检定规程 B。接受美国卫生部和 #38 的资助; 人类服务, NIH, 国家癌症研究所赠款 1 K22 CA207458-01 和法学博士。接受美国卫生部和 #38 的资助; 人类服务, NIH, 国家癌症研究所赠款 1 R01 CA207311-01。

1. 多西紫杉醇浓度的测定导致50% 的菌落形成能力下降 (IC50) 注意: 在这个协议中, 多西紫杉醇 IC50 浓度已被评估使用群体形成能力.用于评估单元生存能力的替代方法 ( 即 MTS 测试) 可根据调查人员和 #39 使用; 首选项. 
<ol> <li> 在75厘米的 2 烧瓶中生长 DU145 或22Rv1 细胞, 并准备足够的10毫米多西紫杉醇稀释的亚砜, 以备将来的实验使用. </li>
	<li> 用平板细胞测定多西紫杉醇的 IC50, 从烧瓶中取出培养基, 用5毫升 PBS 仔细冲洗细胞, 用2毫升0.05% 胰蛋白酶-EDTA 在37和 #176 上孵育3-5 分钟; C 将细胞从烧瓶表面分离. </li>
	<li> 用5毫升新鲜培养基冲洗 trypsinized 细胞, 并在15毫升管中收集细胞悬浮液。颗粒细胞离心 (300 x g, 3 分钟, 室温 (RT)). </li>
	<li> 清除上清液, 重5毫升的新鲜培养基中的细胞颗粒, 并计数细胞。将细胞悬浮液稀释至 2-2. 5 x 10 5 细胞/毫升, 用于 DU145 和 4-4. 5 x 10 5 单元格/ml 22Rv1, 混合好移上下和种子2毫升的悬浮在每一个井的两个6孔板. </li>
	<li> 24 小时后, 当细胞处于 70-80% 汇合处时, 添加剂量升高的多西紫杉醇浓度 1 nm, 2.5 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 250 nm, 500 nm, 和 1000 nm 每一个井。使用多西紫杉醇 ( 图 2A ) 的最高容量处理该控制. </li>
	<li> 72 小时后, 吸药含培养基, 添加2毫升新鲜培养基。在大约4-5 天内, 板块将准备染色. </li>
	<li> 染色的殖民地, 用2-3 毫升 PBS 轻轻地清洗他们和孵化2-3 毫升水晶紫罗兰溶液 (0.1% 瓦特/v 在10% 福尔马林) 在组织培养罩或油烟罩内20分钟. </li>
	<li> 删除染色液和洗涤板2-3 毫升 h 2 O, 删除 h 2 o 和风干板. </li>
	<li> 通过可视化水井来分析结果, 以确定哪一个多西紫杉醇浓度降低 50% (IC50) 细胞活力。在标记笔的帮助下手动计数菌落 (避免两次计数相同的菌落数), 并在图中表示单元生存率的百分比 ( 图 2A )。最后, 以数字图像的板为图形表示 (如 图 3A 所示). </li>
</ol> 2。多西紫杉醇耐药前列腺癌细胞的生成 注意: 使用相同的多西紫杉醇溶液进行细胞治疗 IC50 药物浓度的测定 (提前准备足够的存货进行实验使用)。总是保持一小瓶的细胞, 从前一步, 只是以防万一的东西不能很好地工作。在整个过程中, 父母的细胞应该在一个小烧瓶中平行生长, 并在相应的体积内暴露于车辆 (亚砜) 中, 模仿多西紫杉醇治疗烧瓶中所使用的数量. 
<ol> <li> 在150厘米 2 烧瓶中 DU145 或22Rv1 单元格, 其中包含20毫升介质 (3.5 x 10 6 每瓶 DU145 和 6.5 * 10 6 单元格为 22Rv1)。建议使用10到20烧瓶的细胞在协议的末尾有足够的细胞. </li>
	<li> 24 小时后, 当单元格在70-80% 汇合处 ( 图 2B , 在多西紫杉醇之前), 在该协议的步骤1中确定的 IC50 浓度中添加多西紫杉醇 (5 nM, 用于本协议中描述的单元格). </li>
	<li> 72 小时后, 吸入药物含有培养基, 并添加新鲜, 多西紫杉醇免费媒体 ( 图 2B , 72 小时后多西紫杉醇)。每3-4 天更换一次介质。在大约1-2 周内, 抗性克隆将出现明显的显微镜下 ( 图 2B , 7 和14天后多西紫杉醇). </li>
	<li> 吸气培养基, 用15毫升 PBS 仔细冲洗细胞, 用4毫升0.05% 胰蛋白酶-EDTA 在37和 #176 上孵育3-5 分钟; C 从烧瓶表面分离细胞. </li>
	<li> 重用8毫升的新鲜培养基从烧瓶中 trypsinized 细胞。从所有被处理的烧瓶池细胞。颗粒细胞离心 (300 x g, 3 min, RT). </li>
	<li> 清除上清液, 重20毫升的新鲜培养基中的细胞颗粒, 并将细胞在150厘米 2 烧瓶 (3.5 x 10 6 细胞每瓶 DU145 和 6.5 * 10 6 细胞每瓶 22Rv1). </li>
	<li> 24 小时后, 当细胞在70-80% 汇合处, 再加入 5 nM 多西紫杉醇 (初始 IC50 浓度). </li>
