克肿瘤生长的体内追踪）18,19。 注:的慢病毒滴度&gt; 10 8 TU毫升（每毫升转导单位）推荐/为PDXs成功转导。慢病毒p物品应该假与来自水泡性口炎病毒（VSV-G），允许的各种哺乳动物细胞的转导的包膜ģ糖蛋白。 注:此处使用的高滴度慢病毒颗粒的产生和滴定的协议可在<a href="http://www.kottonlab.com">www.kottonlab.com</a> 。在这个协议中使用的载体是pSIH1-H1-copGFP-T2A-普罗和双启动噬菌体EF1aL萤光素酶UBC-GFP-W。通过用在噬菌体EF1aL-DsRed的-UBC-GFP-W矢量EF1aL启动子下游的荧光素酶基因，达雷尔Kotton，波士顿大学的一种礼物代替DsRed的生成该向量。 </li></ol> 3.患者来源的移植瘤产生<ol><li>通过在免疫缺陷小鼠3,4-乳房脂肪垫植入原发性或转移性乳腺肿瘤生成从乳腺癌患者来源异种移植物（PDX）。在直径1厘米的最大建议大小的肿瘤生成的肿瘤较大为1-ikely含有坏死核心。 注:详细的建立和异种移植方法德罗斯等[18]描述。建立-移植PDXs生长进入&gt;1厘米直径肿瘤植入后4和24周之间发生在免疫功能低下的NOD / SCID / ILIIrg - / - （NSG），这取决于固有的肿瘤生长速率。虽然这个协议描述乳腺癌PDXs的转导，它可以用于适合于在体外通路短期任何肿瘤的病毒转导。 PDXs短期的灵敏度- 体外存活（24 96小时）是固有的每个肿瘤，并且必须通过实验确定。 </li></ol> 4.肿瘤解剖和离解PDX-来源的肿瘤细胞<ol><li>用二氧化碳吸入颈椎脱位执行安乐死PDX小鼠（遵循动物实验伦理委员会的机构准则）。淹没的小鼠在0.2％CH1分钟lorhexidine溶液。上一个夹层板的上方仰卧位设置鼠标，并使用引脚扩展的上肢和下肢固定在板上。 </li><li>使用无菌技术，使用手术刀切肿瘤周围的皮肤。与一组镊子的拉扯皮肤，并使用干净的手术刀，直到肿瘤完全暴露其从肿瘤中分离出来。用干净的一套镊子，手术刀，切除肿瘤，并将其放置在5毫升洗冰介质。 </li><li>取剖分肿瘤成BLS2柜作进一步处理。除去介质，用10ml Hank氏平衡盐钠改性的10mM Hepes（HBSS / HEPES）冲洗肿瘤，两次。 </li><li>滴肿瘤在无菌预先称重60厘米组织培养板。称量装有估计肿块肿瘤板。 </li><li>使用手术刀，剪肿瘤成两半，然后从一个半切割3毫米的光盘，并用10％福尔马林将其放置在容器中24小时。使用此组织验证的异质性肿瘤样本（父母PDX）。添加HBSS /的Hepes的200微升（足以避免组织变干），并剁碎在使用镊子和干净的手术刀的最小可能块剩余肿瘤。 </li><li>转移切碎的肿瘤到无菌的50ml锥形管中。至少添加1毫升消化缓冲液含有每100毫克肿瘤1X抗真菌抗生素。 注:抗生素/抗真菌剂的加入阻止肿瘤细胞的污染体外 。 </li><li>在30℃下消化肿瘤3小时，在125摇动 - 200转。 注意:增加温度高达37℃提高消化效率（更单个细胞随着时间的推移），但它通常是伴随着增加的细胞死亡。对于大多数的肿瘤，在30℃下在可行的解离细胞的足够数量的3小时的结果消化进行到标记步骤。 </li><li>停止消化加入35毫升洗涤培养基（DMEM / F12有5％FBS）。通过70微米尼龙滤网为50 mL的干净Çonical管取出未消化的组织。离心含有消化细胞经过滤的媒体，在400×g离心5分钟。 </li><li>吸上清，重悬沉淀在1ml红血细胞裂解液。在室温下孵育5分钟。 </li><li>加入10 mL洗涤缓冲液（DMEM:F12，5％FBS）停止裂解。通过细胞通过一个40微米尼龙网除去团块。离心机400 XG 5分钟，在冰上上清，到位。 </li><li>悬浮消化在5毫升细胞洗涤缓冲液和使用台盼蓝排除计数都存活和非存活细胞。简要地说，混合单元50和50μl锥虫蓝，用血细胞计数器计数台盼蓝排除细胞。 注:该粗消化产物将包含来自PDX衍生的人肿瘤细胞，以及鼠基质细胞（成纤维细胞，血细胞， 等 ）。消化的细胞现在可以用于标记（步骤6）进行处理，或人类癌细胞可以使用所描述的方法之一来富集参考文献5。 </li></ol> 5.上皮性肿瘤细胞富集<ol><li>消耗使用谱系（林+）细胞耗竭套件20小鼠基质细胞。 