研究TGF-β家族成员是由癌症基因组学中心荷兰的支持。锡济亚·利和匠人通过在莱顿大学的中国留学基金管理委员会4年的研究支持。我们感谢弗雷德·米勒博士（芭芭拉·安Karmanos癌症研究所，底特律，MI，USA）的MCF10A细胞系。

使用转基因斑马鱼的荧光TG（FLI:EGFP）所有的研究株（EGFP）已增强型绿色荧光蛋白标记的脉管系统20，包括住房和实验，进行了按照国际准则，并批准由当地机构委员会在莱顿大学医学中心的动物福利（底儿Ethische Commissie（DEC）。 注意:作为总结在图1中，协议被大致分为四个步骤:胚胎收集（ 图1A），显微注射（ 图1B），筛选（ 图1C），并分析（ 图1D）。 1.准备注射针头<ol><li>准备用硼硅酸盐玻璃微毛细管注射针。把微毛细管到微量牵引装置具有以下设置:空气压力，500，热，650;拉，100;速度，200;时间，40.保存在针保持器板的注射针，直到它们被用于注射。 </li></ol> 2.准备注射的荧光灯，遗传标记乳腺癌细胞<ol><li>培养的人乳腺癌MDA-MB-231的DMEM高葡萄糖培养基中的细胞在37℃的含有L-谷氨酰胺，10％胎牛血清和1:100的青霉素 - 链霉素（青霉素 - 链霉素）。 </li><li>培养物中的乳腺上皮细胞系，MCF10A（M1）和MCF10A-RAS（M2），在37℃在DMEM / F12培养基含有L-谷氨酰胺，5％马血清，20ng / mL的表皮生长因子，10毫克/毫升胰岛素，100纳克/毫升的霍乱肠毒素，0.5mg / mL的氢化可的松，和1:100青霉素 - 链霉素。 </li><li>通过共转染PLV-mCherry的，的pCMV-VSVG 21，pMDLg-RRE（GAG / POL）22和PRSV-REV 22质粒导入HEK293T细胞产生的mCherry慢病毒。收获细胞上清液转染，并储存在-80℃下48小时后。 </li><li>一世nfect MDA-MB-231，M1及M2的细胞在30％汇合时用慢病毒上清液24小时1:1稀释，在5毫微克/ ml聚凝胺存在正常培养基。 </li><li>通过稀释的细胞在96孔板中，这允许分离的细胞克隆的生长，直到获得稳定的mCherry表达细胞系选择单细胞克隆。 </li><li>细胞注射的培养一个T75烧瓶中。收获细胞，在用0.5％胰蛋白酶-EDTA处理80％汇合。洗净用1x PBS 2-3倍的细胞。 </li><li>重新悬浮细胞在约200 PBS的微升。储存在他们4℃注射前不到5小时。 </li></ol> 3.准备斑马鱼胚胎注射<ol><li>建立斑马鱼繁殖对收集胚胎，如通过罗森等人以前的朱庇特的文章。 23。 </li><li>选择在0-4 HPF除去未受精和胚胎异常的胚胎。保持胚胎在Petri DISH全蛋水（60μg/ mL的海盐;〜60个胚/皿）的孵育在28℃。 </li><li> Dechorionate用细镊子胚胎在48高倍视野。 </li><li>通过将它们转移至含有水卵大约2分钟注射前，但不长于2小时注射前40微克/毫升三卡因（3-氨基苯甲酸）麻醉胚胎。 注:三卡因储备溶液（4毫克/毫升，100×）被制备成400毫克三卡因粉末在97.9毫升双蒸水和2.1毫升的1摩尔Tris碱（pH为9），具有调节至7.4的pH值。商店在-20℃冷冻机中。 </li></ol> 4.进样人乳腺癌细胞进入卵周隙<ol><li>负载15μL细胞悬浮液到注射针的。安装针到显微操纵器和断绝与细镊子针尖以获得5-10微米的前端开口的直径。 </li><li>使用气动picopump和机械手进行显微注射。 AdjuST的picopump注入每次400个细胞。在注射之前，通过注射将细胞在含有1％琼脂糖的培养皿中的顶部手动计数细胞数。 </li><li>排队麻醉胚胎（2-3天受精后（DPF））上的平坦的，1％琼脂糖注入板，围绕每个时间10个胚胎。 </li><li>定向注射期间由手注入板放置在优选位置上的胚胎插入针（ 即 ，对角）。 </li><li>指向针尖在注射部位和针尖轻轻插入卵黄囊和斑马鱼胚胎（ 图2A）的周皮之间的卵周隙。 </li><li>注入大约400 mCherry-标记的肿瘤细胞。确保卵黄囊不破裂，避免注入卵黄囊。 </li></ol> 5.注射人乳腺癌细胞到文件<ol><li>如所描述的我准备注射针和斑马鱼的胚胎<html> N协议步骤1，2，和3。 </li><li>使用一个45°针的角度，使该文档可从胚胎的背侧接近。 </li><li>针插入的文件（ 图3A）的起点，只是背侧到管道开始扩大在卵黄囊，并注入大约400个细胞;注射是正确的，如果在管道内的体积的脉冲和卵黄囊后直接膨胀。 注:连续几个注射可以不提取针来进行。 </li><li>转移注入斑马鱼胚胎蛋水。 存在单独的斑马鱼胚胎中作为相当大的变化，并且如胚胎后可出现注射的死亡，相对大量的斑马鱼胚胎（约100），应与癌细胞注射:注意。 </li><li>保持在33℃的斑马鱼的胚胎以容纳鱼和哺乳动物细胞的最佳温度要求。 </li></ol>_title"&gt; 6。屏幕上的注射的胚胎<ol><li>筛选荧光立体显微镜下每条鱼在2小时后注射（HPI）为卵周隙注射（ 图2）或在2-24 HPI的文档注射（ 图2），以确保所有的胚胎都与一个注射类似数量的肿瘤细胞。与注射的错误，如破裂（ 图2B）或卵黄囊的注射（ 图3B）中取出胚胎，并挑选出具有低于注射的细胞（ 图2C和图3B），或上述（ 图2D和图 3B）的阈值的胚胎。只保留与文化大约400个细胞胚胎。 </li><li>排除该小区由已经在循环与细胞取出胚胎在注射过程中直接引入循环的可能性。此外，除去任何胚胎与质量接近文件的小区（ 图2D </stron克&gt;）。 </li></ol> 7.图像和分析转移过程<ol><li>收集几个麻醉胚胎用宽尖端的巴斯德吸管，并将它们传送到聚苯乙烯皿的玻璃底部。 </li><li>去除多余的水，并保持蛋水的数量有限。操纵胚胎成位置与头发循环工具放置一个覆盖在玻璃之上。 </li><li>与水浸泡或长途干目标组合使用倒置共焦显微镜。胚胎这样的感兴趣区域是接近目标的可能位置。 </li><li>立即执行成像麻醉后死亡的风险减少由于液体蒸发。 <ol><li>在对胚胎的相同位置从EGFP标记的脉管系统和mCherry-标记的肿瘤细胞的捕获信号，以通过合并所述两个成像通道共同寄存器注射血管细胞。 </li><li>对于每个斑马鱼胚胎，收集两套不同的图像从头部区域和尾部区域。 </li></ol></li><li>量化播散细胞的数量。 <ol><li>用于卵周隙注射，计数已经从细胞块散布朝向胚胎鱼体的头部和尾部区域4,15内的细胞中每条鱼的数目;该区域是超出了心脏腔的边界正面，对鱼鳔背部的顶部，超越泌尿生殖开放尾端。 </li><li>对于文件注射，计算已侵入尾翼的胶原纤维从循环（MDA-MB-231）的单个细胞或通过在尾部造血组织（CHT）细胞统称（M2）形成簇的数目的数目每个斑马鱼19。 </li></ol></li><li>研究侵袭和转移中，通过使用共聚焦显微镜更详细地（强烈推荐）。 <ol><li>使用低倍率（4X目标），以图像的全体和以获得肿瘤细胞传播模式的概述。 注:较高的放大倍数（20X 40X和目标）是适用于研究区域内和肿瘤周围血管生成和传播细胞在胚体的精确定位。 </li><li>使用488nm的激光来扫描斑马鱼胚胎脉管系统和一个543纳米的激光来扫描标记有红色荧光植入肿瘤细胞。在八到十步扫描每个胚胎获得高品质的图像。扫描和平均每步六倍。 </li></ol></li><li>小心地将胚胎放回蛋水，如果需要进一步的实验。 </li></ol> 8.采用方差（ANOVA）的单向分析进行统计分析，随后是事后分析