	<li> 在3天重复步骤2.3 到 2.6. </li>
	<li> 24 小时后, 当细胞在大约70-80% 汇合, 治疗细胞与 2X IC50 多西紫杉醇浓度 (10 nM 的细胞在本议定书中描述). </li>
	<li> 重复步骤2.3 至 2.8, 继多西紫杉醇浓度 (25 nm、50 nm、100 nm 和 250 nm) 的剂量升级后, 在最终最高浓度下, 在您的烧瓶中生成稳定的细胞数量。对于较高浓度, 剂量-上报协议可以实现长达6月, 直到 1000 nM 浓度达到 ( 图 1A ). </li>
</ol> 3。利用菌落形成法测定多西紫杉醇的功能特性 注意: 在本协议中, 化疗使用了菌落形成分析来评估。用于评估单元生存能力的替代方法 ( 即 MTS 测试) 可根据调查人员和 #39 使用; 首选项. 
<ol> <li> 在6个井板中, 使用2毫升介质, 每井板2000细胞 (DU145 或 22Rv1, 双亲和多西紫杉醇). </li>
	<li> 24 小时后, 增加多西紫杉醇浓度 (亲代细胞: 0.25、0.5、1、2.5 和 5 nM;DR 细胞:50, 125, 250, 500 和 1000 nM)。用于 DU145 和22Rv1 细胞系。将亚甲基亚砜只添加到一个控制在同一体积, 用于最高的多西紫杉醇剂量. </li>
	<li> 后72小时, 吸入药物含有媒体和添加新鲜的多西紫杉醇免费媒体. </li>
	<li> 孵化皿1-2 周, 直到显微镜下的菌落可见. </li>
	<li> 染色的殖民地, 用2-3 毫升 PBS 轻轻地清洗他们和孵化2-3 毫升水晶紫罗兰溶液 (0.1% 瓦特/v 在10% 福尔马林) 在组织培养罩或油烟罩内20分钟. </li>
	<li> 删除染色液和洗涤板2-3 毫升 h 2 O, 删除 h 2 o 和风干板. </li>
	<li> 通过在标记笔的帮助下可视化井和手动计数菌落来分析结果 (避免两次计数相同的菌落数), 并表示图中细胞活力的百分比。采取数字图像的车牌图表示 ( 图 3A ). </li>
</ol> 4。qRT-PCR 分析多西紫杉醇耐药性表型特征的研究 注意: 与其父细胞系相比, 多西紫杉醇耐药 (DR) 细胞表现出不同的表达谱 28 , 29 . 因此, 选择的成功可以通过 qRT PCR 方法对特定标记的引物进行验证 (对于底漆序列, 参考材料部分; 有关详细信息, 请参阅结果部分)。这应该在第三轮之后的每一轮选择之后进行。另外, 也可以通过西方印迹对多西紫杉醇耐药表型进行评价. 
<ol> <li> 使用 rna 提取试剂盒从双亲和多西紫杉醇抗性细胞中提取 rna, 并遵循制造商和 #39 的指令 (请参阅 材料表 和试剂以获取详细信息)。建议至少使用 1-2 x 10 6 单元格. </li>
	<li> 用分光光度计对 260 nm 吸收的 RNA 进行量化。为了最好的纯度, 260 nm/280 纳米吸光度比应接近 2.0. </li>
	<li> 性能rm 反向转录, 以获得互补的 DNA (cDNA) 使用的反向转录试剂盒, 并遵循制造商和 #39; s 的说明 (见 材料表 和试剂的细节), 使用1和 #181; g 的 RNA 在20和 #181;反应混合 (如果需要更高的 cDNA 量, 相应地扩大反应组合). </li>
	<li> 为每种感兴趣的基因准备 qPCR 反应, 如下所示: -2 和 #181; 10 和 #181; m 向前底漆 -2 和 #181; l 10 和 #181; m 反转底漆 -10 和 #181; l SYBR 绿色 PCR 大师组合 -5 和 #181; l H 2 O -1 和 #181; 步骤4.3 的新制备的 cDNA. 
	注: 有关本协议中使用的底漆序列, 请参见 材料表 . </li>
	<li> 将 PCR 程序设置为: -95 和 #176; c 为15分钟 -94 和 #176; c 为三十年代和 #160; #160; #160; 和 #160; #160; | 
	-56、#176; 四十年代、#160、#160、#160、#160、#160、#160; 40 次 -72 和 #176; 三十年代和 #160; #160; #160; #160; 和) #160; | </li>
	<li> 要分析 qPCR 结果并确定父母和 DR 样本之间基因表达的变化, 每一个感兴趣的基因的周期阈值 (Ct) 都是确定的, 平均归入控制 (肌动蛋白三份平均值) 对 dr (ct) 和父母细胞 (ct 父母)。父单元格的值被规范化为1。#916, ct (ct) 的父母是确定的, 以获得最后的 mRNA 折叠的变化和代表在一个图形。增加和 #62; 2 或减少和 #60; 0.5 被认为是重要的和良好的验证建立多西紫杉醇获得耐药表型 ( 图 3B ). </li>
</ol>

