We would like to thank Dr. Konstantin Dobrenis for providing murine microglia for these studies.

1.细胞荧光标记注:标签胶质母细胞瘤细胞系和小胶质细胞用荧光染料9。可替换地，产生如在14中所述组成型表达的荧光蛋白如GFP / RFP的细胞系。 <ol><li>在6孔板板细胞，他们将70 - 对染色天80％融合。对于鼠恶性胶质瘤细胞系GL261和人成胶质细胞瘤细胞系U87，板1×10 6和1.5×10 6个细胞，分别在染色前一个6cm皿24小时。 </li><li>在DMSO准备荧光细胞染色染料溶液，加入5μM染料媒体，无论是罗斯威尔公园纪念研究所培养基（RPMI）或巨噬细胞无血清培养基（MSFM），涡好。 </li><li>孵育细胞用染料在37℃，30分钟和5％ 的 CO 2。 </li><li>除去含染料的介质和新鲜培养基（RPMI或MSFM）添加到细胞中。 </li><li>孵育细胞30分钟。注:细胞现在染色并准备在实验中使用。 </li></ol>预涂2矩阵商会共同培养侵袭试验<ol><li>区分使用佛波醇肉豆蔻乙酸酯（PMA）15 THP-1细胞。 <ol><li>板2 - 3×10 5个 THP-1细胞在1.5ml的RPMI / 10％FBS中的在6孔培养板上。电镀后立即添加100nM的PMA和在37℃，5％CO 2孵育48小时。 注:48小时后，已成功地进行分化的THP-1细胞将粘附并扩散到井服用上的圆形形态的底部。 </li><li>通过用1×PBS洗涤一次除去PMA和用新鲜的RPMI / 10％FBS更换。再等48小时，直到细胞准备在实验中使用。 </li></ol></li><li>通过将它们放置在一个井仅含有500微升的培养基（无血清）和添加的媒体500微升单独顶端平衡矩阵预涂腔室。孵育室在37℃，2小时和5％ 的 CO 2。 <br/&gt;注:预涂室是从制造商那里购买;见材料/设备的表 。 </li><li>轻轻取下细胞（荧光标记的胶质母细胞瘤细胞系和小胶质细胞/巨噬细胞），通过抽吸从细胞中取出介质。洗与2mM EDTA的/ PBS培养皿的细胞。加入500μl2mM EDTA的/ PBS中并孵育5 -在37℃下在细胞培养箱10分钟，5％的CO 2。 </li><li>悬浮细胞在5ml的培养基含有0.3％的牛血清白蛋白（BSA）。 </li><li>离心机在120×g下5分钟。 </li><li>吸上清，重悬沉淀在MSFM / 0.3％BSA的介质（GL261细胞和小胶质细胞）或RPMI / 0.3％BSA中（为U87和THP-1细胞），使得细胞浓度为1×10 6个细胞/ ml 。使用血细胞计数器计数细胞。 </li><li>添加每24孔板的孔500微升媒体/ 0.3％BSA中。 </li><li>从已平衡至少2小时（步骤2.2）腔室移除介质。地方chambeRS以及中含有500微升媒体/ 0.3％BSA（步骤2.7）。 </li><li>如果正在使用，研究其对巨噬细胞作用的抑制剂/小胶质细胞刺激神经胶质瘤侵袭，适当的浓度添加到该底部和腔室的顶部部分两者。 注:CSF-1R的浓度的抑制剂来抑制充分刺激小胶质细胞胶质瘤GL261入侵可以参考9中找到。 </li><li>根据所使用的细胞系，增加细胞在接下来的两个步骤中描述的室顶:鼠标胶质母细胞瘤（2.10.1）和人脑胶质瘤（2.10.2）。 <ol><li>混合1.5×10 5 GL261（成胶质细胞瘤）细胞（150微升）和5×10 4小鼠小神经胶质细胞（50微升）（参见步骤2.6媒体详细信息）。加入300微升MSFM / 0.3％BSA带来体积为500微升。细胞混合物添加到顶部室中，并在37℃，5％的CO 2孵育48小时。 