注:推荐用于高度血管肿瘤和/或肿瘤具有高的基质含量，其中的EpCAM表达是未知的或丢失）。 <ol><li>最多需要10 7活细胞在一个干净的5 15mL的聚丙烯管中，在300 xg离心加2ml EEB离心5分钟。完全移液器关闭上清。 </li><li>每107 个细胞的冰冷建筑节能的40微升悬浮细胞沉淀。 </li><li>添加10微升每10 7个细胞的生物素抗体混合物，轻轻吹打混匀并在4℃下进行10分钟（鸡尾酒包含抗体林+小鼠细胞）孵育。 </li><li>加入30微升每10 7个细胞冷建筑节能中，轻轻吹打混合。 </li><li>加入20μl抗生物素微珠每10 7个细胞拌匀轻轻吹打并在4＃孵育176℃持续15分钟。 </li><li>在4℃下洗用3ml冷EEB，离心细胞以300×g离心5分钟。小心吸上清。悬浮颗粒在500微升冰建筑节能和地点。 </li><li>放置列在磁分离器的磁场。用0.5毫升上皮富集缓冲冲洗柱，并允许它滴落通过。不要让柱干。 </li><li>将新的聚丙烯收集管中列下。慢慢地，添加500微升含标记的细胞在柱（林+标记的细胞将保留在磁列）。收集流出物与未标记的细胞的一小部分，代表富集的谱系阴性（肿瘤）细胞级分。 </li><li>用500μlEBB的冲洗柱3次，并且收集流出物在相同的管作为步骤5.1.8的流出物。保持在冰上流出物并继续标记步骤（6）。 </li><li>可选:以列的磁场之外。将新的聚丙烯管下方并通过加入500微升EEB的，三次和使用所提供的柱塞洗脱LIN +馏分。 </li></ol></li><li>人EpCAM +上皮癌细胞的富集（推荐已知表达CD326 +，并含有高小鼠基质含量瘤）。 <ol><li>最多需要10 7活细胞在一个干净的5 15mL的聚丙烯管中，加入2毫升冷的建筑节能和离心机在300×g离心5分钟。完全移液器关闭上清。 </li><li>每107 个细胞的冰冷EBB的60微升悬浮细胞沉淀。 </li><li>加入20微升每10 7个细胞的EpCAM微珠，轻轻吹打混合，并在4℃孵育30分钟。 </li><li>在4℃下洗用3ml冷EEB，离心细胞以300×g离心5分钟。小心吸上清。在500微升建筑节能的悬浮颗粒和雪藏。 </li><li>放置柱在微型分离器的磁场。冲洗用0.5ml EEB柱，并允许它滴落通过。不要让列干出。 </li><li>将新的聚丙烯收集管中列下。慢慢地，添加500微升含标记的细胞在柱（EpCAM的+标记的细胞将保留在磁列）。收集流出物与未标记的细胞，代表小鼠基质细胞的一小部分。 </li><li>用500μlME缓冲液冲洗柱4次，并收集流出物在相同的管作为步骤5.1.8的流出物。 </li><li>取的磁场以外的列。放置一个新的聚丙烯管下方，并通过加入500微升的ME缓冲，三次洗脱的EpCAM +馏分。用力推动活塞入列冲洗掉磁标记的细胞。收集含有富集的EpCAM +肿瘤细胞的部分污水和保持在冰上。继续第6步。 </li></ol></li></ol> 6.转导PDX源性肿瘤细胞的<ol><li>离心分离肿瘤细胞在300 GX 5分钟，重悬在2毫升微球体MEDI一个。采用台盼蓝排斥在4.11计数活细胞。 </li><li>制备10含8微克/毫升聚凝胺（2每毫升介质微升库存聚凝胺）微球体介质的溶液中。 </li><li>确定所需的2×10 5个存活的肿瘤细胞的体积。离心细胞，在300×g离心5分钟，并在2毫升含介质凝聚胺的重悬沉淀。每孔平板2×10 5个存活的肿瘤细胞在6孔超低附着组织培养板。 注意:这是用于与一个慢病毒载体转导所需的细胞的最佳数目。 小心！慢病毒颗粒和的慢病毒颗粒使用的所有耗材应办理以下重组DNA生物危害的制度程序。 注:原发乳腺癌细胞慢病毒转导强烈支持肌上皮细胞标记21在这期间可能导致管腔细胞和肿瘤细胞的亚群的选择标记较差。 </li> &lt;利&gt;孵育200 MU / ml的神经氨酸酶的病毒，在37℃下，以转导，以增加病毒颗粒的不同的初级细胞亚群的结合和校正这一潜在的偏压21之前1小时。 </li><li>在10 MOI（2×10 6 TU为2×10 5个活细胞）中添加的慢病毒颗粒是否PDX-解离的细胞是从林+小鼠细胞耗尽或EPCAM +细胞富集。如果使用非富集的PDX-解离细胞以30的MOI（6×10 6 TU为2×10 5个活细胞）中添加的慢病毒颗粒。 