<ol>
	<li>Wan, L., Pantel, K., 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=Tumor+metastasis:+moving+new+biological+insights+into+the+clinic.">Tumor metastasis: moving new biological insights into the clinic.</a> Nat. Med. 19 (11), 1450-1464 (2013).</li><li>Obenauf, A. C., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+at+a+distance:+Organ-specific+metastasis.">Surviving at a distance: Organ-specific metastasis.</a> Trends Cancer. 1 (1), 76-91 (2015).</li><li>Saxena, M., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+cancer+metastasis+in+the+mouse.">Rebuilding cancer metastasis in the mouse.</a> Mol. Oncol. 7 (2), 283-296 (2013).</li><li>Teng, Y., Xie, X., Walker, 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=Evaluating+human+cancer+cell+metastasis+in+zebrafish.">Evaluating human cancer cell metastasis in zebrafish.</a> BMC cancer. 13 (1), 453 (2013).</li><li>Konantz, M., Balci, T. B., Hartwig, U. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+xenografts+as+a+tool+for+in+vivo+studies+on+human+cancer.">Zebrafish xenografts as a tool for in vivo studies on human cancer.</a> Ann N Y Acad Sci. 1266 (1), 124-137 (2012).</li><li>Zhao, S., Huang, J., Ye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+zebrafish+from+the+perspective+of+cancer+research.">A fresh look at zebrafish from the perspective of cancer research.</a> J Exp Clin Cancer Res. 34 (1), 80 (2015).</li><li>Stanton, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diethylnitrosamine-induced+hepatic+degeneration+and+neoplasia+in+the+aquarium+fish,+Brachydanio+rerio.">Diethylnitrosamine-induced hepatic degeneration and neoplasia in the aquarium fish, Brachydanio rerio.</a> J. Natl. Cancer Inst. 34 (1), 117-130 (1965).</li><li>Lam, S. H., Wu, Y. L., Vega, V. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+gene+expression+signatures+between+zebrafish+and+human+liver+tumors+and+tumor+progression.">Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.</a> Nat. Biotechnol. 24 (1), 73-75 (2006).</li><li>Spitsbergen, J. M., Kent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+the+art+of+the+zebrafish+model+for+toxicology+and+toxicologic+pathology+research-advantages+and+current+limitations.">The state of the art of the zebrafish model for toxicology and toxicologic pathology research-advantages and current limitations.</a> Toxicol. Pathol. 31, 62-87 (2003).</li><li>Stoletov, K., Klemke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+of+the+day:+zebrafish+as+a+human+cancer+model.">Catch of the day: zebrafish as a human cancer model.</a> Oncogene. 27 (33), 4509-4520 (2008).</li><li>Lee, L. M., Seftor, E. A., Bonde, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+human+malignant+melanoma+cells+transplanted+into+zebrafish+embryos:+assessment+of+migration+and+cell+division+in+the+absence+of+tumor+formation.">The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.</a> Dev. Dynam. 233 (4), 1560-1570 (2005).</li><li>Mizgirev, I., Revskoy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+clonal+zebrafish+lines+and+transplantable+hepatic+tumors.">Generation of clonal zebrafish lines and transplantable hepatic tumors.</a> Nat. Protoc. 5 (3), 383-394 (2010).</li><li>Mizgireuv, I. V., Revskoy, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantable+tumor+lines+generated+in+clonal+zebrafish.">Transplantable tumor lines generated in clonal zebrafish.</a> Cancer Res. 66 (6), 3120-3125 (2006).</li><li>Stoletov, K., Montel, V., Lester, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+the+dynamic+tumor+cell-vascular+interface+in+transparent+zebrafish.">High-resolution imaging of the dynamic tumor cell-vascular interface in transparent zebrafish.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (44), 17406-17411 (2007).</li><li>Rouhi, P., Jensen, L. D., Cao, Z., 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=Hypoxia-induced+metastasis+model+in+embryonic+zebrafish.">Hypoxia-induced metastasis model in embryonic zebrafish.</a> Nat. Protoc. 5 (12), 1911-1918 (2010).</li><li>Drabsch, Y., He, S., Zhang, 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=Transforming+growth+factor-&#946;+signalling+controls+human+breast+cancer+metastasis+in+a+zebrafish+xenograft+model.">Transforming growth factor-&#946; signalling controls human breast cancer metastasis in a zebrafish xenograft model.</a> Breast Cancer Res. 15 (6), R106 (2013).</li><li>He, S., Lamers, G. E., Beenakker, J. W., 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=Neutrophil-mediated+experimental+metastasis+is+enhanced+by+VEGFR+inhibition+in+a+zebrafish+xenograft+model.">Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model.</a> J. Pathol. 227 (4), 431-445 (2012).</li><li>Nicoli, S., Presta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish/tumor+xenograft+angiogenesis+assay.">The zebrafish/tumor xenograft angiogenesis assay.</a> Nat. Protoc. 2 (11), 2918-2923 (2007).</li><li>de Boeck, M., Cui, C., Mulder, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad6+determines+BMP-regulated+invasive+behaviour+of+breast+cancer+cells+in+a+zebrafish+xenograft+model.">Smad6 determines BMP-regulated invasive behaviour of breast cancer cells in a zebrafish xenograft model.</a> Sci. Rep. 6, 24968 (2016).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Dev. Biol. 248 (2), 307-318 (2002).</li><li>Stewart, S. A., Dykxhoorn, D. M., Palliser, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9 (4), 493-501 (2003).</li><li>Dull, T., Zufferey, R., Kelly, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72 (11), 8463-8471 (1998).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteries+and+veins:+making+a+difference+with+zebrafish.">Arteries and veins: making a difference with zebrafish.</a> Nat. Rev. Genet. 3 (9), 674-682 (2002).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nat. Rev. Drug Discov. 4 (1), 35-44 (2005).</li></ol>