注:如我所描述的小胶质细胞的小鼠从新生小鼠中分离N + 1 6。 </li></ol></li></ol>混合7.5×10 4 U87（胶质瘤）细胞（75微升）和2.5×10 4 THP1（巨噬细胞）细胞（25微升）。加入400微升RPMI / 0.3％BSA带来500微升的体积。细胞混合物添加到顶部室中。孵育细胞在37℃，5％CO 2的24小时。 <ol><li>孵化后，从室顶部细胞轻轻吸并在PBS在3.7％的甲醛将修复室。允许腔室保持在固定剂在室温下（或过夜，在4℃）15分钟。轻轻吸删除固定液，用500微升1×PBS代替。继续进行图像分析第4节。 注:实验可以在这一刻被暂停，并保存在1X PBS成像。荧光染料信号可以为固定后长达2周进行检测。 </li></ol> 3.侵袭试验"3D"-embedding胶质瘤和巨噬细胞/小胶质细胞在矩阵<ol><li>在4℃下过夜解冻矩阵组合。对执行中的冰罩采用矩阵所有进一步的操作。 </li><li>制备10毫克/毫升基质在介质上冰的0.3％的BSA浓度（MSFM或RPMI）。 注:基质的库存浓度通常为大约15毫克/毫升。 </li><li>以制备用于悬浮细胞（标记为在步骤1）中的矩阵，通过抽吸从细胞中除去介质。洗与2mM EDTA的/ PBS培养皿的细胞。加入500μl2mM EDTA的/ PBS中至细胞并孵育5 -在37℃在培养箱中10分钟，5％的CO 2。 </li><li>悬浮细胞在5ml含0.3％BSA的介质。 </li><li>离心机在120×g下5分钟。 </li><li>从颗粒吸出上清液。在媒体/ 0.3％BSA中重悬沉淀，使得细胞浓度为1×10 6个细胞/ ml。使用血细胞计数器计数细胞。 </li><li>添加1.5×10 5 GL261细胞（150微升）+ 5×10 4小胶质细胞（在50μl）至1.5 ml微量离心管中。加入2×10 5个 GL261细胞（200微升）到一个单独的1.5毫升离心管。 注:单独的没有小胶质细胞GL261作为对照。 </li><li>自旋细胞离心，在120×g下5分钟。 </li><li>悬浮细胞在200冰微升冷的矩阵。 </li><li>板50微升（50,000个细胞）到8微米孔径室刀片的顶室的中心。 </li><li>孵育在37℃下30分钟为发生聚合反应。 </li><li>加入200μl无血清培养基的上部腔室和700微升含有血清的细胞生长培养基到下孔。 </li><li>孵育在37℃，5％CO 2的48小时。 </li><li>孵化后，从室顶部细胞轻轻吸并在PBS在3.7％的甲醛将修复室。允许腔室保持在固定剂在室温下（或过夜，在4℃）15分钟。轻轻吸删除固定液，用500微升1×PBS代替。请进入第4 FOR图像分析。 </li></ol> 4.成像测定<ol><li>卸下3.7％的甲醛商会和洗井含有新鲜500μl的1X PBS。 注意:不要让室内干燥。 </li><li>使用棉签轻轻刮过滤表面清洗从过滤器（侧朝向腔室的内部）的顶部部分的非侵入性的细胞。轻轻地启动和逐步增加的压力。做每室这至少3次。 </li><li>放置室中任一玻璃底培养皿中或在24孔板离开。 </li><li>将样品在配备有摄像头和图像采集软件9,10啶或激光共聚焦荧光显微镜的阶段。 </li><li>就拿几个有代表性的领域10X 20X或图像。 </li><li>使用图像分析软件，如ImageJ的，计数已越过过滤器的底面9,10胶质母细胞瘤细胞的数量。 </li><li>如果成像"3D上述4.6 - 4.2"侵袭测定（在第3节中描述），使用荧光激光共聚焦显微镜5-6产生为Z堆叠一系列要图像上的过滤器的下侧的侵入性细胞群，遵循协议的步骤。 </li></ol>