<ol>
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作者声明他们没有竞争的金融利益。

在这篇手稿中, 我们描述了一种方法来产生多西紫杉醇耐药的前列腺癌细胞模型系统, 允许研究的机制, 有助于获得化疗表型。虽然这种方法是高度可靠和可重复性的, 但应该考虑一些潜在的限制。由于只有一小部分的细胞的大部分人口将展出化疗28, 建议开始与大量的细胞 (我们通常板20烧瓶150厘米2, 占约 1.2 x 108细胞)。应考虑该议定书的关键步骤, 以确保程序的成功。首先, 这是非常重要的使用相同的多西紫杉醇新鲜准备的股票长期使用 (10 毫米股票等分可用于多年时, 储存在适当的-80 ° c)。多西紫杉醇分的微小变化会影响 IC50 的结果、细胞线定时的生成和菌落形成的结果。其次, 在每个单独的单元格中开始确定 IC50 的协议是至关重要的, 因为在使用新的一批单元格时可能会发生变化。第三, 监测药物治疗的有效性, 必须经常检查显微镜下的细胞, 以便及时发现和纠正任何错误。最后, 我们建议始终保持一股中间步骤的情况下, 污染或意想不到的问题, 可能会影响细胞的生存能力在协议的中间。重要的是, 我们的协议的目的是产生耐药性的模式, 通过暴露前列腺癌细胞的高剂量多西紫杉醇72小时后, 长期恢复期, 而不是较低的恒定浓度的药物试图模仿最好的临床使用此紫杉代理15,16进行前列腺癌治疗的情况报告。
此外, 应该指出, 耐药细胞表现出很高的可塑性, 这可能导致逐渐丧失获得的耐药性的特性随着时间的推移。因此, 不建议在文化中延长这些细胞的使用超过2-3 月。如果需要永久性的供应, 最好不断地生成新的抗性细胞。然而, 如果大量的细胞被培养了很长时间, 菌落形成分析和 qPCR 可以用来重新评估他们的抵抗力。另一种方法是通过对72小时 (500-1, 000 nM) 高剂量多西紫杉醇的细胞进行治疗, 从而提高抗衰退性。经过一、两周的恢复期后, qPCR 和菌落形成分析可重新表型。也有可能, 虽然不建议, 储存等分的处理细胞在液氮 (在 FBS, 10% 亚甲基亚砜)。请注意, 在冻融循环后, 化疗表型应始终用上述方法进行重新评估。
尽管有这些警告, 我们28、29和其他30、31、32、33都能够成功地使用这些模型系统来识别关键的新型蜂窝和分子机制导致肿瘤启动能力升高, 细胞存活, 多西紫杉醇耐药细胞的侵袭性。利用这些癌细胞模型系统, 我们发现细胞表现出未分化的表型, 特征是缺乏上皮和前列腺相关的分化标记和过度表达的可发育 (缺口和刺猬) 信号通路, 生存化疗暴露28。在第二项研究中, 使用这些模型与公开提供的患者基因数据集24,25, 我们发现先驱转录因子 GATA2 是高度表达的转移性去势耐药性抗化疗的前列腺癌细胞。GATA2 通过直接激活 IGF2-dependent 下游激酶信号网络29来提高细胞的存活率。值得注意的是, 这些研究有助于鉴别 GATA2 在晚期转移性前列腺癌中的新的关键作用34。
耐药性的发展是一个不限于多西紫杉醇和前列腺癌的问题, 因为它在许多其他类型的癌症治疗中普遍存在, 包括多种化疗药物。实际上, 对实验模型的可用性也有类似的限制, 可以想象, 我们的协议可用于研究其他癌症类型及其各自的化疗机制。
本协议所述的多西紫杉醇耐药细胞的生成可作为一种功能性工具, 用于鉴别化疗的相关分子和细胞机制。这为寻找新的目标打开了大门, 对高恶性前列腺癌的新治疗方法的发展可能会阐明, 再次推动我们对这种疾病的了解, 以及我们如何治疗病人的界限。