作者什么都没有透露。

在这里，我们描述了两个方法来研究乳腺癌细胞中的Tg（FLI1:EGFP）的侵入行为斑马鱼的胚胎，同卵周隙和Doc注射。通过注射标记有化学染料或荧光蛋白引入转基因斑马鱼胚胎癌细胞，侵袭和转移的动态和空间特性可以清楚地在实时的在荧光显微镜下的单细胞或群集水平跟踪。在大多数情况下，转移的斑马鱼中的快速进展确保所述测定法可以在1周内移植之后进行。此外，强大的统计数据可以?...

在临床上明显的癌转移包括一系列被称为"转移级联"复杂和多步事件。级联已经被广泛审查，并可以分割成连续的步骤:局部浸润，血管内，传播，逮捕，外渗，和殖民1，2。更好地了解癌症转移的发病机理和潜在的治疗策略体内的发展需要癌细胞扩散的强大的主机型号。啮齿动物模型已经非常成熟和被广泛用于评估转移3，但是这些方法效率低和道德的限制和昂贵的最前沿模型，以确定一个特定的操作是否会影响转移表型。需要其他高效，可靠，低成本的模式，以快速访问（EPI）的遗传变化或pharmacolog的潜在影响的iCal化合物。由于其较高的遗传同源性，人类和他们的胚胎斑马鱼（ 斑马鱼 ）的透明度已经成为一个重要的脊椎动物模型，并正在越来越多地应用于发育过程，微生物-宿主相互作用，人类疾病，药物筛选等方面的研究4。建立在斑马鱼的癌转移模型可以提供一个答案的啮齿动物模型5,6的缺点。 虽然自发瘤形成在野生斑马鱼7几乎看不到，有几种技术长期以诱导所希望的癌症在斑马鱼。致癌物诱导的基因中的突变或信号传导途径的激活可以在组织学和分子模型癌变，在斑马鱼7，8，9模仿人类疾病。通过德向前荷兰国际集团优势的多样化和反向癌基因或肿瘤抑制基因的基因操作，（转基因）斑马鱼也启用的癌症形成和维持6,10潜力的研究。在斑马鱼的诱发癌症模型涵盖广泛，包括消化，生殖，血液，神经系统和上皮6。 在癌症研究斑马鱼的利用最近扩大归因于这个有机体建立人肿瘤细胞异种移植模型中。这首先与成功，2005年11嫁接在斑马鱼胚胎的囊胚阶段的人类转移性黑素瘤细胞的报道。几个独立的实验室已经通过在不同位点和发育阶段引入哺乳动物癌细胞系不同范围到斑马鱼验证了该开创性工作的可行性5 &lt;/ SUP&gt;。例如，胚盘和囊胚期的囊胚注射附近;注射入卵黄囊，卵周隙，居维叶（DOC）的导管，和6-H-后主静脉〜5天龄的胚胎;和注射到30日龄免疫抑制幼虫的腹膜腔中已经执行5,12。此外，还报告了斑马鱼12,13同种异体肿瘤移植。对使用异种移植物的巨大优势是，移植的癌细胞可以很容易地荧光标记，并从正常细胞区分开来。因此，调查的microtumor地层14，细胞侵入和转移15，16，17，肿瘤诱导的血管生成15，1的动态行为8，和癌细胞和宿主之间的相互作用因子17可以在活鱼体被清晰可见，特别是当转基因斑马鱼线被施加5。 通过斑马鱼异种移植物模型的高电位启发来评价转移，我们证明在TG（FLI:EGFP）的尾翼区域不同的乳腺癌细胞系的经血管外渗性质通过文件注射16斑马鱼胚胎。转化生长因子β（TGF-β）16和骨形态发生蛋白的作用（BMP）在促/抗乳腺癌细胞的侵袭和转移19个信号传导途径进行了研究在该模型中。此外，我们还利用概括移植斑马鱼模型与卵周隙注射各种乳腺癌细胞系的血管内的能力进入流通。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>MP Biomedicals</td>
			<td>AGAF0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary</td>
			<td>Harvard Apparatus</td>
			<td>300038</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera enterotoxin&#160;</td>
			<td>Calbiochem</td>
			<td>227035</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>SP5 STED</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-high glucose media containing L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 media containing L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>21041025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools Inc</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Merck Millipore</td>
			<td>01-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereo microscope</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cell line</td>
