Name: 3-d cell culture system for studying invasion and evaluating therapeutics in bladder cancer
Uploaded: 2018-09-13T14:45:00
Description: Watch this Scientific Journal Video about 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer at JoVE.com

作者感谢霍华德-克劳福德博士 (密歇根大学) 实验室的技术支持, 为这项研究提供材料和设备, 艾伦凯莱赫技术支持。
这项工作由密歇根大学 Rogel 癌症中心核心赠款 CA046592-26S3、nih K08 CA201335-01A1 (进工党)、BCAN YIA (进工党)、nih R01 CA17483601A1 (DMS) 资助。

1. 生长中的癌症球体
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从细胞线生长
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<li>培养人膀胱癌细胞在常规黏附细胞培养情况下和维护在37°c 孵化器供应与 5% CO2。保持细胞在 < 90% 融合。 注: 所使用的培养基是 Dulbecco 的改良最小必需培养基 (DMEM), 含有4.5 克/升 d-葡萄糖, l-谷氨酰胺, 110 毫克/升丙酮钠, 并在本议定书中提供10% 胎牛血清 (血清)。</li>
		<li>在实验开始前的一天, 胰蛋白酶处理 (通过使用0.25% 胰?...

<ol>
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在这里, 我们描述了一个3维的肿瘤球体模型, 允许实时观察膀胱癌侵袭, 这是至关重要的癌症进展和转移。该系统可以将各种基质和细胞成分结合在一起, 使研究者能够更好地复述发生膀胱癌侵袭的组织微环境。膀胱癌球体可以从各种来源, 如细胞系 (包括基因修饰细胞线, 用于检查影响侵袭过程的信号通路) 和小鼠原发性肿瘤 (这里描述)。它也有可能适应人类原发性肿瘤的分析。根据所使用的成像?...

侵袭是癌症进展的一个关键步骤, 这是转移的需要, 并与较低的生存和预后较差的患者。在人类膀胱癌中, 泌尿道最常见的恶性肿瘤每年导致大约16.5万人死亡, 癌症分期、治疗和预后直接关系到入侵1的存在或缺失。大约75% 的膀胱癌病例为非肌肉侵入, 并以局部切除术进行管理。相反, 肌肉侵袭性膀胱癌 (约25% 的所有病例) 是积极的肿瘤, 转移率高, 并以积极的综合治疗2,3治疗。因此, 了解触发入侵的分子通路, 对于更好地表征侵入性进展的危险和发展能够预防侵入性进展的治疗干预是至关重要的。
肿瘤侵袭性进展发生在复杂的三维 (3) 环境中, 与其他肿瘤细胞、基质、基底膜和其他类型的细胞 (包括免疫细胞、成纤维细胞、肌肉细胞和血管) 相互作用。内皮细胞。渗透性支持 (例如Transwell) 检测系统通常用于定量癌细胞入侵4, 但这些系统是有限的, 因为它们不允许对入侵过程进行实时的显微监测和对样品进行进一步染色和分子分析的检索具有挑战性。研制一种3维膀胱肿瘤球体系统来研究入侵是可取的, 因为它允许在体外系统的方便的情况下纳入已定义的微环境成分。
在本议定书中, 我们描述了一个系统, 以检查人体膀胱癌细胞的侵袭过程, 使用3维球形入侵检测, 包括胶原基凝胶基质和共焦显微镜, 以允许调查人员监测细胞活力和实时入侵 (图 1A)。该系统是多才多艺的, 可以修改, 以询问各种基质/肿瘤的设置。它可以纳入大多数膀胱癌细胞系或原发性膀胱肿瘤和其他基质细胞, 如癌症相关的成纤维细胞和免疫单元5,6,7。该协议描述了由 type-1 胶原组成的基质, 但可以被修改以加入其他分子, 如纤维粘连蛋白, 层粘连蛋白, 或其他胶原蛋白。侵入过程可以遵循72小时或更长的时间取决于显微镜和系统使用的能力。3维基质中嵌入的肿瘤的固定和免疫荧光染色允许对侵入细胞中的蛋白质上调进行审问, 从而提供通常不存在或难以收集的关键信息。使用其他3维文化模型。该系统还可用于屏蔽入侵的化合物, 并描绘受此类化合物影响的信号通路。

<table border="0" cellpadding="0" cellspacing="0" style="width:1511px;" width="1511">
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			<td height="21" style="height:21px;width:527px;">Human bladder cancer cell lines UM-UC9, UM-UC13, UM-UC14, UM-UC18, 253J</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td style="width:612px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">DMEM cell culture medium</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">A3803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS), pH 7.4&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar Ultral-low attachment 6-well cluster&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">3471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional inverted microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">491206-0001-000</td>
			<td>General use for cell culture and checking spheroids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen type 1 from rat tail, high concentration&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-Tek II Chambered Coverglass</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">LSM800</td>
			<td>A confocal miscoscope with climate chamber, multi-location imaging, and Z-stack scanning function&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat micromtome</td>
			<td>Leica Biosystems</td>
			<td style="width:169px;">CM3050 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zen 2 Image processing software&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde solution</td>
			<td>Electron Microscopy Sciences</td>
			<td align="right" style="width:169px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImmEdge Hydrophobic Barrier PAP Pen</td>
			<td>Vector Laboratories&#160;</td>
			<td style="width:169px;">H4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">O.C.T compound&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">23730571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 solution&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">62249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-ATDC (Trim29) antibody</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">HPA020053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Cytokeratin 14 antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab7800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Vimentin antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab24525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

3-d cell culture system for studying invasion and evaluating therapeutics in bladder cancer

膀胱癌是一个重要的健康问题。据估计, 今年美国将有超过1.6万人死于膀胱癌。虽然75% 的膀胱癌是无创性的, 而且不太可能转移, 但有25% 的进展是侵入型生长模式。多达半数的侵袭性癌症患者将会产生致命的转移性复发。因此, 了解膀胱癌侵袭性进展的机制对于预测患者预后和预防致死性转移是至关重要的。在本文中, 我们提出了一个三维的癌症入侵模型, 允许合并肿瘤细胞和基质成分, 模仿在体内的条件发生在膀胱肿瘤微环境。该模型提供了利用时间推移成像实时观察侵入过程的机会, 并利用共焦荧光成像和筛查化合物, 以阻断入侵的可能性, 对所涉及的分子通路进行审问。虽然本议定书的重点是膀胱癌, 但也有可能采用类似的方法来检查其他肿瘤类型的侵袭和运动。

成功地创建浸润性膀胱癌肿瘤球体需要形成适当大小的肿瘤球体从细胞系或原发肿瘤。图 2A显示适当大小的球体开发从四人膀胱癌细胞线 (UM-UC9, UM-UC13, UM-UC14, 253J 和 UM-UC18)。图 2B显示一个肿瘤球体的 BBN 产生的小鼠膀胱肿瘤嵌入胶原基质。这些球体被嵌入如上文所述, 有代表性的图像被捕获使用一个显?...

Watch this Scientific Journal Video about 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer at JoVE.com

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

控制膀胱癌的过程代表了生物标志物和治疗发展的机会。在这里, 我们提出了一个膀胱癌入侵模型, 其中包括3维的肿瘤球体文化, 时间推移成像和共焦显微术。该技术可用于定义侵入过程的特征和筛选治疗药物。

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Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

基于插入 - 滤池3D文化系统原发性前列腺细胞分化

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...