Name: generation of tumor organoids from genetically engineered mouse models of prostate cancer
Uploaded: 2019-06-13T14:00:00
Description: Watch this Scientific Journal Video about Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer at JoVE.com

作者要感谢斯坦福大学的Calvin Kuo实验室提供HEK293细胞,这些细胞通过HA-小鼠Noggin-Fc或HA-mouse Rspo1-Fc进行稳稳的转染。我们还要感谢唐院长博士允许我们进入他实验室的荧光解剖显微镜。这项工作得到了国家癌症研究所的CA179907对D.W.G.的支持。罗斯威尔公园综合癌症中心的共享资源得到了国家卫生研究院癌症中心支持赠款 CA016056 的支持。

此处描述的动物程序是经纽约布法罗罗斯威尔公园综合癌症中心实验室动物资源部机构动物护理和使用委员会 (IACUC) 批准进行的。
注:雄性小鼠被解剖以分离前列腺或前列腺肿瘤,以生成器官应至少达到性成熟年龄 - 约8-10周的年龄。小鼠的特定年龄可能因研究而异。选择年龄时需要考虑的一些因素包括前列腺细胞群的年龄依赖性变化、特定启动子驱动Cre转基因的年...

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前列腺肿瘤切除和器官生成协议中的关键步骤 去除非前列腺组织和小鼠前列腺肿瘤的精细解剖对于癌症器官的最佳生成至关重要,因为非前列腺上皮细胞和正常的前列腺上皮细胞都会生成有机体。特别是对于固体前列腺肿瘤,分离活肿瘤区域以去除坏死组织的污染至关重要,这样可以减少活细胞的数量。在有机物生成期间,应努力监测胶原酶的组织消化,因为长时间接触胶原酶会限制细?...

自20世纪80年代末以来,通过同源重组来改变基因的能力极大地推动了生物系统1的研究。诱导、组织或细胞特异性的推进系统和位点特异性重组酶,如Cre-loxP,通过促进对基因修饰的时空控制,2、3、 4.这些基因策略的结合创造了广泛的实验模型系统5,6,7。
基因工程小鼠模型(GEMMs)是评估单个基因或基因组如何影响哺乳动物发育和疾病的一个整体工具。在临床前癌症研究中,GEMM是研究癌症发展、进展和治疗的最具有生理相关性和严谨性的方法。我们的实验室专门生产癌症 GEMM 并将其定性。
在美国男性中,诊断出最高度的非皮癌是前列腺癌(PCa)。大多数 PCa 患者患有低风险疾病和高生存可能性,但当疾病在晚期诊断或有针对性的激素治疗导致进展到侵略性、不可治愈的 PCa 时,存活率急剧下降子类型9,10.我们的实验室已经开发出利用一个或多个肿瘤抑制基因的絮状等位基因的GEMM。肿瘤抑制基因表达的重组和丧失特别发生在前列腺,因为我们引入了一个转基因与Cre重组酶下游的前列腺启动子只激活在前列腺上皮细胞11,12.我们还培育了我们的 GEMM,以包含一种称为mT/mG的 Cre 报告者转基因,该转基因在缺乏 Cre 和绿色荧光蛋白 (GFP) 表达的细胞中诱导番茄荧光蛋白表达,在 Cre13的细胞中。虽然这种方法的介绍和我们具有代表性的结果表明,我们在实验室中研究的GEMM,该协议可用于从任何小鼠模型生成前列腺癌有机体。然而,正如我们在代表性结果部分详细讨论的那样,我们已经观察到某些肿瘤特征是前列腺癌有机体生成的最佳选择。
在过去的十年里,从上皮源组织培养细胞的新方法使我们在体外器官系统模型的能力上有了重大进步。术语"3D细胞培养"被归因于参与建立和维护器官的技术,一般可以定义为由细胞组成的结构,这些细胞由器官特异性细胞系驱动的二次结构驱动特征16.这些新方法不同于经典的二维细胞培养,因为细胞不需要转化或永生,无法长期生长;因此,正常细胞的3D培养可以与病变细胞进行比较。这在癌症研究中特别有价值,因为通常没有正常的细胞控制培养物。此外,有机体自发地形成具有适当分化细胞类型的二次组织架构,使它们成为比2D细胞系17更好的模型系统来理解体外癌症。我们的实验室从从我们的 PCa GEMM 中分离出肿瘤问题的 3D 有机体线,以补充我们的体内数据,并进行在 GEMM 中不可行的实验。
在本文中,我们提出了PCa GEMM的完整尸体解剖的书面和视觉协议,包括解剖不同的小鼠前列腺叶和转移性病变。我们描述并展示了一种从小鼠前列腺肿瘤中产生器官的分步方法,该方法基于Drost等人先前发表的从正常小鼠前列腺上皮组织18中衍生器官的规程。

