Name: preclinical assessment of the bioactivity of the anticancer coumarin ot48 by spheroids, colony formation assays, and zebrafish xenografts
Uploaded: 2018-06-26T13:00:00.000+00:00
Duration: 9 min 20 s
Description: Watch this Scientific Journal Video about Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts at JoVE.com

首尔大学的研究由韩国 MEST 的国家研究基金会 (NRF) 支持, 用于肿瘤微环境全球核心研究中心 (GCRC) 赠款, [赠款编号 2011-0030001];由汉城国立大学研究补助金和由脑韩国 (BK21) 加节目。

1. 菌落形成测定
<ol><li>培养 A549 细胞在2万个细胞/cm2在15毫升 RPMI1640 细胞培养培养基补充10% 个血清 (胎牛血清) 和1% 个抗生素在 75 cm2瓶在 CO2个孵化器在37°c 和 5% CO2。</li>
	<li>在实验当天, 使用 hemocytometer 确定细胞浓度。以 400 x g为7分钟, 在1.5 毫升离心管中收集5万个细胞, 并在100毫升管中以1.5 µL 的1x 无菌 PBS (磷酸盐缓冲盐水) 重新悬浮细胞。</li>
	<li>将1.1 毫升半固态纤维素培养基 (MCBM) 在15毫升管中, 用 multipipette。为每个条件准备一个管。</li>
	<li>使用 P200 微添加110µL 的 MCBM 1.1 毫升的血清。</li>
	<li>种子细胞在1000细胞/毫升。收集2.4 µL 细胞悬浮液从股票溶液 (5万细胞100µL 1x PBS) 使用 P2 微和增加1.1 毫升 MCBM 补充10% 血清。</li>
	<li>在添加细胞后, 将管子涡旋1分钟, 以最高的速度垂直排列, 以使 MCBM 和细胞混合良好。</li>
	<li>添加测试化合物 (单独 OT48 或与 A1210477 组合)。准备一个单独的管作为一个控制溶剂使用 (这里二甲基亚砜)。根据化合物的浓度和每个条件添加的体积, 溶剂控制将包括最高浓度的溶剂用于稀释的试验化合物, 以评估溶剂所造成的不利影响。</li>
	<li>涡流管1分钟。</li>
	<li>对于每种化验, 添加1毫升的混合物与 P1000 微到中心井的12井细胞培养板。</li>
	<li>用1毫升的无菌水或 1x PBS 填充空水井, 以稳定湿度。</li>
	<li>孵化培养皿10天在孵化器在37°c 和 5% CO2。</li>
	<li>经过10天的孵化, 添加200µL 的 MTT (methylthiazolyldiphenyl-四氮唑溴) 股票溶液 (5 毫克/毫升), 以达到最终浓度的1毫克/毫升。</li>
	<li>孵化10分钟在孵化器在37°c 和 5% CO2。生存的殖民地将变成紫罗兰。</li>
	<li>使用数码相机拍摄图像, 使用白色背景板 (白径托盘)。使用以下设置: 方法, 数字化;曝光类型, 精度;曝光时间, 手动, 1 秒;灵敏度/分辨率高分辨率。以 tiff 格式保存图像。</li>
	<li>使用 ImageJ 软件量化可行的殖民地。选择菜单 "图像 |类型 |8位 "。选择菜单 "调整 |门槛 "。设置阈值以控制和保持恒定的所有条件;选择菜单 "分析 |分析粒子 "。使用统计软件保存数据并分析图形表示。</li>
</ol>2. 球形地层测定
<ol><li>培养 A549 细胞在2万个细胞/cm2在 75 cm2瓶与15毫升的 RPMI 1640 中等补充与10% 血清和1% 抗生素。</li>
	<li>在实验当天, 准备一个96井的 U 型底板。</li>
	<li>板1万细胞入井已经包含兴趣化合物 (这里50µM OT48 并且/或者20µM A1210477)。调整最终体积到100µL 细胞培养培养基。形成具有完整外边界的均匀球体的细胞数被认为足以形成球体。</li>
	<li>孵化3天在孵化器在37°c 和 5% CO2。</li>
	<li>经过3天的孵化, 捕捉图像的球体使用光显微镜与4x 放大倍数。</li>