			<td>American Type Culture Collection</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>SigmaAldrich</td>
			<td>227035</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>SigmaAldrich</td>
			<td>I-6634</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF10A (M1) cell line</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)&#160;</td>
		</tr>
		<tr>
			<td>MCF10Aras (M2) cell line</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)&#160;</td>
		</tr>
		<tr>
			<td>MDA-MB-231 cell line</td>
			<td>American Type Culture Collection</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual micromanipulator&#160;</td>
			<td>World Precision Instruments</td>
			<td>M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-tip Pasteur pipette (0,5-20 ul)</td>
			<td>Eppendorf</td>
			<td>F276456I</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-VSVG plasmid</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PLV-mCherry plasmid</td>
			<td>Addgene</td>
			<td>36084</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg-RRE (gag/pol) plasmid</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Dr. Rob Houben (Leiden University Medical Center, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Pneumatic picoPump</td>
			<td>World Precision Instruments</td>
			<td>SYS-PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>SigmaAldrich</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 4 software</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-REV plasmid</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg (fli:EGFP) zebrafish strain</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Dr. Ewa Snaar-Jagalska (Institute of Biology, Leiden University, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Tris-base&#160;</td>
			<td>SigmaAldrich</td>
			<td>11814273001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (3-aminobenzoic acid)</td>
			<td>SigmaAldrich</td>
			<td>A-5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5%)</td>
			<td>ThermoFisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene (60 &#215; 15 mm)</td>
			<td>SigmaAldrich</td>
			<td>P5481-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene dish with glass bottom</td>
			<td>WillCo</td>
			<td>GWST-5040</td>
			<td></td>
		</tr>
	</tbody>
</table>