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The authors have nothing to disclose.

高级别星形细胞瘤和胶质母细胞瘤的高度创伤性使这些脑癌非常致命的。因此至关重要它是理解胶质母细胞瘤侵袭的分子和细胞机制。很多人都了解胶质母细胞瘤入侵已经17的过程。使用本文详述的测定形式，我们的实验室已表明在小鼠和人模型肿瘤相关巨噬细胞可以通过5刺激神经胶质瘤细胞侵袭 - 10倍。这个共培养模型忠实复制成胶质细胞瘤细胞的物理上与巨噬细胞/小胶质细胞相互作?...

胶质母细胞瘤是一种积极的人脑肿瘤与来自诊断1,2的时间的大约12个月的中位生存期。胶质母细胞瘤是最致命和临床具有挑战性的癌症之一，因为它是难治标准化疗和手术切除。胶质母细胞瘤的扩散性质，使肿瘤细胞在整个大脑的正常传播使晚期肿瘤几乎不可能完全手术切除。这种高度侵入性的方面是胶质母细胞瘤等先进星形细胞瘤的标志特征。因此，许多研究的重点是胶质母细胞瘤细胞侵袭的分子机制。胶质母细胞瘤肿瘤微环境在建立恶性肿瘤3-6至关重要的作用。肿瘤相关巨噬细胞/被证明小胶质细胞负责促进胶质母细胞瘤侵袭7,8。大部分这些研究然而用测量的巨噬细胞/小胶质细胞的作用测定其物理上胶质瘤细胞将它们分开。我们的实验室已着手产生提高检测这让我们能够研究胶质母细胞瘤入侵是如何依赖于共同培养巨噬细胞/小胶质细胞和入侵期间使我们能够像它们之间的物理相互作用。 经典实验测定细胞侵袭包括"标准"Boyden小室趋化和化学侵袭格式。这里待研究的细胞铺在含有对具有指定尺寸（一般为0.4和8微米的直径）的孔的底部的聚碳酸酯过滤器的塑料室中。细胞侵袭的方法，包括一个物理屏障，通常由细胞外基质蛋白质。在化学侵袭测定中，所使用的优选的基质是Matrigel中（以下简称为"矩阵"），通过Engelbreth-Holm的-群（EHS）分泌的胞外基质蛋白混合物小鼠肉瘤细胞和大部分由山口的拉根Ⅳ型和层粘连蛋白。然后将腔室放置在包含有或没有被怀疑刺激侵袭生长因子的细胞培养基的组织培养孔。具有较高侵袭能力通过细胞外基质涂覆的过滤器在较高的频率会侵入并附着到过滤器的下侧的细胞。我们已在以评估小胶质细胞和肿瘤相关巨噬细胞对成胶质细胞瘤细胞侵袭的作用改性该测定。 我们已经能够确定使用该纸张胶质可由5刺激两成胶质细胞瘤细胞系的侵袭中描述的共培养测定法- 10倍9,10。这反映了在胶质母细胞瘤的动物模型中观察到。此外，我们开发了一种三维侵袭测定，其中成胶质细胞瘤细胞和巨噬细胞之间的相互作用/小胶质细胞，可以更直接地检测。胶质母细胞瘤细胞浸润由macropha刺激的程度在3D试验GES /小胶质细胞可媲美什么是使用矩阵涂层的腔体的方式看到。类似的分析是以前开发的入侵在11-13研究与巨噬细胞乳腺癌的相互作用。本文中所描述这两种方法应在解剖胶质母细胞瘤细胞的巨噬细胞/小胶质细胞刺激的入侵的分子机制（S）的能力帮助。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Corning BioCoat Matrigel Invasion Chamber: With BD Matrigel Matrix</td>
			<td>Corning/Fisher Scientific</td>
			<td>Cat: 354481</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage Serum Free Media (MSFM) (500 ml)</td>
			<td>Life Technologies</td>
			<td>12065-074</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red CMTPX Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C2925</td>
			<td></td>
		</tr>
		<tr>
			<td>GL261 cell line</td>
			<td>National Cancer Institute (NCI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U87 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium (500 ml)</td>
			<td>Life Technologies/Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polycarbonate membrane cell culture inserts (8 &#181;M pore) 48 per pack.</td>
			<td>Corning</td>
			<td>CLS3422</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, PathClear</td>
			<td>Trevigen</td>
			<td>3445-005-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>Cat: 10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, Fraction V, Heat Shock Treated</td>
			<td>Fisherscientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma Aldrich</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture assays to study macrophage and microglia stimulation of glioblastoma invasion