</ol>3. 斑马鱼的移植试验
注意: 这种技术方法被可视化为示意图 (图 1)。
<ol><li>
库存试剂的制备
	<ol>
<li>准备1升 30x Danieau 的溶液: 1740 毫米氯化钠, 21 毫米氯化钾, 12 毫米 MgSO4∙7H2O, 18 毫米钙 (无3)2, 150 毫米 HEPES。使用 ph 计调整到缓冲液的 ph 值为7.6。</li>
		<li>准备 1% (50x) tricaine 溶液: 在2毫升蒸馏水中溶解20毫克 3-氨基苯甲酸磺酸乙酯 (tricaine)。</li>
		<li>制备1% 苯酚红 pbs 溶液: 在1.5 µL 管中加入2µL 酚红钠盐溶液, 以198µL 消毒 pbs 溶液。</li>
		<li>准备3% 纤维素: 添加3克纤维素到100毫升 1x Danieau 的解决方案。</li>
	</ol>
</li>
	<li>
斑马鱼卵的生产与孵化
	<ol>
<li>将一只雌性和一只雄性斑马鱼分开, 用紫外线灭菌和过滤的自来水在28.5 摄氏度的黑暗中填充。经过24小时的分离, 混合两个性别的鱼开始交配。将受精卵从交配池转移到含有5毫升新鲜 Danieau 溶液的培养皿中。</li>
		<li>用5毫升的 Danieau 溶液洗鸡蛋三次。对于洗涤, 小心地用吸管吸入鸡蛋, 并转移到5毫升的新鲜溶液。在28.5 摄氏度孵育8小时。<ol>
<li>在 Danieau 的溶液中孵化8小时后, Danieau 的溶液中含有 0.03% 1 苯基2硫脲 (PTU) 以抑制色素沉着。整理出不透明的卵 (未受精或含有未发育的胚胎) 以防止污染。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
胚 dechorionation
	<ol>
<li>24 h 后受精 (hpf), dechorionate 斑马鱼胚胎使用锋利的钳。孵化胚胎在 Danieau 的解决方案与 0.03% PTU 在28.5 °c, 直到 48 hpf。</li>
	</ol>
</li>
	<li>
复方处理细胞制剂
	<ol>
<li>种子 A549 细胞在2万个细胞/cm2在 25 cm2培养瓶。在 24 h 以后播种, 增加化合物 (这里 OT48 在50µM 和并且/或者 A1210477 在20µM) 为 24 h 在孵化器在37°c 和 5% CO2。设置每种化合物的治疗时间, 以维持细胞的生存能力, 但要将它们用于细胞死亡。这个时间点需要根据细胞的生存能力, 形态学和分子标记单独建立。</li>
		<li>2 h 在治疗结束前, 染色细胞与4µM 荧光细胞追踪染料 CM-Dil 在37°c 和 5% CO2。经过2小时的孵化, 胰蛋白酶处理细胞与0.05% 胰蛋白酶-EDTA, 稀释 106细胞在50µL 苯酚红 PBS 溶液注射前。</li>
	</ol>
</li>
	<li>
微注射癌细胞对异种异体形成的研究
	<ol>
<li>麻醉斑马鱼在一个 0.02% tricaine 溶液5分钟, 直到他们定居在一个琼脂板块。</li>
		<li>通过微拉拔 (程序设置: 300, 热: 260, 拉:60, 威:50, 时间: 200) 拉动1.0 毫米 (外径) 玻璃毛细管)。用注射器切开 microcapillary, 以产生锋利的边缘。负载 microcapillaries 与20µL 细胞/苯酚红 PBS 溶液。设置注射压力和时间, 以分配 2 nL 每注入。</li>
		<li>将100–200细胞注入蛋黄囊中, 通过3到5注射注射6到 10 nL。注射后, 允许鱼恢复10至30分钟5毫升新鲜 Danieau 的解决方案包含 PTU 溶液。调度鱼在24井板与1毫升 Danieau 的解决方案包含 PTU 溶液是和孵化72小时在28.5 摄氏度。</li>
		<li>经过72小时的孵化, 麻醉鱼在一个 0.02% tricaine 的解决方案和固定在玻璃滑动与下降3% 纤维素。以620到 750 nm 的波长荧光显微镜拍摄5x 图片。</li>
		<li>用 ImageJ 软件量化荧光肿瘤的大小。选择菜单 "分析 |措施 "。保存与荧光信号对应的值, 并用统计软件分析图形表示。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

