Name: characterization of the effects of migrastatic inhibitors on 3d tumor spheroid invasion by high-resolution confocal microscopy
Uploaded: 2019-09-16T13:45:00
Description: Watch this Scientific Journal Video about Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy at JoVE.com

我们要感谢克里斯·琼斯教授为KNS42细胞系做出了贡献。Zeiss LSM880 与 AiryScan 的共聚焦显微镜与 AiryScan 一起，是赫德斯菲尔德创新和孵化项目 （HIIP） 的一部分，该项目由利兹市地区企业合作伙伴 （LEP） 增长交易资助。显微镜图像的信用图3：卡尔蔡司显微镜有限公司，microscopy@zeiss.com。

1. 生成细胞类球体
第1天
<ol><li>根据被调查的细胞系的要求准备标准培养基。</li>
	<li>使用无菌处理技术在组织培养罩中执行所有组织培养相关的步骤。</li>
	<li>胰腺化并计数癌细胞。每盘使用20 mL的细胞悬浮液。将细胞悬浮液保存在明确标记的无菌通用管中。</li>
	<li>向每个井添加预定数量的单元。所需的细胞初始数量和最终球体大小取决于被调查的细胞系的增?...

<ol>
	<li>Gandalovi&#269;ov&#225;, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migrastatics-anti-metastatic+and+anti-invasion+drugs:+promises+and+challenges.">Migrastatics-anti-metastatic and anti-invasion drugs: promises and challenges.</a> Trends in Cancer. 3 (6), 391-406 (2017).</li><li>Delgado-L&#243;pez, P. D., Corrales-Garc&#237;a, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+in+glioblastoma:+a+review+on+the+impact+of+treatment+modalities.">Survival in glioblastoma: a review on the impact of treatment modalities.</a> Clinical and Translational Oncology. 18, 1062 (2016).</li><li>Foreman, P. 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F., Futrega, K., Clements, J. A., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microwell-mesh:+a+high-throughput+3D+prostate+cancer+spheroid+and+drug-testing+platform.">The microwell-mesh: a high-throughput 3D prostate cancer spheroid and drug-testing platform.</a> Scientific Reports. 8, 253 (2018).</li><li>Jonkman, J., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+way+you+slice+it-a+comparison+of+confocal+microscopy+techniques.">Any way you slice it-a comparison of confocal microscopy techniques.</a> Journal of Biomolecular Techniques. 26 (2), 54-65 (2015).</li><li>Pont&#233;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+human+glia+cells+in+culture.">Neoplastic human glia cells in culture.</a> Human Tumor Cells in Vitro. , 175-206 (1975).</li><li>Takeshita, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+an+established+human+glioma+cell+line,+KNS-42.">Characteristics of an established human glioma cell line, KNS-42.</a> Neurologica Medico Chirurgica. 27 (7), 581-587 (1987).</li><li>Bance, B., Seetharaman, S., Leduc, C., Bo&#235;da, B., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+acetylation+but+not+detyrosination+promotes+focal+adhesion+dynamics+and+cell+migration.">Microtubule acetylation but not detyrosination promotes focal adhesion dynamics and cell migration.</a> Journal of Cell Science. 132, (2019).</li><li>Bacon, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+3+indirectly+modulates+tubulin+acetylation.">Histone deacetylase 3 indirectly modulates tubulin acetylation.</a> Biochemical Journal. 472, 367-377 (2015).</li><li>Jackman, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+infiltration+in+paediatric+glioma+using+histone+deacetylase+inhibitors,+a+promising+approach.">Tackling infiltration in paediatric glioma using histone deacetylase inhibitors, a promising approach.</a> Neuro-Oncology. 20, i19-i19 (2018).</li><li>Bhandal, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+glioma+migration+with+the+histone+deacetylase+inhibitor+MI192.">Targeting glioma migration with the histone deacetylase inhibitor MI192.</a> Neuro-Oncology. 19, i12-i12 (2017).</li><li>Del Duca, D., Werbowetski-Ogilvie, T. E., Del Maestro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+preparation+from+hanging+drops:+characterization+of+a+model+of+brain+tumor+invasion.">Spheroid preparation from hanging drops: characterization of a model of brain tumor invasion.</a> Journal of Neuro-oncology. 67 (3), 295-303 (2004).</li><li>Bender, B. F., Aijian, A. P., Garrell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics+for+spheroid-based+invasion+assays.">Digital microfluidics for spheroid-based invasion assays.</a> Lab on a Chip. 16 (8), 1505-1513 (2016).</li><li>Vinci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+establishment+and+analysis+of+three+dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biology. 10, 29 (2012).</li><li>H&#228;rm&#228;, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+dynamic+morphological+drug+responses+in+3D+organotypic+cell+cultures+by+automated+image+analysis.">Quantification of dynamic morphological drug responses in 3D organotypic cell cultures by automated image analysis.</a> PLOS ONE. 9 (5), e96426 (2014).</li></ol>

