Name: a 3d spheroid model as a more physiological system for cancer-associated fibroblasts differentiation and invasion in vitro studies
Uploaded: 2019-08-08T15:00:00
Description: Watch this Scientific Journal Video about A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies at JoVE.com

我们感谢芭芭拉·斯考丁在准备BM2和Lebein-1方面的帮助。我们感谢奥格内斯·诺埃尔分享了她在球形测定方面的专长。我们感谢索尼娅·舍尔哈斯和迈克尔·舍弗斯在S2条件下处理慢病毒转染方面的帮助。我们感谢萨宾·冯·吕登在准备胰腺癌组织CAF方面所助一的协助。
根据REA赠款协议(316610)向J.A.E.提供的成果,导致这些结果的研究得到了欧洲联盟第七框架方案FP7/2007-2013/的人民方案(玛丽·居里行动)的资助。此外,J.A.E.和A.C.M.C.在卓越细胞集群(EXC 1003-CiM)内由德国基金会(DFG)提供财政支持。该项目还得到了威廉·桑德·斯蒂夫通的支持(授予:2016.113.1 至 J.A.E.)。

1. 3D球体作为体外模型,用于使用细胞基质相互作用的不同TGF-#1抑制化合物进行成纤维细胞/CAF分化
<ol><li>混合6mg/mL甲基纤维素溶液的1部分和细胞培养基培养基的3部分,MEM补充1%的热灭活胎儿牛血清(FBS)和1%青霉素/链霉素,以获得球形形成溶液。</li>
	<li>在球形形成溶液中重新悬浮新解冻的不朽正常成纤维细胞 (iNF)、不朽的 CAF (iCAF) 或 integrin #3+1 KO iCAF(通过至多 25)。如果制备一个96孔板,制备过量的球形...

<ol>
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开发适当的体外模型来研究 CAF 分化是一项具有挑战性的任务。采用不同方法后,得出三维球形模型是更实用、生理和临床相关的模型,可以研究胰腺癌细胞与永生性CAF之间的相互作用。该模型防止成纤维细胞的自发分化,由于技术事实压力,如细胞培养塑料的刚度,至少在短期内培养条件(高达48小时)。虽然这些研究中使用的球形的确切刚度没有被测量,但伊托等人使用机器人集成微流体芯?...