MCBM 获得的菌落形成率取决于细胞类型。通常, 对于非黏附细胞来说, 与黏附细胞相比, 菌落数量要高得多。我们观察到, A549 细胞在10天后形成了30到40个菌落。以前我们已经报告了不同的白血病细胞, 殖民地的数量在200到 2509之间。我们的研究结果表明, 单独的 OT48 并没有显著减少菌落数量。然而, 结合 BH3 模拟 A1210477 A549 细胞的菌落形成能力协同降低。此前我们报道, 另一种 hydroxycoumarin 衍生物 OT52 也协同 BH3 mimetics 抑制 A549 细胞的菌落形成能力。建议的播种浓度可能从 1 x 103到 1.5 x 10 3不等。CFA 使用 MethoCult 是一种有价值的技术, 通常用于测量肿瘤细胞系的肿瘤发生, 但有一定的局限性。通常在实验后恢复存活的菌落是不方便的, 而细胞一旦从 MCBM 和 RPMI1640 培养基中分离出来就不会生长良好。然而, 它是一个公认的前临床技术, 以更好地了解癌症进展的机制, 在3D 的环境。3D 生长条件准确地反映了体内环境下肿瘤形成的自然环境。然而, 这种方法是缓慢的, 劳力密集, 不适合高通量筛选10。
SFAs 是用黏附细胞进行的, 在癌症药物发现中得到广泛的普及, 因为它们表现出细胞存活率增加、肿瘤形态学和缺氧核心等生理特性, 而在体内情况更具代表性。11,12。由 U 底板技术获得的球体的整体尺寸、质地和完整性可能会因细胞系而异。此外, 用于形成球体的细胞的起始浓度也可能影响其整体发育。结果表明, OT48 与 BH3 mimeticA1210477 结合, 抑制了球体的完整性, 降低了 A549 细胞的球体形成能力。以前, 我们报告了另一个 hydroxycoumarin 衍生物与 BH3 mimetics9抑制 A549 球体。该技术的主要优点是重现性和成本效益。其他技术, 如液体叠加技术也经常用于开发3D 球体的癌细胞。在这种技术细胞生长在一个非黏附细胞培养皿。细胞团簇然后被收集和放置在悬浮培养形成球体。然而, 与此技术相关的主要限制之一是细胞团簇转移到悬浮培养中, 而不同细胞系的耐受性并不良好。为了提高复合筛选的吞吐量, 这种3D 的养殖系统, 一些公司正在开发特定的基质, 低附着检测板和脚手架, 以改善3D 结构的形成13。随着进一步的技术进步, 开发一个更具代表性的3D 肿瘤模型, 将有助于更好地理解新化合物的药理学的特定类型的癌症。
斑马鱼的移植被认为是最具成本效益的动物化验, 通常用于抗癌药物的发展9,14。我们优化了斑马鱼体内系统的各种固体和血液癌类型以及患者派生细胞。使用斑马鱼进行药物筛选有许多优点。原则上, 药物可以直接添加到水中, 不需要注射。此外, 这种方法还提供有关药物候选者的 ADME (吸收、分布、新陈代谢和排泄) 特性的信息。然而, 这一技术的主要局限性之一是某些分子不能溶于水, 因此不能立即适用于这种方法的研究。为了克服这一局限性, 癌细胞在细胞被植入的阶段被注射到斑马鱼体内, 但仍保持生存能力。结果表明, OT-48 或 BH3 模拟 A1210477 治疗的 A549 细胞与 A549 和 OT48 的组合相比, 并没有强烈降低 A1210477 细胞的肿瘤形成能力。这一观察进一步证实了我们的体外CFA, 其中结合显示了显著的抗癌效果相比, 个别治疗。总之, 这项技术可以帮助快速筛选药物在临床前的设置。

癌症是由细胞突变引起的, 因此生化信号通路被打乱, 触发不受控制的细胞分裂和抵抗细胞死亡。肿瘤会影响消化、神经、循环系统和随后邻近组织的生理功能1。尽管广泛的研究工作, 癌症仍然是最普遍的威胁生命的疾病在世界2。精密医学已被公认为未来癌症治疗的基础。新的分子实体与现有药物进行例行测试, 以改善临床结果。
然而, 与开发新的有效靶向疗法相关的一个重要的限制是, 通常使用的化验方法未能模拟药物暴露的确切生物学结果3。癌症药物发现仍然主要依赖于检测在2D 单层培养的癌细胞系中的治疗药物的功效, 这在临床试验中难以证实4。因此, 发展更好的癌症模型, 更好地模仿在体内5的肿瘤的本土特征, 是一个越来越大的兴趣。近年来, 对3D 培养模型的兴趣越来越大, 导致了改进的方法, 以生成3D 肿瘤模型5。
在这里, 我们提出了一个方法与三种不同的3D 细胞培养技术, 允许提高对 hydroxycoumarin OT486的效力的理解, 结合 BH3 mimetics 更深入的体内测定。我们的方法包括结合菌落和 SFAs 与一个在体内的肿瘤形成试验, 基于斑马鱼模型, 以进一步验证 OT48 和 BH3 mimetics 联合在肺癌细胞和监测癌症进展在生活中的效果生物。
菌落形成测定通常用于评估抗癌药物对固体和造血肿瘤的疗效。该化验确定细胞无限增殖的能力, 形成殖民地7。抗癌剂对细胞菌落形成能力的影响由菌落数量和/或大小的减少决定。
球体代表体外肿瘤模型, 作为一种低成本的抗癌药物筛选平台。球体是悬浮或嵌入在3D 基质中的细胞聚集物。该方法广泛应用于药物筛选和肿瘤生长、增殖和免疫相互作用的研究8。
为了充分了解新药的特性, 对啮齿动物进行体内实验是非常必要的。然而, 这种传统的方法是昂贵和费时。近年来, 斑马鱼 (斑马斑马) 成为了一个广泛研究的实验室有机体, 更便宜, 更快地提高。在斑马鱼开发的肿瘤代表了3D 细胞培养方法, 但在体内设置的脊椎动物9。
总之, 我们在这里使用三种不同的3D 文化方法, 包括 CFAs, SFAs 和斑马鱼在体内肿瘤形成, 以证明 hydroxycoumarin OT48 的抗癌能力的肺癌 A549 细胞模型结合 BH3 mimetics。