细胞周期控制的丢失导致的增殖率增加是癌症的一个标志1。这种功能性的特性使癌细胞在 s-和 M 阶段的细胞周期的关键过程中具有独特的依赖性, 从而导致了各种细胞周期扰动药物的形成, 引起 DNA 损伤或有丝分裂缺陷。虽然许多 anti-mitotic 剂, 如有丝分裂激酶或运动蛋白抑制剂, 目前正在开发和测试的临床试验, 传统微管靶向剂 (mta) 继续是唯一的临床认可的方法靶向肿瘤有丝分裂2,3,4。mta 不稳定 (长春, 长春碱, 长春瑞滨) 或稳定性 (紫杉衍生的药物紫杉醇, 多西紫杉醇或 Cabazitaxel) 微管, 从而防止形成一个功能分裂纺锤5,6。这些代理触发主轴组件检查点7,8, 这会导致培养细胞中的有丝分裂停止和最终细胞死亡9,10和肿瘤中的有丝分裂缺陷11 ,12 , 因此对治疗多种癌症非常有用, 其中前列腺癌的患者有13。
前列腺癌是最常见的癌症, 是导致男性癌症相关死亡的主要原因14。多西紫杉醇等有丝分裂剂改善前列腺癌患者的生存率, 是治疗本病的主要15,16,17。不幸的是, 尽管最初的肿瘤萎缩, 肿瘤细胞往往发展抗药性在治疗期间。一些细胞机制已经牵连到导致化疗的发展, 包括解除对凋亡和炎症通路的放松, 或像 ATP 结合盒式转运器的药物外排泵的活化/过度表达p-糖蛋白 (P-gp/ABCB1), 后者的结果是输出的化疗药物的细胞18,19。对紫杉的抗性也与其他原因相关, 如蛋白 isotypes 或蛋白突变表达的变化, 这就阻止了药物与其目标2021之间的交互。此外, 抗病性一直与基因组不稳定积累和肿瘤非均质22,23有关。然而, 导致紫杉抵抗的机制还没有完全被理解, 因为缺乏治疗策略来防止其出现。因此, 迫切需要产生实验模型, 协助解剖这些机制, 并确定新的治疗方法, 防止对这些药物的抗药性的发展。
在这篇文章中, 我们描述了一种方法, 允许产生多西紫杉醇耐药 (DR) 前列腺癌细胞。我们将进一步描述如何通过菌落形成检测和定量 rt-pcr 来验证这些细胞的抗性状态。这种方法产生的细胞具有综述的优势, 可以在公开提供的人类肿瘤样本数据库中发现许多分子特征, 并增加了提供多功能性以进行多种实验分析的好处。比肿瘤标本所允许的时间长。这些癌症模型的治疗耐药性可以扩展许多周的组织培养, 并在其他方法, 可以遗传操作, 可视化的显微镜方法和分析的基因表达。此外, 它们还可以在小鼠体内xenotrasplanted, 进一步分析肿瘤的形成和生长的作用。目前, 研究前列腺癌耐药性的主要替代方法是直接分析肿瘤标本。虽然这些样本提供了一个非常强大的系统收集有关基因表达和突变状态的特定肿瘤的信息, 他们的访问可能是非常有限的, 他们很难保持进一步的操纵和实验分析,可以很容易地用此协议生成的单元格行24,25。重要的是, 在未来, 替代方法, 如人类衍生 organoids 直接从病人的生成将是一个非常强大的工具和替代目前的方法26,27。因为他们将在3D 的背景下重述肿瘤在其原始环境中的情况, organoids 将是细胞系和人类肿瘤之间的完美模型。然而, 本协议中所描述的实验性细胞模型仍然更能维持并适于更快的分析。通过研究这些细胞系的结果可以推断和证实在人类样本和3D 文化。因此, 这项协议可能是感兴趣的研究人员寻求细胞模型研究化疗机制在任何细胞系, 因为我们的协议可以适应研究的其他药物和癌症类型。这里描述的方法很容易应用于任何实验室, 价格适中, 并允许对耐药性分子和细胞机制的初步研究。

