Name: labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies
Uploaded: 2016-11-30T15:00:00.000+00:00
Duration: 9 min 53 s
Description: 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

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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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=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nat Med. 17 (11), 1514-1520 (2011).</li><li>Kabos, 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=Patient-derived+luminal+breast+cancer+xenografts+retain+hormone+receptor+heterogeneity+and+help+define+unique+estrogen-dependent+gene+signatures.">Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures.</a> Breast cancer research and treatment. 135 (2), 415-432 (2012).</li><li>Zhang, X., 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+renewable+tissue+resource+of+phenotypically+stable,+biologically+and+ethnically+diverse,+patient-derived+human+breast+cancer+xenograft+models.">A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models.</a> Cancer Res. 73 (15), 4885-4897 (2013).</li><li>Lum, D. 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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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat Rev Cancer. 11 (2), 135-141 (2011).</li><li>Powell, 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=p53+deficiency+linked+to+B+cell+translocation+gene+2+(BTG2)+loss+enhances+metastatic+potential+by+promoting+tumor+growth+in+primary+and+metastatic+sites+in+patient-derived+xenograft+(PDX)+models+of+triple-negative+breast+cancer.">p53 deficiency linked to B cell translocation gene 2 (BTG2) loss enhances metastatic potential by promoting tumor growth in primary and metastatic sites in patient-derived xenograft (PDX) models of triple-negative breast cancer.</a> Breast Cancer Res. 18 (1), (2016).</li><li>DeRose, Y. 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=Patient-derived+models+of+human+breast+cancer:+protocols+for+in+vitro+and+in+vivo+applications+in+tumor+biology+and+translational+medicine.">Patient-derived models of human breast cancer: protocols for in vitro and in vivo applications in tumor biology and translational medicine.</a> Curr Protoc Pharmacol. , (2013).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. 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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><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&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; 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>30 &amp;#176;C shaker</td><td>NewBrunswick Scientific CO. INC</td><td>Series 25 Incubator Shaker</td><td></td></tr><tr><td>70 &amp;#956;m 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&amp;nbsp;</td><td>NDC# 30798-624-35</td><td></td></tr><tr><td>Red blood&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp;</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

12.1K 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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

科研

癌症研究

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

医学

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

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 ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

检查肺转移瘤的肿瘤细胞播散

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

作者聚焦:使用新的体内技术解码转移转移播种以追踪乳腺癌扩散 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

使用光转换追踪肺转移瘤的肿瘤细胞播散

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

<meta charset="utf8"></head><body>乳腺癌患者来源的异种移植物的高通量解离和原位植入

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

乳腺癌患者来源的异种移植物的高通量解离和原位植入

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

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

摘要

探索更多视频

此视频中的章节

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

研究使用正交乳腺癌模型的三阴性乳腺癌

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

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

使用光转换追踪肺转移瘤的肿瘤细胞播散

使用光转换追踪肺转移瘤的肿瘤细胞播散

使用光转换追踪肺转移瘤的肿瘤细胞播散

乳腺癌患者来源的异种移植物的高通量解离和原位植入

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

摘要

探索更多视频

此视频中的章节

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

研究使用正交乳腺癌模型的三阴性乳腺癌

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

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

使用光转换追踪肺转移瘤的肿瘤细胞播散

使用光转换追踪肺转移瘤的肿瘤细胞播散

使用光转换追踪肺转移瘤的肿瘤细胞播散

乳腺癌患者来源的异种移植物的高通量解离和原位植入

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移