我们感谢斯瓦蒂·阿鲁尔博士（MD安德森癌症中心）提供了 让60（n1046）。我们还要感谢大卫·雷纳博士（休斯顿得克萨斯A&M健康科学中心生物科学与技术研究所）的 林-1 菌株。最后，我们感谢丹妮尔·加辛博士和她的实验室（德克萨斯大学麦戈文医学院）提供了一些试剂。一些蠕虫菌株由国家卫生研究院基础设施项目办公室（P40 OD010440）资助的CGC提供。这项研究得到了德克萨斯州癌症预防和研究所（CPRIT）向JF汉考克提供RP200047的支持。

1. 线虫生长介质 （NGM） 板准备
<ol><li>在 2 L 埃伦迈尔烧瓶中加入 2.5 克辣椒和 3 克 NaCl 到 970 mL 的去离子水。使用磁搅拌棒搅拌内容。此后，在烧瓶中加入20克醋。在 121 °C 和 15 磅/在2 的压力下将烧瓶的含量高压 30 分钟。消毒后，将烧瓶放在搅拌盘上，让中等温度冷却至 50 °C。</li>
	<li>要准备 NGM 板，请在冷却介质中添加以下试剂:25 mL 的 1 M 磷酸钾缓冲器 （pH = 6.0），1 mL 的 1 M MgSO4，1mL 的 1 M CaCl2，1 mL （5 毫克/毫升在 95% 乙醇） 胆固醇， 1 mL（乙醇中10%v/w）尼他汀，1 mL 25毫克/mL链霉素。</li>
	<li>在层压流下，将冷却的介质倒入 60 mm x 15 mm 无菌培养皿中。让板凝固 2 小时。这些板可以在4°C下保存1个月。</li>
</ol>2. 传播 C.埃莱根斯
<ol><li>将 100 μL 隔夜生长的 大肠杆菌 OP50 点到每个 NGM 板的中心，让板在层压罩中干燥 24 小时。随后，这些板可以储存在聚苯乙烯容器中。 注: 大肠杆菌 OP50培养在板播种前生长在卢里亚-贝尔塔尼 （LB）介质的37°C介质中。培养植物可以在4°C下储存1~2个月，用于板的定期播种。</li>
	<li>使用无菌蠕虫采摘，在解剖显微镜下从以前生长的盘子中收集 10/12 重力成年蠕虫。将这些蠕虫转移到新鲜的 NGM 板中，用 大肠杆菌 OP50 播种，并在 20 °C 下孵育板 24 小时。</li>
	<li>24小时后，使用无菌蠕虫采摘从板中去除成年蠕虫。在 20 °C 下孵育板 3 天。胚胎会发育成重力成年蠕虫。</li>
</ol>3. 准备同步 的 C. 埃莱根斯 文化
<ol><li>通过用 M9W 清洗 2 到 4 个盘子，将重力成年蠕虫收集到 15 mL 圆锥管中。<ol>
<li>M9W 的准备:溶解 5 克 NaCl，6 克 Na2HPO4，3克 KH2PO4在去离子水中溶解到最终体积为 1 L. 自动清洁溶液在 121 °C 在 15 磅/在2的压力 30 分钟。在冷却溶液中加入 1 mL 的 1 M MgSO4。</li>
	</ol>
</li>
	<li>通过在 450 x g 下离心 1 分钟来对蠕虫进行离心。在不干扰蠕虫颗粒的情况下对超强的超强体进行排泄。</li>
	<li>将 400 μL 的 8.25% 次氯酸钠（家用漂白剂）和 5 N NaOH 的 100 μL 组合在一起，准备蠕虫裂解溶液。将此溶液添加到蠕虫颗粒中，然后轻拂管子以混合管子中的内容。通过在解剖显微镜下观察蠕虫的裂解，防止管内胚胎过度出血。</li>
	<li>在锥形管中加入 10 mL 的 M9W，当 70% 的成年蠕虫裂解时稀释裂解混合物。</li>
	<li>以 450 x g 离心管 1 分钟。用 10 mL 的 M9W 替换超高纳特。</li>
	<li>重复步骤 3.5 两次。</li>
	<li>完成洗涤步骤后，加入 3/5 mL 的 M9W 以重新悬念蛋丸。在室温下（RT）以18转速在管旋转器上旋转管子过夜。</li>
	<li>过夜孵化后，从旋转器中取出管子，以 450 x g 离心 1 分钟离心将 L1 幼虫颗粒化。将 M9W 吸气至 250 μL 留在管中。</li>
	<li>摇动管子以重新使用幼虫。将三滴含有幼虫的 5 微升滴加入培养皿盖上。使用解剖显微镜计算每滴蠕虫的数量，并确定 1 μL 中的蠕虫数量。</li>
</ol>4. 药物检测的准备
注:此检测中的步骤显示在 图 1中。
<ol><li>在 50 mL 圆锥形管中生长 30 mL 大肠杆 菌 OP50，在 150 rpm 的轨道摇床中在 37 °C 的夜间生长。</li>