<table><tbody><tr><td>&lt;strong&gt;Materials required for colony formation assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-Streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>1.5ml tube</td><td>Extragene</td><td>Tube-170-C</td><td>RT</td></tr><tr><td>15 ml tube</td><td>Hyundai Micro</td><td>H20015</td><td>RT</td></tr><tr><td>12 well plate</td><td>SPL</td><td>30012</td><td>RT</td></tr><tr><td>MethoCult</td><td>StemCell technologies</td><td>4230</td><td>-20 C&amp;deg;</td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide (MTT powder)</td><td>Sigma</td><td>M5622</td><td>4 C&amp;deg;</td></tr><tr><td>LAS4000</td><td>GE Healthcare Technologies</td><td></td><td>RT</td></tr><tr><td>&lt;strong&gt;Materials required for spheroid formation assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25-300-054</td><td>4 C&amp;deg;</td></tr><tr><td>Corning costar ultra low attachemnt 96 well plate</td><td>Corning</td><td>3474</td><td>RT</td></tr><tr><td>Microscopy</td><td>Nikon</td><td>Eclipse TS100</td><td>RT</td></tr><tr><td>&lt;strong&gt;Materials required for zebrafish xenografts&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T25</td><td>SPL</td><td>70025</td><td>RT</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>Cell culture flask T175</td><td>SPL</td><td>71175</td><td>RT</td></tr><tr><td>1.5ml tube</td><td>Extragene</td><td>Tube-170-C</td><td>RT</td></tr><tr><td>24 well plate</td><td>SPL</td><td>30024</td><td>RT</td></tr><tr><td>Petridish</td><td>SPL</td><td>10100</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25-300-054</td><td>4 C&amp;deg;</td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>71382</td><td>RT</td></tr><tr><td>Potassium chloride (KCL)</td><td>Sigma-Aldrich</td><td>P9541</td><td>RT</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO4.7H2O)</td><td>Sigma-Aldrich</td><td>M2773</td><td>RT</td></tr><tr><td>Calcium nitrate tetrahydrate (Ca(NO3)2)</td><td>Sigma-Aldrich</td><td>C1396</td><td>RT</td></tr><tr><td>HEPES solution</td><td>Sigma-Aldrich</td><td>H0887</td><td>RT</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma-Aldrich</td><td>E10521</td><td>RT</td></tr><tr><td>Phenol Red solution</td><td>Sigma-Aldrich</td><td>P0290</td><td>RT</td></tr><tr><td>Methylcellulose</td><td>Sigma-Aldrich</td><td>M0512</td><td>RT</td></tr><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</td><td>RT</td></tr><tr><td>CM-Dil dye</td><td>Invitrogen</td><td>C7001</td><td>-20 C&amp;deg;</td></tr><tr><td>Glass capillary</td><td>World Precision Instruments</td><td>TW 100F-4</td><td>RT</td></tr><tr><td>Micropipette puller</td><td>Shutter instrument, USA</td><td>P-97</td><td>RT</td></tr><tr><td>Micro injector</td><td>World Precision Instruments</td><td>PV820</td><td>RT</td></tr><tr><td>Syringe</td><td>KOVAX</td><td>1ml</td><td>RT</td></tr><tr><td>Micro loader</td><td>Eppendorf</td><td>5242956003</td><td>RT</td></tr><tr><td>Glass slide</td><td>Marienfeld</td><td>HSU-1000612</td><td>RT</td></tr><tr><td>Fluorescence microscopy</td><td>Leica</td><td>DE/DM 5000B</td><td>RT</td></tr></tbody></table>

preclinical assessment of the bioactivity of the anticancer coumarin ot48 by spheroids, colony formation assays, and zebrafish xenografts