胶质母细胞瘤（IV级胶质瘤）是一个非常积极的人类癌症1年后诊断中位生存期。尽管引起胶质母细胞瘤的分子事件的了解增多，这种癌症仍然是非常难治常规治疗。高档脑肿瘤的手术切除是很少完整由于胶质瘤细胞的浸润性高的性质。因此，这削弱胶质母细胞瘤细胞侵袭的治疗方法是一个有吸引力的选择。我们的实验室和其他人已经表明，肿瘤相关巨噬细胞和小胶质细胞（脑居民巨噬细胞）强烈刺激胶质母细胞瘤入侵。本文中所描述的协议被用来模拟使用体外培养测定胶质母细胞瘤，巨噬细胞/小胶质细胞的相互作用。这种方法可以极大地方便，使这个恶性behav的扰乱与巨噬细胞的沟通药物的开发和/或发现IOR。我们已经建立了两个强大的共培养入侵检测，其中的小胶质细胞/巨噬细胞刺激5胶质瘤细胞侵袭 - 10倍。不具有和具有巨噬细胞/小胶质细胞接种于涂覆基质 - 聚碳酸酯室插入标记有荧光标记物或组成型表达的荧光蛋白胶质母细胞瘤细胞或嵌入在三维基质。细胞的侵袭是通过使用荧光显微镜图像和计数仅侵袭细胞在过滤器的下侧进行评估。使用这些测定，若干药理学抑制剂（JNJ-28312141，PLX3397，吉非替尼，和塞马莫德），已经鉴定阻断巨噬细胞/小胶质细胞刺激胶质母细胞瘤侵袭。

使用此处所述的方法中，我们已经表明，小胶质细胞和巨噬细胞可基本上刺激成胶质细胞瘤细胞的侵袭。两种不同的侵袭测定法采用与图1中描绘在图2中，组成型表达的荧光蛋白mCherry GL261细胞涂布在有和没有小胶质细胞48小时预涂布腔室。然而，当培养与小胶质细胞的侵袭能力增加了大约10倍，GL261细胞在自己的微创。 <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

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 ...

coculture-assays-to-study-macrophage-microglia-stimulation

Research

JoVE Journal

Cancer Research

13.9K 次观看.  Kean University. Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

视频: Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

脱细胞和细胞肺模型研究肿瘤转移

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

科研

癌症研究

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

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

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 ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

多维共同培养系统模拟肺鳞癌进展

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

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 ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells

The major cause of cancer-related deaths is metastasis formation (i.e., when cancer cells spread from the primary tumor to distant organs and form secondary tumors). Delamination, defined as the degradation of the basal lamina and basement membrane, is the initial process that facilitates the transmigration and spread of cancer cells to other tissues and organs. Scoring the delamination capacity of cancer cells would indicate the metastatic potential of these cells.
We have developed a standardized method, the ex ovo CAM-Delam assay, to visualize and quantify the ability of cancer cells to delaminate and invade, thereby being able to assess metastatic aggressiveness. Briefly, the CAM-Delam method includes seeding cancer cells in silicone rings on the chick chorioallantoic membrane (CAM) at embryonic day 10, followed by incubation from hours to a few days. The CAM-Delam assay includes the use of an internal humidified chamber during chick embryo incubation. This novel approach increased embryo survival from 10%-50% to 80%-90%, which resolved previous technical problems with low embryo survival rates in different CAM assays.
Next, the CAM samples with associated cancer cell clusters were isolated, fixed, and frozen. Finally, cryostat-sectioned samples were visualized and analyzed for basement membrane damage and cancer cell invasion using immunohistochemistry. By evaluating various known metastatic and non-metastatic cancer cell lines designed to express green fluorescent protein (GFP), the CAM-Delam quantitative results showed that the delamination capacity patterns reflect metastatic aggressiveness and can be scored into four categories. Future use of this assay, apart from quantifying delamination capacity as an indication of metastatic aggressiveness, is to unravel the molecular mechanisms that control delamination, invasion, the formation of micrometastases, and changes in the tumor microenvironment.

The CAM-Delam assay to evaluate the metastatic capacity of cancer cells is relatively fast, easy, and cheap. The method can be used for unraveling the molecular mechanisms regulating metastasis formation and for drug screening. An optimized assay for analyzing human tumor samples could be a clinical method for personalized cancer treatment.