	<li>旋转一夜之间生长 大肠杆菌 OP50培养在4000 x g 10分钟颗粒细胞。取出超高颗粒，在 M9W 的 3 mL 中重新使用颗粒以集中培养物。</li>
	<li>在为药物检测准备工作解决方案之前，在 100 mL 的 M9W 中加入 0.1 mL（95% 乙醇中的 5 毫克/毫升）胆固醇。</li>
	<li>通过稀释每种药物加车辆（二甲基硫氧化物）来准备每个实验药物的工作解决方案:DMSO）在4.8ml M9W补充胆固醇，以获得适当的浓度。溶解 DMSO 在 4.8 mL 的 M9W 补充胆固醇，以准备车辆控制。</li>
	<li>在每个含有车辆控制或药物和漩涡管的管中加入 200 μL 浓缩 大肠杆菌 OP50 培养物，以确保它们混合。</li>
	<li>要执行实验，在 12 井组织培养板中将每个工作药物溶液或车辆控制中的 2 mL 添加到每个井中。重复测试每个药物浓度和车辆控制。</li>
	<li>使用无菌微管添加每口井 100 L1 幼虫（参见同步 C. elegans 培养步骤）。将蠕虫体积限制在 10 μL 以内。</li>
	<li>在 20 °C 下孵育板。</li>
	<li>如果需要，在检测的第 3 天用 50 微升 10 倍浓缩 大肠杆菌 OP50 补充井。</li>
</ol>5. 为显微镜准备的阿加罗斯垫
<ol><li>在 5 mL 的去离子水中溶解 0.1 克藻醇，准备 2% 的葡萄糖 w/v 溶液。加热微波炉中的内容以溶解藻子。20 张幻灯片可以从 5 mL 的阿加罗斯溶液中准备。</li>
	<li>将实验室胶带条沿两个玻璃幻灯片放置。胶带将充当隔板，限制玫瑰垫的厚度。之后，在带胶带的幻灯片之间放置第三个干净的玻璃滑梯。</li>
	<li>要制作一个玫瑰垫，将 100 μL 的熔融玫瑰点到干净的滑梯中心。将另一个干净的玻璃滑梯穿过，放在玫瑰的顶部，轻轻向下按压以形成垫子。当垫子凝固时，移除顶部滑动。</li>
</ol>6. 观察 让-60、让-23和林-1 菌株中的Muv表型
注:只有候选药物，抑制Muv表型在 let-23 和 让-60 菌株将使用 林-1 菌株进行检测，以确定抑制是否发生在RAS或EGFR的水平。
<ol><li>当达到生命周期的适当阶段（让-60和让-23菌株3天，林-1菌株5天），从孵化器中取出板。并使用移液器在 15 mL 圆锥管中收集蠕虫。</li>
	<li>以 450 x g 离心管 1 分钟。移除 M9W 而不干扰蠕虫颗粒，并添加 5 mL 的新鲜 M9W。</li>
	<li>重复步骤 6.2 两次。</li>
	<li>在不干扰蠕虫颗粒的情况下，通过吸入去除所有剩余的 M9W。此后，加入 500 μL 的 2 mM 钠阿齐德或 2 mM 四米盐酸四米索。允许管子在RT孵育15分钟2mM钠阿齐德或2mM四米盐酸四米索将麻醉蠕虫成像。 注意:在处理钠阿齐德时，一定要佩戴个人防护装备 （PPE）。解决方案必须在化学罩下准备。</li>
	<li>将麻醉蠕虫悬架的 10 μL 添加到玫瑰垫的中心。将#1.5 盖片轻轻放在蠕虫悬架上。如果需要，用指甲油固定盖唇，以防止干燥。</li>
	<li>使用DIC显微镜观察让-60（n1046），让-23（sa62）和林-1（sy254）菌株。在 10 倍和 20 倍放大倍数下对蠕虫进行成像。</li>
	<li>对于 让 60 （n1046） 和 让 23 （sa62）， 根据 Muv 表型的存在或不存在给成年蠕虫打分。而对于 lin-1 菌株，使用高分辨率DIC显微镜计算L4幼虫腹侧采用1°或2°细胞命运的VPC数量。</li>
	<li>在为每个菌株和药物治疗评分后，执行学生 t 测试，以确定治疗之间的统计差异。</li>
</ol>

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作者声明没有相互竞争的经济利益。

我们描述使用蠕虫的检测是简单和廉价的，以确定EGFR和RAS功能的抑制剂。 C. elegans 是药物发现有吸引力的模型，因为由于生命周期短（3 天在 20 °C）和产生大量幼虫的能力，在实验室中很容易生长。更重要的是，EGFR-RAS-ERK MAPK通路在进化和功能上得到保护，哺乳动物提供了一个基因可传导系统来分析EGFR和RAS抑制剂的影响。此外，蠕虫的透明性使研究者能够可视化不同的结构，以及绿色荧光...