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			<td height="21" style="height:21px;width:217px;">0.25 % Trypsin+2.21 mM EDTA</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">25-053</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">1 1/4 in, 23 gauge, disposable syringe needles</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">Z192430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">10 % neutral buffered formalin</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">HT501128</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">32 % paraformaldehyde</td>
			<td style="width:177px;">Electron Microscopy Services</td>
			<td align="right" style="width:119px;">15714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">A83-01</td>
			<td style="width:177px;">MedChemExpress</td>
			<td style="width:119px;">HY-10432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Advanced DMEM/F12+++</td>
			<td style="width:177px;">Gibco</td>
			<td align="right" style="width:119px;">12634</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:217px;">Analytical balance</td>
			<td style="width:177px;">Mettler Toledo</td>
			<td align="right" style="width:119px;">30216623</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">B27 (50X)</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">17504044</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Collagenase II</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">17101015</td>
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			<td height="21" style="height:21px;width:217px;">Dissecting Board</td>
			<td style="width:177px;">Thermo-Fisher</td>
			<td style="width:119px;">36-1</td>
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			<td height="147" style="height:147px;width:217px;">EHS Sarcoma matrix, Pathclear Lot#19814A10</td>
			<td style="width:177px;">Manufactured by Trevigen</td>
			<td style="width:119px;">Requistitioned from the National Cancer Institute at the Frederick National Laboratory</td>
			<td style="width:167px;">Holder of grants from the National Cancer Institute can request matrix</td>
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			<td height="21" style="height:21px;width:217px;">HEPES (1M)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">25-060</td>
			<td></td>
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			<td height="42" style="height:42px;width:217px;">human recombinant Epidermal growth factor (EGF)</td>
			<td style="width:177px;">PeproTech</td>
			<td>AF-100-15</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">L-glutamine (200 mM)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">25-005</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">N-Acetyl-L-Cysteine</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">A9165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Penicillin-Streptomycin</td>
			<td style="width:177px;">Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Precision balance</td>
			<td style="width:177px;">Mettler Toledo</td>
			<td align="right" style="width:119px;">30216561</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Scalpel #23</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">504176</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Scalpel Handle #7, 16 cm</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">500238</td>
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			<td height="21" style="height:21px;width:217px;">Single-edge carbon razor blade</td>
			<td style="width:177px;">Fisherbrand</td>
			<td style="width:119px;">12-640</td>
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			<td height="42" style="height:42px;width:217px;">Stainless steel dissecting scissors, 10 cm, straight</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">14393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Stainless steel Iris forceps, 10 cm, curved tip, serrated</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">15915</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Stainless steel Nugent utility forceps, straight tip, serrated</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">504489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Y-276632 (Rock Inhibitor)</td>
			<td style="width:177px;">APExBIO</td>
			<td style="width:119px;">A3008</td>
			<td></td>
		</tr>
	</tbody>
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generation of tumor organoids from genetically engineered mouse models of prostate cancer