<table><tbody><tr><td>DU145 cells</td><td>ATCC</td><td>HTB-81</td><td></td></tr><tr><td>22Rv1 cells</td><td>ATCC</td><td>CRL-2505</td><td></td></tr><tr><td>Docetaxel</td><td>Selleck Chemicals</td><td>S1148</td><td>in DMSO (10mM)</td></tr><tr><td>Tissue culture flask 150cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Falcon</td><td>355001</td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>11875093</td><td>Supplemented with 10% FBS and 1% Penicilin/Streptomycin</td></tr><tr><td>Foundation B Fetal Bovine Serum</td><td>Gemini</td><td>900208</td><td></td></tr><tr><td>1X Phosphate-Buffered Saline (PBS)</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>0.05%Trypsin-EDTA</td><td>Gibco</td><td>25300-062</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>SuperScript III first strand synthesis system for RT-PCR</td><td>Invitrogen</td><td>18080051</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>Invitrogen</td><td>4367659</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma-Aldrich</td><td>C0775</td><td></td></tr><tr><td>10% Formalin Solution</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Primers for qRT-PCR:</td><td>Life technologies</td><td></td><td></td></tr><tr><td>CK19 F: CTGCGGGACAAGATTCTTGGT</td><td></td><td></td><td></td></tr><tr><td>CK19 R: CCAGACGGGCATTGTCGAT</td><td></td><td></td><td></td></tr><tr><td>CDH1 F: GGAAGTCAGTTCAGACTCCAGCC</td><td></td><td></td><td></td></tr><tr><td>CDH1 R: AGGCCTTTTGACTGTAATCACACC</td><td></td><td></td><td></td></tr><tr><td>ABCB1 F: TGTTCAAACTTCTGCTCCTGA</td><td></td><td></td><td></td></tr><tr><td>ABCB1 R: CCCATCATTGCAATAGCAGG</td><td></td><td></td><td></td></tr><tr><td>GATA2 F: CATCAAGCCCAAGCGAAGA</td><td></td><td></td><td></td></tr><tr><td>GATA2 R: TTTGACAGCTCCTCGAAGCA</td><td></td><td></td><td></td></tr><tr><td>ACTIN F: CATGTACGTTGCTATCCAGGC</td><td></td><td></td><td></td></tr><tr><td>ACTIN R: CTCCTTAATGTCACGCACGAT</td><td></td><td></td><td></td></tr></tbody></table>