注:MOI增加允许有效转导，即使在大量的碎片，并在粗提取物的死细胞的存在。保持一个孔与未标记的细胞，作为活力和转导效率的对照。 </li><li>涡流与细胞混合病毒。孵育在37℃，时间长达96小时，5％CO 2。加入500μl新鲜的微球体媒体​​转染后24小时。细胞不会阿塔克h至板，不吸媒体。 </li></ol> 7.转导效率和标记的细胞重新植入免疫缺陷小鼠的评价<ol><li>监视可追踪标记（GFP）使用荧光显微镜在10倍放大倍率，每24小时的表达。 注:GFP表达可以观察到早24小时感染后，但在大多数PDXs GFP表达是后72小时（ 图1）清晰可见。 </li><li>通过将总井内评估的GFP +细胞的百分比估计转导的效率。 注意:由于培养物含有肿瘤细胞，残余基质细胞和死细胞在不同程度，具有低至10％的GFP +细胞的孔可以在宿主小鼠被植入。 </li><li>传送转导的细胞从6孔板入15ml锥形管中。加入1 ml微球体媒体​​到井收集留下的所有单元格。广场上冰。 </li><li> 10毫升HBSS / HEPES添加到转导的细胞和centrifuge在300×g离心5分钟，4℃。小心吸上清，重悬在冰上50微升地下室矩阵提取物（BME）。 注:BME等分必须在冰上解冻，1 - 使用2小时前。 BME将固化在室温下;保持在冰上在任何时候。 </li><li>加载BME嵌入细胞变成0.5 ml胰岛素注射器，保持在冰上，并提请动物设施进行再植入老鼠NSG。 </li><li>麻醉女4 - 8周老NSG鼠，用5分钟5％异氟醚/ 95％的氧气，其次是2％异氟醚/ 98％的氧气，或根据批准的协议批准的动物研究伦理。 </li><li>当动物是响应对疼痛刺激（脚趾捏），清洁用优碘，随后用乙醇擦拭物注射区，并在4 个乳腺脂肪垫注射50微升的细胞。提供小鼠镇痛，以减轻注射不适在批准的协议表示。 </li><li>使细胞形成肿瘤（2 - 12周）和监测GFP和/或使用可调光系统和/或体内的荧光素酶的成像系统（ 图2）的荧光素酶的活性。 注:按照麻醉，镇痛，肿瘤负荷测量和安乐死的标准准则批准的机构动物研究伦理委员会。 </li></ol> 8.标记PDXs质量控制<ol><li>通过在标记的肿瘤评估萤光素酶和GFP表达确定标记的效率。 注:按照机构动物实验伦理委员会批准体内生物发光成像程序（ 图2，图3）。 <ol><li>解剖从安乐死的小鼠的肿瘤时肿瘤大小已经达到1.0 - 1.5厘米直径的（或根据批准的方案，如在4描述之前）。解剖应该下一个可调谐光系统（或等效系统，使GFP荧光评估）来执行。 </li><li>从每个肿瘤定性估计的PEGFP +肿瘤用显微镜rcentage。如果低于100％，解剖仅GFP +部分。解剖3毫米磁盘并在10％福尔马林石蜡包埋和组织学分析确定，保存一小片用于RNA或其它所需的分析，和尽可能多的肿瘤保存尽可能在含有90％FBS / 10％个体1.5毫升冷冻管DMSO冻存。 </li></ol></li><li>重新植入件标记的肿瘤成新的收件人NSG鼠18扩大和实验研究转移使用。 </li><li>执行从肿瘤转导前和在每一代得到石蜡包埋的组织的标准免疫组织化学染色后，以验证标记PDXs保持组织学类似于原始PDXs。 注:用于质量控制的标记与每个PDXs变化。乳腺癌PDXs，雌激素受体的表达，孕酮受体，表皮生长因子受体2（HER2），表皮生长因子受体1（HER1或EGFR），PA正角蛋白（泛CK）被推荐为初步筛选。 </li></ol> 9.实验转移模型，标PDXs <ol><li>下面在第4所描述的步骤，从一个标记PDX解离的肿瘤细胞。如果肿瘤是高度血管或含有丰富的间质鼠标，如4.5或4.6的说明执行癌细胞的富集。 </li><li>计数活细胞用台盼蓝染色，并稀释250,000个细胞在100微升PBS每小鼠（ 钙 ，镁离子免费）。保持细胞在冰上。对于多个小鼠注射，调整相应的数字（并准备至少一个额外注射过量以弥补移液错误）。 </li><li>带来细胞在冰上到适当的动物操作室以及根据批准的IACUC协议进行心内注射。 注:详细对于心内注射癌细胞协议已经在别处22,23说明。 </li><li>跟踪和量化转移性使用生物发光成像遍布时间。可视化使用生物发光和/或尸检24分离的器官的GFP成像（ 图6）在不同的器官的宏观转移。 </li></ol>