肿瘤微环境是一个非常复杂的利基,对于肿瘤细胞的维持和进展极为重要。它不仅由癌细胞形成,而且由基质成纤维细胞形成。肿瘤细胞被一个频闪包围,它特异性,不同于正常组织的频闪2。拉米宁-332是一种细胞外基质蛋白,在不同肿瘤的频闪中异位表达,如胰腺腺癌3。此外,ECM的生化成分及其生物物理特性,如刚度和张力,在肿瘤体积4内发生变化。这种肿瘤频闪,或"反应频闪",是由成纤维细胞适应邻近的癌细胞和招募其他重要的玩家,为肿瘤进展发展一个有利和支持的环境造成的。基质成纤维细胞的分化导致癌症相关成纤维细胞(CAF)。这些细胞可以使用不同的标记物进行识别,如β-平滑肌活动(βSMA)5或神经/胶质抗原2(NG2)6。
最合适的体外模型,以CAFs重述肿瘤微环境(TME)是难以选择的。对于这种模型系统,必须考虑以经济高效和可重现的方式模拟TME生理参数的方法。在TME中,不同的过程,如增殖、分化、迁移和入侵不同的细胞类型发生。这些细胞过程可以单独使用不同的方法执行。然而,实验条件必须考虑细胞与肿瘤频闪ECM的相互作用,因为基底的刚度会影响CAF分化过程。R.G. Wells评论了基质刚度对细胞行为的影响,并强调在体外培养细胞中观察到的细胞骨骼组织和分化状态可能是技术事实7。不同的刺激似乎涉及CAF分化,包括机械张力5,7。为了避免这种情况,2D 软基板可能是分化研究的可能方法,因为它们避免了硬培养盘塑料的问题。可以种植成纤维细胞的软二维表面可以是胶原蛋白-I涂层聚丙烯酰胺凝胶,据此,通过聚丙烯酰胺和凝胶交联结剂的浓度来操纵凝胶刚度。富_SMA富应力纤维的附着力和形成在成纤维细胞中随着凝胶刚度8而增强。这些结果强调了软基体支架对于更多生理体外分化模型的重要性。然而,在我们手中,这些凝胶的实验重现性和成像是具有挑战性的。为了克服这些缺点,我们改变了2D软基板系统为3D球形模型进行分化和入侵研究。该模型在临床上更相关,类似于体外有机体,在体内细胞-细胞相互作用、ECM生产和沉积以及细胞行为9中重述。
当细胞缺乏附着基板时,形成球形。当细胞没有粘合表面时,它们聚合成或多或少的球形结构。如果球体由一种类型的细胞组成,它们称为同源体;如果由两种或两种以上的不同细胞类型组成,它们就形成杂血球体。
在球体制备的不同方法中,我们使用非粘附圆底96孔板执行协议。它在费用方面非常有效。在这里,我们生产成纤维细胞的同源体,CAF 或 CAF 缺乏集成 _3 亚单位来检查 CAF 或整数 #3 KO CAF 和胰腺管癌细胞(AsPC-I 和 PANC-I)的分化过程和杂物类,以研究入侵到周围的矩阵。
这些研究的目的是使用从人类胰腺癌活检中分离的原发性CAF。然而,获得细胞的活检很少,因此,这些研究中使用的CAF已经使用含有HTERT的扁豆病毒永生。它们被称为iCAF,它们的正常对应物,即原发性人类胰纤维细胞,被称为iNF。人类胰纤维细胞和胰腺管癌细胞AsPC-I和PANC-I已上市。
该协议用于研究拉明宁-332-一元相互作用在CAF分化过程中的影响。为了证明这种相互作用的特异性及其功能,使用了抑制剂化合物:BM2,一种单克隆抗体,它阻断了结合位点层的层素-332+3链10,或lebein 1,一种蛇毒衍生化合物,可阻断拉米宁结合一元,[3]1,#6+1和#7+111,12。
对于入侵测定,细胞被转用于含有cDNA编码的慢病毒,其中mCherry(iCAFs和integrin+3 KO iCAFs)或GFP(AsPC-I和PANC-I)用于区分杂血球体中不同的细胞类型。细胞转导使其永生和/或用荧光蛋白(mCherry和GFP)表达标记,在先前的研究13中进行了描述,应查阅,以了解更多信息。