invasive behavior of human breast cancer cells in embryonic zebrafish

在许多情况下，癌症病人不要原发性肿瘤的死亡，而是转移的原因。尽管众多的啮齿动物模型可用于体内研究癌症转移，需要其它有效的，可靠的，低成本的模型来快速访问的（EPI）的遗传变化或药理学化合物的潜在影响。因此，我们说明和解释采用注入斑马鱼胚胎，以支持这一目标的人类乳腺癌细胞异种移植模型的可行性。在显微镜下，荧光蛋白或化学标记的人乳腺癌细胞移植到转基因斑马鱼胚胎，TG（FLI:EGFP），在受精后48个小时的卵周隙或居维叶（DOC）的导管。不久之后，癌细胞侵袭，传播和转移在活鱼体内的时空处理在荧光显微镜下观察。使用不同的注射部位， 即模型，每ivitelline空间或文件是彼此互补，这反映了早期（血管内步骤）和晚期事件的多步转移级联的（外渗工序）。此外，瘤周和瘤内血管生成可与注入卵周隙被观察到。整个实验时间不超过8天。这两种模式相结合的细胞标记，微移植和荧光成像技术，使癌转移的快速评估响应于遗传学和药理学操作。

在具有周隙注射胚胎异种移植物模型的斑马鱼，标记的癌细胞在鱼体内的血行播散被认为是活性迁移。这个过程可以被检测到并在荧光显微镜下进行定量，如上述方法中所述。为了说明这一点异种移植模型中，我们遵循不同的乳腺癌细胞系的具有根据在体外和体内小鼠研究，包括良性正常乳腺上皮M1细胞，HRAS转化癌前已知的（或不加）浸润/转移潜在传...

Watch this Scientific Journal Video about Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish at JoVE.com

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

在这里，我们将介绍使用两个不同的注射部位， 即卵周隙和居维叶管道，调查侵入行为，并分别评估人类乳腺癌细胞的血管内和外渗潜力，异种移植模型斑马鱼。

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

invasive-behavior-of-human-breast-cancer-cells-in-embryonic-zebrafish

Research

JoVE Journal

Cancer Research

16.5K 次观看.  Leiden University Medical Center. 在这里，我们将介绍使用两个不同的注射部位， 即卵周隙和居维叶管道，调查侵入行为，并分别评估人类乳腺癌细胞的血管内和外渗潜力，异种移植模型斑马鱼。 

视频: Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

科研

癌症研究

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

乳腺癌相关的淋巴水肿的超声评价

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

测试癌细胞的斑马鱼的血管侵袭能力（<em&gt;斑马鱼</em&gt;）

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

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 ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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

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

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

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

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

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...