基于同源重组来修饰基因的方法大大推进了生物学研究。基因工程小鼠模型(GEMM)是研究哺乳动物发育和疾病的严格方法。我们的实验室已经开发出几个前列腺癌(PCa)的GEMM,这些GemM缺乏使用位点特异性Cre-loxP重组酶系统和前列腺特异性促进剂表达的一个或多个肿瘤抑制基因。在本文中,我们将介绍我们这些PCa GEMM的尸检方法,主要侧重于小鼠前列腺肿瘤的解剖。过去十年开发的新方法促进了上皮衍生细胞的培养,以三维在体外模拟器官系统。我们还详细介绍了一种3D细胞培养方法,用于从小鼠PCa GEMM生成肿瘤器官。临床前癌症研究一直以二维细胞培养和细胞系衍生或患者衍生异种移植模型为主。这些方法缺乏肿瘤微环境,这是临床前使用这些技术的限制。GEMM 在生理上更相关,有助于了解肿瘤发生和癌症进展。肿瘤有机体培养是一种体外模型系统,可概括肿瘤结构及细胞系特征。此外,3D细胞培养方法允许正常细胞的生长,以便与肿瘤细胞培养进行比较,使用2D细胞培养技术很少可能。结合在临床前研究中使用 GEMM 和 3D 细胞培养,有可能提高我们对癌症生物学的理解。

图2A显示了在前前列腺区域带有大量充满液体的原发性前列腺肿瘤的小鼠的代表性尸检图像。相比之下,图2B显示了具有代表性的小鼠的尸检图像,其前列腺肿瘤较大,个别前列腺区域无法区分。荧光解剖图像显示相同的固体前列腺肿瘤从图2B表达GFP,表明肿瘤细胞表达Cre(图2C)。</str...

Watch this Scientific Journal Video about Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer at JoVE.com

