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 hydroxyco...

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?...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.4K 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

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

抗癌金属配合物的合成与细胞毒性评价MTT法

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

科研

化学

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

癌症研究

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

质谱法和基于发光的-途径表征阶段我代谢能力的<em&gt;体外</em&gt;细胞培养

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

生物化学

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

医学

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

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

摘要

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抗癌金属配合物的合成与细胞毒性评价MTT法

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

质谱法和基于发光的-途径表征阶段我代谢能力的

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酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

使用患者源性肿瘤类器官的高通量 体外 检测

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光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其^对32 P-postlabeling 的测定

使用患者源性肿瘤类器官的高通量体外检测