<ol>
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H., Matsen, C., Welm, A. L., Welm, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+human+primary+tumorgraft+models:+comparisons+with+traditional+oncology+preclinical+models+and+the+clinical+relevance+and+utility+of+primary+tumorgrafts+in+basic+and+translational+oncology+research.">Overview of human primary tumorgraft models: comparisons with traditional oncology preclinical models and the clinical relevance and utility of primary tumorgrafts in basic and translational oncology research.</a> Curr Protoc Pharmacol. , (2012).</li><li>Marangoni, 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=A+new+model+of+patient+tumor-derived+breast+cancer+xenografts+for+preclinical+assays.">A new model of patient tumor-derived breast cancer xenografts for preclinical assays.</a> Clin Cancer Res. 13 (13), 3989-3998 (2007).</li><li>Garrido-Laguna, 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=Tumor+engraftment+in+nude+mice+and+enrichment+in+stroma-+related+gene+pathways+predict+poor+survival+and+resistance+to+gemcitabine+in+patients+with+pancreatic+cancer.">Tumor engraftment in nude mice and enrichment in stroma- related gene pathways predict poor survival and resistance to gemcitabine in patients with pancreatic cancer.</a> Clin Cancer Res. 17 (17), 5793-5800 (2011).</li><li>Landis, M. D., Lehmann, B. D., Pietenpol, J. A., Chang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+breast+tumor+xenografts+facilitating+personalized+cancer+therapy.">Patient-derived breast tumor xenografts facilitating personalized cancer therapy.</a> Breast Cancer Res. 15 (1), 201 (2013).</li><li>Norum, J. H., Andersen, K., Sorlie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+the+intrinsic+subtypes+of+breast+cancer+in+the+quest+for+precision+therapy.">Lessons learned from the intrinsic subtypes of breast cancer in the quest for precision therapy.</a> Br J Surg. 101 (8), 925-938 (2014).</li><li>Tentler, J. 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=Patient-derived+tumour+xenografts+as+models+for+oncology+drug+development.">Patient-derived tumour xenografts as models for oncology drug development.</a> Nat Rev Clin Oncol. 9 (6), 338-350 (2012).</li><li>Zhang, 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=Patient-derived+xenografts+of+triple-negative+breast+cancer+reproduce+molecular+features+of+patient+tumors+and+respond+to+mTOR+inhibition.">Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition.</a> Breast Cancer Res. 16 (2), R36 (2014).</li><li>Liu, 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=Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models.">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc Natl Acad Sci U S A. 107 (42), 18115-18120 (2010).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cancer+stem+cell+metastasis+in+xenograft+animal+models.">Analysis of cancer stem cell metastasis in xenograft animal models.</a> Methods Mol Biol. 568, 7-19 (2009).</li><li>Thibaudeau, 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=Mimicking+breast+cancer-induced+bone+metastasis+in+vivo:+current+transplantation+models+and+advanced+humanized+strategies.">Mimicking breast cancer-induced bone metastasis in vivo: current transplantation models and advanced humanized strategies.</a> Cancer Metastasis Rev. 33 (2-3), 721-735 (2014).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. 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The authors have nothing to disclose.