<table>
	<tbody>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>SIGMA-ALDRICH</td>
			<td>D9542-10</td>
			<td></td>
		</tr>
		<tr>
			<td>6F12 anti-Laminin &#946;3 subunit Mouse (monoclonal)</td>
			<td></td>
			<td></td>
			<td>Homemade</td>
		</tr>
		<tr>
			<td>A3IIF5 Anti-&#945;3 integrin subunit Mouse (monoclonal)</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. M. Hemler, Dana Faber-Cancer Institute, Boston</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>SIGMA-ALDRICH</td>
			<td>32201</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraction V - BSA</td>
			<td>AppliChem</td>
			<td>A1391</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 Goat (polyclonal) anti-Mouse</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 Goat (polyclonal) Rabbit</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-laminin &#947;2 subunit Mouse (monoclonal)</td>
			<td>Santa Cruz</td>
			<td>sc-28330</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NG2 Rabbit (polyclonal) Millipore, AB5320</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#945;-SMA-Cy3 Mouse (monoclonal)</td>
			<td>SIGMA-ALDRICH</td>
			<td>C6198</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 cell line</td>
			<td>ATCC</td>
			<td></td>
			<td>Kindly given by prof. Jorg Haier&#39;s Lab</td>
		</tr>
		<tr>
			<td>Bench centrifuge</td>
			<td>Fisher Scientific</td>
			<td>50-589-620</td>
			<td>Sprout</td>
		</tr>
		<tr>
			<td>BM2 anti-laminin &#945;3 subunit Mouse (monoclonal)</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Patricia Rousselle, CNRS, Lyon</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Fluka</td>
			<td>21074</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Multifuge 1S-R</td>
		</tr>
		<tr>
			<td>Centrifuge tubes 50 mL</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Roche</td>
			<td>11088831001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-I, rat tail</td>
			<td>Gibco</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 700 and 800</td>
		</tr>
		<tr>
			<td>DMEM (High glucose 4.5 g/L)</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>FACSCaliburTM</td>
		</tr>
		<tr>
			<td>Gelifying matrix</td>
			<td>ThermoFisher Scientific</td>
			<td>A1413202</td>
			<td>Matrigel, Geltrex</td>
		</tr>
		<tr>
			<td>Goat IgG, isotype</td>
			<td>DAKO</td>
			<td>X 0907</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>SIGMA-ALDRICH</td>
			<td>12449-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Primary Pancreatic Fibroblasts</td>
			<td>PELOBiotech</td>
			<td>PB-H-6201</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heraeus</td>
			<td>B6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin-332</td>
			<td>Biolamina</td>
			<td>LN332</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>SIGMA-ALDRICH</td>
			<td>M4655</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, 96 wells, U-bottom</td>
			<td>Greiner Bio-One</td>
			<td>650101</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG, isotype</td>
			<td>SIGMA-ALDRICH</td>
			<td>I8765</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi axle rotating mixer</td>
			<td>CAT</td>
			<td>RM5 80V</td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-I</td>
			<td>ATCC</td>
			<td></td>
			<td>Kindly given by Prof. Jorg Haier&#39;s Lab</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Riedel-de Ha&#235;n</td>
			<td>16005</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Reverse Transcription Kit</td>
			<td>Qiagen</td>
			<td>205310</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG, isotype</td>
			<td>Invitrogen</td>
			<td>10700</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes, 1.5 mL</td>
			<td>Greiner Bio-One</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR cycler</td>
			<td>Qiagen</td>
			<td></td>
			<td>Rotor-Gene Q</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor Gene SYBR Green PCR Kit</td>
			<td>Qiagen</td>
			<td>204074</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Lonza</td>
			<td>BE12-702F</td>
			<td>Add glucose to 4.5 g (0.2 um filter) and 1% sodium pyruvate</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>SIGMA-ALDRICH</td>
			<td>X100RS</td>
			<td></td>
		</tr>
		<tr>
			<td>V&#243;rtex</td>
			<td>Scientific Industries</td>
			<td></td>
			<td>Vortex-Genie 2</td>
		</tr>
		<tr>
			<td>&#956;-Slide Angiogenesis, uncoated</td>
			<td>Ibidi</td>
			<td>81501</td>
			<td></td>
		</tr>
	</tbody>
</table>

a 3d spheroid model as a more physiological system for cancer-associated fibroblasts differentiation and invasion in vitro studies

确定体外研究的理想模型至关重要,主要是研究细胞分化等生理过程。在肿瘤频闪中,宿主成纤维细胞受到癌细胞的刺激以分化。因此,他们获得一个表型,有助于肿瘤微环境,并支持肿瘤进展。通过使用球体模型,我们建立了这样的3D体外模型系统,其中我们分析了层蛋白-332及其受体一体体[3]1在分化过程中的作用。这种球形模型系统不仅以更准确的方式再现肿瘤微环境条件,而且是一个非常通用的模型,因为它允许不同的下游研究,如细胞内和细胞外免疫荧光染色标记,以及沉积的细胞外基质蛋白。此外,还可以用该模型对qPCR、流式细胞测定和细胞入侵进行转录分析。在这里,我们描述了一个球形模型的协议,以评估CAF的整数α3+1及其异位沉积配体,拉米宁-332,在分化和支持胰腺癌细胞入侵中的作用。

这一实验设计的结果发表在马丁斯·卡瓦科等人13号上,建议进一步阅读从这些实验中得出的结论。
图1,免疫荧光球体的代表性图像,显示了不朽正常成纤维细胞和永垂的CAF的整数+3亚单位的免疫染色(图1A),以及免疫荧光qPCR 的定量 (图 1B) 和整数 -...