generation of prostate cancer cell models of resistance to the anti-mitotic agent docetaxel

微管靶向剂 (mta) 是治疗范围广泛的肿瘤的主干。然而, 获得的抗化疗药物是一种常见的疾病进展机制和预后决定因素的特点恶性肿瘤。在前列腺癌 (PC), 抗 mta, 如紫杉多西紫杉醇规定治疗失败, 以及进展到致命阶段的疾病, 是由一个糟糕的预测和高死亡率的定义。尽管研究了几十年, 但对后天抵抗的一系列机制还没有完全理解, 因此对新的治疗策略的发展有很大的限制, 这些战略可以使患者在这些先进阶段受益。疾病.在本议定书中, 我们描述了一代多西紫杉醇耐药前列腺癌细胞系模仿晚期前列腺癌的致命特征, 因此可以用来研究的机制, 获得化疗产生。尽管细胞模型存在潜在的局限性, 如随着时间的推移阻力特性的丧失, 这种方法所产生的多西紫杉醇抗性细胞系已经成功地应用于最近的研究中, 并提供了促进我们在致命的前列腺癌中获得化疗的分子理解。

这项协议将导致产生多西紫杉醇耐药 (DR) 前列腺癌细胞。在图 1A和图 1B中的协议步骤的示意图表示中总结的完成时间范围可以从4到7月不等。时间投资可能会因选择的细胞线和协议中所描述的药物浓度范围而不同。重要的是, 每一个细胞线的多西紫杉醇 IC50 浓度的测定, 使药物浓度的设计在协议中使用的细胞的选择 (图 2A)。一旦协议开始, 在显微镜下的不同时间点可以观察到 DU145 和22Rv1 细胞的初始多西紫杉醇效应, 以监测该协议是否正常工作。建议的时间点, 以监测多西紫杉醇治疗过程是: 前多西紫杉醇暴露 (基线) 后, 多西紫杉醇暴露72小时, 7 天14天。注意, 只有少数肿瘤细胞存活的多西紫杉醇暴露和生成克隆, 这可以在显微镜下观察 (图 2B)。
在图 3A中显示了父母和新生成的多西紫杉醇耐药细胞 (DR) 的代表性菌落形成分析和量化图。请注意, 所产生的多西紫杉醇耐药细胞可以生存的药物浓度高达1000倍以上的父母细胞。
最后, 通过 qRT PCR (图 3B) 可以验证多西紫杉醇耐药表型。请注意, 与亲本细胞相比, 多西紫杉醇耐药细胞显示了 ABCB1 和 GATA2 的特征, 并对 CK19 和 CDH1 进行了调节。这些基因被选择进行验证, 因为我们已经在我们以前发表的著作 GATA2 上调和下调的 CK19 与 CDH1 一起在 DR 单元格模型28,29中进行了实验演示。ABCB1 基因也被选择作为一个主要基因, 已经显示了其他人一致上调在耐药细胞18,19。然而, 重要的是要注意, 其他基因可以选择根据文献, 并根据细胞模型和药物选择产生耐药细胞由研究员。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56327/56327fig1.jpg"> 
图 1: 生成多西紫杉醇耐药前列腺癌细胞模型的时间线.(A) 关于生成多西紫杉醇耐药细胞模型的协议时间线的代表图。插图描述了多西紫杉醇治疗、验证步骤和每个部分所需的时间。(B) 方案的验证步骤的示意图表示, 包括对父母和多西紫杉醇耐药细胞的功能菌落形成测定 72 h (红色) 的多西紫杉醇浓度, 代表菌落百分率图和 qRT PCR 用于表型验证。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56327/56327fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56327/56327fig2.jpg"> 
图 2: 生成多西紫杉醇耐药前列腺癌细胞模型系统.(A) 图, 描述在父母前列腺癌细胞系 DU145 和22Rv1 中多西紫杉醇 IC50 的测定 (《议定书》1步)。所需的细胞活力和多西紫杉醇剂量升高的预期百分比包括在内。有代表性的 DU145 和22Rv1 细胞活力图表明 5 nM 作为 IC50 后72小时的多西紫杉醇暴露包括在内。实验是 triplicates 的, 数据代表了平均± SD. (B) 典型的 DU145 和22Rv1 细胞在多西紫杉醇世代抵抗期间的明亮场显微图像 (协议的步骤 2)。红色箭头表示该协议完成后, 多西紫杉醇抗性克隆。缩放条形图 = 50 nm。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56327/56327fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56327/56327fig3.jpg"> 
图 3: 多西紫杉醇耐药细胞模型的功能和表型特征.(A) 具有代表性的菌落形成测定方法治疗的父母和多西紫杉醇抗性细胞的显示 72 h. 每种治疗浓度的菌落百分比均在包含图中表示。实验是 triplicates 的, 数据代表了平均± SD. 刻度条 = 5 mm (B), 显示了 ABCB1、GATA2、CK19 和 CDH1 的 qRT PCR 法定量化的 DR/双亲相对 mRNA 的增加。所有 qPCR 的原始数据都被规范化为肌动蛋白 (详见协议)。实验是 triplicates 的, 数据代表了平均± SD #60; 0.05。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56327/56327fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel at JoVE.com