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

介绍了一种小鼠前列腺癌模型的尸检和解剖方法,重点是前列腺肿瘤解剖。提出了小鼠前列腺肿瘤器官生成分步方案。

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

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

与传统细胞培养物相比，小鼠器官培养物更类似于体内细胞组织。在癌症研究中，器官比2D细胞系更模拟原始肿瘤，并可用于测试体外潜在的癌症疗法，以开发新的药物治疗。对于雄性小鼠泌尿系统进行整体提取，抓住膀胱两侧的脂肪垫，向上拉，露出睾丸。仔细解剖每个睾丸远离泌尿生源区域的其余部分。轻轻抬起膀胱，使泌尿生源区域一起升高，露出下面的尿道。握住膀胱时，将剪刀对着背前列腺的底面，以将尿道切除。整个泌尿生源区域将从腹腔中释放。切除泌尿生源系统后，位于泌尿生源系统后面和脊柱两侧的骨盆淋巴结将暴露。去除淋巴结定向淋巴组织下的直钳并拉起。接下来，抓住直钳和切口的直肠，拉上直肠，解开整个结肠和小肠，寻找转移性病变的中肠淋巴结。当整个氦气被移除后，继续拉上十二指肠，露出胃。切食道完全切除胃。如果观察到淋巴结中有任何转移性病变，请仔细解剖这些远离肠道的结，以储存在PBS中。当胃被切除后，脾脏可以从腹部的后侧收获，储存在4%的半成甲醛中。取出肝脏以储存在PBS中。从脊柱的两侧取出肾脏以及任何含有转移性病变的肾脏淋巴结。要露出胸腔，小心地沿着肋骨笼切割隔膜。胸腔内释放的负压会暴露心脏和肺部。向上拉胸骨，进一步打开胸腔。扫描胸腔的腹腔沿肋骨笼的转移性病变在胸淋巴结中，如果存在解剖。在仍然拿着胸骨时，切断腹骨笼以进入心脏和肺部，并拉上心脏切到肺部下方。要完全切除心脏和肺，切断气管内所有前血管。小心地将心脏转移，而不会损坏肺组织或肺转移性病变，将其转移到 PBS 容器中。用于前列腺肿瘤的解剖，将泌尿生源区域置于解剖显微镜下。使用一对直钳和一对弯曲钳子将泌尿生体区域翻转到其后侧表面。找到可由尿道的粉红色-红色识别的近位前列腺区域。牢牢抓住尿道，让尿生体组织用弯曲的钳子操作。找到具有开创性的囊泡的底座，以便小心地去除。然后使用钳子的弯曲侧去除血管延迟和尽可能多的脂肪和结缔组织。在仍然牢牢地握住尿道和近角前列腺区域时，使用一把细尖剪刀将膀胱从尿道上取出。用直钳牢牢抓住近端前列腺区域和尿道，用钳子的弯曲侧抓住前前列腺区域，将组织从膀胱和前列腺的其余部分牢牢拉开。将前前列腺组织放在PBS中，并定位腹前列腺区域。以相同方式切除腹前列腺区域。如果横向区域可以与后侧区域区分开来，请删除两侧的侧前列腺区域。然后去除后前列腺区域，将近位前列腺区域放在4%的半成甲醛中。切碎后，将肿瘤片放入15毫升的消化缓冲液管中，在37摄氏度下进行一个半小时至两个小时的消化。在消化结束时，通过离心沉淀消化的组织，并丢弃上流液。轻拂管子以松开细胞颗粒。将细胞重新吸收在一毫升预热的三普辛中，辅以10微摩尔Y-27632 ROCK抑制剂，在37摄氏度下5分钟。在孵化结束时，用标准的P1000移液器尖端对组织浆液进行五次三化。将管子返回水浴，再进行五分钟的孵化和三角化。对于基质圆顶电镀洗消化的细胞在9毫升的冷介质，然后收集细胞离心。将细胞重新用一毫升新鲜介质进行计数。数了之后，再次通过离心收集细胞。在适当的基质体积中稀释肿瘤细胞。小心地将200微升的基质细胞溶液放入每井55摄氏度加热的六井板中。让圆顶在室温下凝固两分钟，然后将盘子倒置在 37 摄氏度的培养箱中 20 分钟。当圆顶完全凝固后，在每一井中加入两毫升前列腺有机体介质，并辅以安激素R1881和 ROCK抑制剂。将板返回细胞培养箱。荧光解剖图像通过实体前列腺肿瘤显示绿色荧光蛋白或GP表达，表明肿瘤细胞表达Cre。来自同一动物的肝脏和肺部表现出表达GP的转移性肿瘤，证明这些肿瘤起源于原发性前列腺肿瘤。此小鼠的骨盆淋巴结表示 GFP 而不是番茄，表示此转移性肿瘤已超过淋巴结，且未保留任何正常组织。在第一天，从实体前列腺肿瘤产生的培养小器官开始形成与番茄和GGFP表达细胞存在于肿瘤器官培养。然而，当前列腺肿瘤器官完全形成时，第七天及以后，器官表达GP，但不是番茄，表明器官起源于Cre表达肿瘤细胞，而不是正常的上皮细胞。在由充满液体的前列腺肿瘤产生的小器官培养的第一天，番茄和GGFP表达细胞都存在于器官培养中。然而，来自充满液体前列腺肿瘤的器官在第七天及以后表达GP或番茄，表明由不表达Cre的细胞形成的器官。为了在收获前列腺组织时保持方向，请时刻注意膀胱和尿道的位置。器官生成后可用于成像应用、分子生物学分析和药物筛选。

Research

JoVE Journal

Genetics

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

科研

遗传学

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

同步视频脑电图-心电图监测对小鼠癫痫模型 Neurocardiac 功能障碍的鉴别

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

RNA 下一代测序和生物信息学管道, 以识别点对位的表达 Line-1

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...