调节生物体内发育事件的细胞通路在所有元子中都得到了高度保护。其中一个途径是EGFR-RAS-ERK线粒体活性蛋白激酶（MAPK）信号级联，这是控制细胞增殖、分化、迁移和生存的关键途径1，2。此信号通路中的缺陷可能导致病理或疾病状态，如癌症。表皮生长因子受体（EGFR）在人类肿瘤中表现出高度表达，包括50%的口腔鳞状细胞癌，并有助于恶性肿瘤3、4、5的发展。而三个RAS同位素H-，K-和N-RAS的突变是多种人类癌症恶性转化的主要驱动因素。在这三种RAS同位素中，K-RAS的致癌突变最为普遍，为6、7、8。要使EGFR和RAS发挥作用，它们必须定位到等离子膜（PM）。防止这些分子的本地化到PM可以完全废除这个信号通路9，10的生物活性。因此，抑制这些蛋白质的本地化到PM是一种治疗策略，以阻止下游信号和由此产生的不良后果。使用高含量筛选检测，芬迪林，L型钙通道阻滞剂，被确定为K-RAS活性的抑制剂11。在芬迪林的存在下，K-RAS 到 PM 内部传单的纳米集群显著减少。此外，K-RAS从等离子体膜重新分配到内质视网膜（ER）、高尔吉仪器、内分泌体和细胞溶胶。更重要的是，胰腺癌、结肠癌、肺癌和子宫内膜癌细胞系的增殖被芬迪林11抑制下游信号所阻断。这些数据表明，芬迪林作为一种特定的K-RAS抗癌疗法，导致RAS蛋白对PM的误定位。
线虫 卡诺哈布德炎埃莱甘斯 已在发展的背景下进行了广泛的研究。许多控制蠕虫发展的信号通路都是进化的，在功能上是保守的。例如，EGFR 介解 RAS 激活和随后激活的 ERK MAPK 信号级联在蠕虫12中保存。级联由以下蛋白质表示:LET-23 > LET-60 > LIN-45 > MEK-2 >MPK-1。LET-60 与 RAS 同源，而 LET-23 与 EGFR 同源。在蠕虫中，这种途径调节外阴13的发育。外阴是蠕虫腹壁上的上皮孔径，允许产卵。蠕虫外阴的形成取决于外阴前体细胞 （VPC） 暴露在 EGFR-RAS-MAPK 信号级联激活梯度下。在正常发育过程中，近亲VC从淋瑙锚细胞接收强信号，以分化成1°和2°细胞的命运，从而产生功能性外阴12。而离散性VC区分成3°细胞的命运，融合到皮下同步，不会形成外阴由于耗尽的信号。在没有信号的情况下，所有 VPC 都区分为 3° 细胞命运，从而形成无外阴。然而，构成信号导致形成一个或多个非功能性外阴，由于所有VPC的诱导，以承担1°和2°细胞的命运。
导致外阴诱导缺陷或过度的突变已经为代表该通路的蛋白质编码的许多基因被识别出来。有缺陷的外阴诱导导致无外阴（Vul）表型，而过度的外阴诱导导致多外阴（Muv）表型，其表现是在整个心室壁上出现大量非功能性异位伪外阴。让-60（n1046）菌株表达的Muv表型是由于RAS功能突变的增加，而在让-23（sa62）菌株中，它是由于EGFR14，15的激活突变。这些突变菌株中的强Muv表型已被药理干预所困扰，通过MEK-1抑制剂U0126 16、17治疗让-60（n1046）蠕虫就证明了这一点。有趣的是，我们已经表明，R-芬迪林和抑制剂，影响苯丙胺代谢抑制在蠕虫18的Muv表型。为了证明这些抑制剂块让60信号在RAS的水平，林-1空菌株已被使用17。Lin-1是一种类似Ets的抑制转录因子，在外阴19的发育中起到抑制作用。在让-60（n1046）蠕虫中，Muv表型的强烈回归，对lin-1空蠕虫没有影响，这表明这些抑制发生在RAS的水平。
在此协议中，我们演示了使用C. elegans作为模型来识别 RAS 和 EGFR 蛋白的抑制剂。使用液体基检测，我们通过抑制让-60（n1046）和让-23（sa62）突变菌株的C.elegans的Muv表型来证明R-芬迪林的抑制作用。这种检测验证了在抗癌治疗药物发现初始阶段使用C.elegans作为工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Millipore Sigma&#160;</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0118-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Agar&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0145-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco LB Broth</td>
			<td>Fisher Scientific</td>
			<td>DF0446-17-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP510-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>ICN10138201</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP213-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Acros organics</td>
			<td>AC455500050</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium pPhosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Fendiline</td>
			<td></td>
			<td></td>
			<td>Commercially Synthesized (Pharmaceutical grade)</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Millipore Sigma&#160;</td>
			<td>S2002-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>8.25% Sodium Hypochlorite&#160;</td>
			<td></td>
			<td></td>
			<td>Bleach</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr>
			<td>(&#8722;)-Tetramisole Hydrochloride</td>
			<td>Millipore Sigma&#160;</td>
			<td>L9756</td>
			<td></td>
		</tr>
		<tr>
			<td>UO126 (MEK inhibitor)</td>
			<td>Millipore Sigma&#160;</td>
			<td>19-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical Sterile Polypropylene Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 10mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 25mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-575</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 1.5&#160; 18 mm X 18 mm Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12-541A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish with Clear Lid (60 x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid (100X15mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Plain Glass Microscope Slides (75 x 25 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>12- Well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>50-197-4804</td>
			<td></td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strain</td>
			<td>Genotype</td>
			<td>Transgene</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>MT2124 &#160;</td>
			<td>let-60(n1046) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>MT7567</td>
			<td>lin-1(sy254) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>PS1839</td>
			<td>let-23(sa62) II.</td>
			<td></td>
			<td>CGC</td>
		</tr>
	</tbody>
</table>