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

抗肿瘤治疗有助于疾病的进展和死亡。确定阻力的机械基础对于改善治疗反应是至关重要的。这份手稿详细的协议, 以产生紫杉抗性细胞模型的前列腺癌 (pc), 以帮助解剖的路径涉及的进展, 多西紫杉醇耐药性的 pc 患者。

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

generation-prostate-cancer-cell-models-resistance-to-anti-mitotic

Nanyang Technological University

Research

JoVE Journal

Cancer Research

13.2K 次观看.  Icahn School of Medicine at Mount Sinai. 抗肿瘤治疗有助于疾病的进展和死亡。确定阻力的机械基础对于改善治疗反应是至关重要的。这份手稿详细的协议, 以产生紫杉抗性细胞模型的前列腺癌 (pc), 以帮助解剖的路径涉及的进展, 多西紫杉醇耐药性的 pc 患者。

视频: Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

科研

癌症研究

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

雄激素依赖性和去势抗性前列腺癌的荧光自体小鼠模型

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

遗传学

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

前列腺有机体培养作为工具，将基因型和突变谱转化为药理反应

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

医学

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

人前列腺癌的临床前原位小鼠模型

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Crystal Violet Staining: A Technique to Visualize Drug-Resistant Cancer Cells Using Colony Formation Assays

Source: Mohr, L. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/56327">Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell viability assay using a crystal violet staining procedure for chemotherapeutic drug-resistant cancer cells.

结晶紫染色:一种使用集落形成测定法可视化耐药癌细胞的技术

Source: Mohr, L. et al., Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel. J. Vis. Exp. (2017)
In this video, ...

JoVE Encyclopedia of Experiments

Biological Techniques

Staining Techniques

Cell staining

Generating Chemoresistant Prostate Cancer Cells: A Procedure for Obtaining Drug-resistant Cancer Cells In Vitro

Source: Mohr, L. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56327" target="_blank">Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel.</a> J. Vis. Exp. (2017)
This video describes a protocol for generating taxane-resistant cell models of prostate cancer. These models can help study the pathways involved in progression to Docetaxel resistance in prostate cancer patients.

产生化疗耐药性前列腺癌细胞:体外获得耐药癌细胞的程序

Source: Mohr, L. et al. Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel. J. Vis. Exp. (2017)
This video ...