该协议中的关键步骤: 使用高滴度的慢病毒颗粒（&gt; 10 8 TU /毫升）是这个协议的成功的一个关键步骤，如允许体外转期间媒体组成的严格控制。虽然生产高滴度的病毒颗粒的多种方法得到了很好的描述18,19;该协议使用于中详细描述产生的慢病毒颗粒<a href="http://www.kottonlab.com">www.kottonlab.com</a> 。对于肿瘤的消化和分解癌细胞的温和方法是成?...

源自患者的肿瘤异种移植物（PDXs），其中，手术切除肿瘤样品被移入直接进入免疫妥协小鼠的发展，提供了超过标准细胞系异种移植模型几个优点，并代表在癌症研究1,2的一大进步。 PDXs可以保持，并通过与在第一通道生长的肿瘤的遗传和生物学特性最小改变连续传代扩大;和更准确地反映肿瘤的异质性比来自人肿瘤细胞系3-8衍生异种移植物。这些模型现在广泛用作个性化癌症治疗9,10一个平台，如在药物开发6,11的临床前平台和作为研究癌症生物学4,12的实验工具。 最PDXs植入皮下传播，其中使用卡尺可行允许肿瘤生长的测量随着时间的推移。然而，转移性疾病已经越来越难以用PDXs建模。专门为乳腺癌，转移性能力不同器官异种移植物已被描述3,5,13，但自发传播到转移部位的频率极低。凡报道，转移性负担的定性和定量依赖于靶器官验尸费力组织学检查。表达生物发光（荧光素酶，卢克）或荧光癌细胞系（绿色荧光蛋白，GFP）基因记者常用于乳腺癌转移到脑，肺，骨和肝腔内后，尾静脉，intrafemoral和脾注射的实验模型中使用14-16。虽然这些模型绕过从原发性肿瘤传播，它们是研究器官趋向性和转移性定植的机制有价值。然而，从初级病人肿瘤和PDXs来源的细胞可以具有低转染或转导率全光照g标准程序。一个替代方案是建立在体外 17，其可使用常规的组织培养协议来然后标记的PDX-来源的细胞系。然而，这种方法不适合用于标记最PDXs，为此细胞系派生是困难的，可以改变细胞的表型。在这里，我们提出了一个协议对PDX-解离的肿瘤细胞与合适的用于体内成像的慢病毒载体的转导。此外，我们将介绍使用分离的吕克 - 绿色荧光蛋白标记的PDX细胞心内注射免疫功能低下小鼠的实验性转移。 用于与基因-报道表达慢病毒PDX-解离组织体转导的基本协议先前已经描述18。在当前的协议中，我们描述了其他的方法来富集人类肿瘤细胞，将获得接近100％的转导效率，以及用于检测试验乳腺癌使用标记PDXs的转移。这个协议可以适于标记与各种发光和荧光标记PDXs的多种癌症类型，以及基因的表达（ 即 ，shRNA的目的基因的敲低）的调制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>Hyclone</td>
			<td>SH30023.01</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>BD Biosciences</td>
			<td>354060</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>354001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco/Thermo Fisher</td>
			<td>17504-44</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fungi-antibiotics</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies</td>
			<td>AM-105-500</td>
			<td>Digestion Buffer</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS Red Ca++/Mg++ free</td>
			<td>Hyclone</td>
			<td>SH30031.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Hyclone</td>
			<td>SH30258.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex</td>
			<td>Cultrex</td>
			<td>3433-005-01</td>
			<td>Basement Matrix Extract (BME)</td>
		</tr>
		<tr>
			<td>30C shaker</td>
			<td>NewBrunswick Scientific CO. INC</td>
			<td>Series 25 Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>70um filters</td>
			<td>Falcon</td>
			<td>7352350</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpels</td>
			<td>Fisher</td>
			<td>22079690</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorhexidine disinfectant</td>
			<td>Durvet&#160;</td>
			<td>NDC# 30798-624-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood&#160; cell lysis reagent</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuraminidase</td>
			<td>Sigma</td>
			<td>N7885-1UN</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (CD326+) microbeads*</td>
			<td>Miltenyil Biotec</td>
			<td>130-061-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lineage cell depletion Kit, mouse*</td>
			<td>Miltenyil Biotec</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator&#160;</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini MACS Magnetic Stand</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-201</td>
			<td>MS or LS columns can be used, adjust to number of cells.</td>
		</tr>
		<tr>
			<td>Illumatool Tunable light system</td>
			<td>Lightools research</td>
			<td>Various</td>
			<td>For in vivo fluorescence imaging</td>
		</tr>
		<tr>
			<td>Xenogen IVIS200 imaging device</td>
			<td>Xenogen</td>
			<td>Various</td>
			<td>For in vivo luminiscence imaging</td>
		</tr>
		<tr>
			<td>Human Cytokeratin Clone MNF116 Monoclonal antibody</td>
			<td>DAKO</td>
			<td>M0821</td>
			<td>Pan-cytokeratin&#160;</td>
		</tr>
		<tr>
			<td>Epidermal Growth factor receptor antibody</td>
			<td>Cell signaling</td>
			<td>4267S</td>
			<td>EGFR</td>
		</tr>
	</tbody>
</table>

labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

该方法描述了使用高滴度慢病毒载体pSIH1-H1-copGFP-T2A-普罗和噬菌体EF1aL萤光素酶 - UBC-GFP-W PDX-解离的乳腺癌细胞的转导。这些载体表达，允许估计在体外转导的效率的荧光标记物，感染（ 图1a）后早24小时。对于大多数PDXs，绿色荧光蛋白的表达将被延迟到感染（ 图1b）后72小时，此时细胞聚集体的形成通常观察到。可以（使用林+细胞耗竭， <...

Watch this Scientific Journal Video about Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies at JoVE.com

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

我们描述了源自患者的异种移植物（PDXs）用表达绿色荧光蛋白和荧光素酶记者慢病毒颗粒的稳定标记的方法。该方法允许用于跟踪PDXs的生长在主站点，以及检测使用体内成像系统自发和实验转移。

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

labeling-breast-cancer-patient-derived-xenografts-with-traceable

Research

JoVE Journal

Cancer Research

12.0K 次观看.  University of Colorado Anschutz Medical Campus. 我们描述了源自患者的异种移植物（PDXs）用表达绿色荧光蛋白和荧光素酶记者慢病毒颗粒的稳定标记的方法。该方法允许用于跟踪PDXs的生长在主站点，以及检测使用体内成像系统自发和实验转移。 

视频: Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

科研

癌症研究

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

患者衍生的中枢神经系统转移异种移植模型的建立与利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

斑马鱼拉瓦尔异种草和肿瘤行为分析的一代

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

患者来源乳腺类器官的建立和培养

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...