在体外和体内对新型治疗药物的临床前筛选是肿瘤药物发现的重要工具。尽管人类癌细胞系对 2D (维) 单层细胞培养中的治疗化合物有反应, 但为了了解药物在生理相关模型中的功效, 开发了3D 培养系统。近年来, 在临床前研究中观察到一个范式的转变, 以验证新分子在3D 培养系统中的效力, 更精确地模仿肿瘤微环境。这些系统以生理上相关的方式描述疾病状态, 有助于获得更好的机械洞察力和对特定分子药理效力的理解。此外, 随着目前在体内肿瘤模型改良方面的发展趋势, 斑马鱼已经成为一种重要的脊椎动物模型, 用来评估体内瘤的形成, 并研究治疗药物的作用。在这里, 我们研究了 hydroxycoumarin OT48 单独或联合 BH3 mimetics 在肺癌细胞系 A549 使用三种不同的3D 培养系统, 包括菌落形成检测 (CFA), 球形形成试验 (林业) 和在体内的斑马鱼移植。

在图 2中, 肺癌细胞系 A549 被播种在 MCBM 中, 在单独 OT48 治疗后形成菌落, 或与 BH3 模拟 A1210477 结合在指定的浓度。结果表明, 在10天的孵化后, 该组合显著减少了菌落数量、大小和总表面积。
在图 3中, A549 细胞单独 OT48 或与 BH3 模拟 A1210477 结合进行治疗, 并允许用 U 型底板技术形成球体。经过3天的孵化, 球体的图像被拍摄。利用图像 J 和3D 图像生成了球体的量化。
在图 4中, A459 细胞单独 OT48 或结合 BH3 模拟 A1210477 24 小时, 并注入斑马鱼卵黄囊中。72小时后, 荧光肿瘤的量化与化合物的抑制电位有关。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57490/57490fig1.jpg"> 图 1: 斑马鱼异种移植试验的总体过程示意图.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57490/57490fig1large.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/57490/57490fig2.jpg"> 图 2: hydroxycoumarin OT48 和 BH-3 模拟 A1210477 对 A549 菌落形成的协同抑制作用.(A) 用50µM OT48 和/或 A1210477 20 µM 处理的 A549 菌落形成试验的图像。(BD)菌落数量、平均面积和总表面积的量化。实验实现了三项。进行了特别分析。统计意义在 p 值在0.05 以下被评估并且由以下传奇代表: * p ≤0.05, ** p ≤0.01, ** p ≤0.001, *** p ≤0.0001;Dunnett后的专题分析;Sidak)。所有直方图都代表了至少三项独立实验的平均值。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57490/57490fig2large.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/57490/57490fig3.jpg"> 图 3: OT48 和/或 A1210477 对 A549 细胞形成球体的能力的影响.A549 肿瘤球体3天后的影像。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57490/57490fig4.jpg"> 图 4: 化合物对 A549 肿瘤肿块形成的抑制作用.(A) 图片显示 OT48 和/或 A1210477 对 CM Dil 染色 A549 细胞的肿瘤形成能力的抑制作用。(B) 量化肿瘤的形成。进行了特别分析。统计意义在 p 值在0.05 以下被评估并且由以下传奇代表: * p ≤0.05, ** p ≤0.01, ** p ≤0.001, *** p ≤0.0001;Dunnett后的专题分析;Sidak)。所有直方图都代表了至少十项独立实验的平均值。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57490/57490fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts at JoVE.com

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

在这里, 我们提出了应用3D 培养和斑马鱼的抗癌香豆素的临床前筛选。

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids

Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.3K 次观看.  Seoul National University. 在这里, 我们提出了应用3D 培养和斑马鱼的抗癌香豆素的临床前筛选。

视频: Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

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 ...

科研

癌症研究

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

医学

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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

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

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

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

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

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

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

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

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

斑马鱼拉瓦尔异种草和肿瘤行为分析的一代

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

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

利用斑马鱼幼虫进行高效的体内肿瘤研究

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

作者聚焦:利用斑马鱼肿瘤异种移植推进癌症治疗

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

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此视频中的章节

测试癌细胞的斑马鱼的血管侵袭能力（

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

斑马鱼拉瓦尔异种草和肿瘤行为分析的一代

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

利用斑马鱼胚胎对化合物毒性的快速评价