identification of egfr and ras inhibitors using caenorhabditis elegans

表皮生长因子受体（EGFR）及其下游效应器RAS的血浆膜定位变化与包括癌症在内的多种疾病有关。自由活的线虫 C.elegans 具有进化和功能保存的EGFR-RAS-ERK MAP信号级联，是外阴发育的核心。RAS同源LET-60和EGFR同源LET-23功能突变的增益诱导这些蠕虫的腹壁产生可见的非功能性异位伪外阴。以前，这些蠕虫中的多外阴（Muv）表型已被小化学分子抑制。在这里，我们描述了在液体基检测中使用蠕虫来识别消除EGFR和RAS蛋白活动的抑制剂的协议。通过此检测，我们显示 K-RAS 的间接抑制剂 R-fendiline 抑制 了在 let-60 （n1046） 和 让-23 （sa62） 突变蠕虫中表达的 Muv 表型。检测简单、价格低廉，设置不费时，可作为发现抗癌治疗的初始平台。

我们首先证明，与DMSO治疗的蠕虫相比，R-fendiline能够抑制 让-60（n1046） 突变菌株中的Muv表型。我们的数据显示，R-芬迪林能够以依赖剂量的方式阻断 让-60（n1046） 中的Muv表型（图2A，B）。然而，在 林-1 空突变菌株中观察到Muv表型的未逆转，以响应R-芬迪林浓度的增加（图2B）。数据表明，R-芬迪林块激活 让60 信?...

Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

基因可处理的线虫 卡诺哈布迪炎的叶原体 可以用作药物发现一个简单和廉价的模型。这里描述的是一个协议，以确定抗癌治疗，抑制RAS和EGFR蛋白的下游信号。

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

identification-of-egfr-and-ras-inhibitors-using-caenorhabditis-elegans

Research

JoVE Journal

Cancer Research

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

2.9K 次观看.  University of Texas Health Science Center. 基因可处理的线虫 卡诺哈布迪炎的叶原体 可以用作药物发现一个简单和廉价的模型。这里描述的是一个协议，以确定抗癌治疗，抑制RAS和EGFR蛋白的下游信号。

视频: Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

切片乳腺全身为病变鉴别

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

科研

癌症研究

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

建立双抗 EGFR-使用 tki 药物治疗和满足使用 tki 药物治疗在肺腺癌细胞体外与 2 步剂量升级程序

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

Homeobox genes are regulatory genes often associated with tumors in the adult organisms. We investigated their comparative expression by immunohistochemical and real-time PCR analysis, in normal and inflammatory nasal mucosae and in sinonasal neoplasms in order to use them as possible diagnostic and therapeutic targets.

otx1 和 otx2 作为鼻窦癌和嗅觉神经母细胞瘤的两种可能的分子标记物的鉴定

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

Methods Article

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