Watch this Scientific Journal Video about A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies at JoVE.com

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

该协议的目标是建立一个3D体外模型,以研究肿瘤状环境中癌症相关成纤维细胞(CAFs)的分化,可在免疫荧光、转录等不同分析系统中解决。分析和生命细胞成像。

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

在球形中培养成纤维细胞比在硬塑料上常用的 2D 系统好得多。它允许研究成纤维细胞在肿瘤样环境中的分化。在这种方法中，细胞培养塑料工作的刚性对成纤维细胞分化没有影响。相反，可以在体外分析细胞基质相互作用对成纤维细胞肿瘤诱导分化的影响。要开始这个程序，获得细胞培养基，这是 MEM 辅以 1% 热灭活 FBS 和 1% 青霉素链霉素。此外，获得每毫升6毫克甲基纤维素溶液。将甲基纤维素的一部分与细胞培养基的三部分混合，以准备球体形成溶液。重新暂停新鲜解冻的不朽的原生成纤维细胞或其他细胞类型，如球形形成解决方案中的文本协议中所列。如果细胞因子或集成蛋白抑制剂包含在研究中，请将这些化合物添加到细胞悬浮液中，以便在形成过程中将其插入球体。在适当情况下，添加抑制剂化合物和每毫升10毫微克的TGFβ一，以触发不朽的正态成纤维细胞的分化。将 100 微升的球体形成溶液分配到圆形底部 96 多井板的每个井中。在每个井上下移液几次混合。然后，在37摄氏度的细胞培养箱中孵育细胞培养箱中的板，用5%的二氧化碳孵育24小时，使每井中形成一个球体。首先，切断移液器尖端的末端，以扩大孔。孵育完成后，使用这种切割移液器收集球类到一个1.5毫升的反应管每个实验条件或每个蛋白质要分析。在 1000 倍 G 下将球体离心约 30 到 60 秒。通过移液小心去除含有上流剂的甲基纤维素，确保不干扰颗粒球状物。将球体转移到反应管时应特别小心。通过切割尖端，确保没有剪切漂移力。收集所有球体也很重要。在洗涤步骤中，确保不会因吸气而丢失球体。要洗球类，添加50微升的一个XPBS。在 1000 倍 G 下离心 30 至 60 秒。然后通过移液小心地取出 PBS。根据要染色的蛋白质，在室温下用50微升4%半甲醛和PBS固定球体20分钟。如前所述，用 PBS 清洗球体。在室温下用0.1%三吨X和PBS对细胞进行四分钟的渗透。然后，如前面所述，在 PBS 中清洗球体三次。将含有5%FBS和2%BSA的PBS加入球体，在室温下孵育一小时，以阻止非特异性蛋白质相互作用位点。现在，在4摄氏度下用30微升原抗体和PBS孵育球体，用2.5%FBS和1%BSA。第二天，如前所述，在PBS中清洗球体三次。在此之后，在室温下用30微升的二次抗体和PBS孵育球类，用2.5%FBS和1%BSA孵育90分钟。如前所述，在 PBS 中清洗球体三次。接下来，用30微升的Dappy染色溶液孵育细胞，在室温下孵育4分钟。如前所述，将 PBS 中的球体洗涤一次。使用玻璃巴斯德移液器，将球体放在玻璃滑梯上，并滴一滴 PBS。使用激光扫描显微镜以 10 倍的放大倍率通过荧光显微镜对球体进行成像。在Z堆栈的不同光学平原上拍摄球体的不同光学横截面。与不朽癌症相关成纤维细胞的同球体相比，正常胰腺成纤维细胞同球体免疫荧光染色的代表性图像显示，分化细胞中的α三亚基信号增加。然后用3D球体的Z堆栈图像量化球体中细胞的荧光信号，由qPCR确定整数α三亚基基因的转录水平。所有这些结果表明，与正常细胞相比，Integrin alpha 3在不朽的癌症相关成纤维细胞中是受调节的。这证明，集成α三β一可以被认为是胰腺成纤维细胞分化的标记。这种方法非常适合在器官和癌症质量中通常发现的俘点自我组织，其中额外的细胞基质僵硬决定细胞的命运。球类培养是一种多功能模型，可与其他分析测定相结合后，球体分离，如数量实时PCR和全细胞测量。用于固定球体的甲醛是一种危险的化学剂。应佩戴手套和眼罩以进行保护。

Research

JoVE Journal

Cancer Research

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, ...

科研

癌症研究

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 ...

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. ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A 3D Organotypic Melanoma Spheroid Skin Model

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

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

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 ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

三维流状入侵检测中的全人肿瘤基基质

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion 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 ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A 3D Spheroid Model for Glioblastoma

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...