介绍了一种利用高分辨率共聚焦显微镜鉴定压凝药物活性的癌细胞球体的新方法。使用低粘附板比其他技术，如挂滴15，促进了产生可重复和均匀的球体用于胶原蛋白迁移和入侵测定的手段。该协议中的关键点是，在细胞球体嵌入胶原体基质之前，从96孔板中去除生长介质，并仔细处理此后含有球体的胶原蛋白塞。数字微流体16等新技术现已推出，虽然价格较高?...

用于临床前药物发现和潜在抗癌药物的开发的三维球形技术正日益受到传统药物筛选的青睐;因此，有较多的米格雷静态 - 迁移和入侵预防 - 药物的发展。癌症治疗这些发展背后的理由很明确：3D球形测定是筛选潜在抗癌药物的更现实的方法，因为它们比细胞单层培养更忠实地模仿3D肿瘤结构，更准确地概括药物-肿瘤相互作用（动力学），并允许在肿瘤相关环境中描述药物活性。此外，许多癌症类型对化学毒性药物的抗药性上升，以及癌症患者因癌细胞迁移到遥远肿瘤部位的能力而发生转移而死亡率高，支持了化疗药物的加入在未来的临床癌症试验1中，将癌细胞的迁移潜力作为辅助治疗。高侵入性癌症尤其如此，如高等级胶质细胞瘤（GBM）。GBM 管理包括手术、放疗和化疗。然而，即使结合治疗，大多数患者在初步诊断后1年内复发，中位存活期为11-15个月2，3。在过去几年中，3D 技术领域取得了巨大进步：旋转式系统、微结构结构和 3D 支架以及其他单个检测正在不断改进，以便进行大规模的常规测试4 5，6，7.但是，必须以有意义的方式分析从这些检测中获得的结果，因为使用 2D 图像分析系统分析 3D 生成数据的尝试通常会妨碍数据解释。
尽管在图像采集速度和光毒性降低方面更可取，但大多数广域系统仍然受到分辨率8的限制。因此，除了与药物疗效相关的数据读出外，如果使用宽场系统成像，药物作用对迁移细胞的3D细胞结构的详细影响将不可避免地丧失。相反，共聚焦激光扫描显微镜 （CLSM） 可捕获高质量光学切片图像，这些图像可在使用计算机软件在 3D 采集后重建和渲染。因此，CLSM 易于适用于成像复杂的 3D 细胞结构，从而能够询问抗压格雷抑制剂对 3D 结构的影响，并深入分析细胞迁移机制。这无疑将指导未来的止血药物开发。这里描述了球体生成、药物治疗、染色方案以及高分辨率共聚焦显微镜表征的组合工作流程。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>for glioma invasion assay; this is offered by many distributors/manufacturers and will need to be determined for both the type of assay intended and cell lines used. For glioma cancer cell lines Collagen rat tail type 1 ( e.g. Corning) is the preferred choice. Collagen should be stored at 4oC, in the dark, until required. It is not advisable to mix collagen from different batches as this may affect the consistency of the polymerized collagen.&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>various</td>
			<td>various</td>
			<td>for microscopy imaging</td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>Sigma</td>
			<td>D5648</td>
			<td>needed at 5X concentration for collagen solution for glioma invasion assay;&#160; this may be purchased in powdered form, made up in double distilled water and, depending upon final composition of the growth medium, completed with any additives required. The complete 5X solution should be filtered through a syringe filter system (0.22 microns) before use.</td>
		</tr>
		<tr>
			<td>Foetal calf serum</td>
			<td>Sigma</td>
			<td>F7524-500ML</td>
			<td>needed for cell culture of glioma cell lines</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>various</td>
			<td>various</td>
			<td>for microscopy imaging</td>
		</tr>
		<tr>
			<td>High glucose DMEM</td>
			<td>Gibco</td>
			<td>41965062</td>
			<td>needed for cell culture of glioma cell lines</td>
		</tr>
		<tr>
			<td>Inhibitor</td>
			<td>Tocris</td>
			<td>various</td>
			<td>various - according to experimental design; inhibitors can be purchased from manufacturers such as Selleckchem and Tocris. These manufacturers offer detailed description of inhibitor characteristics, links to associated references and suggestion of working concentrations. As with all inhibitors, they may be potentially toxic and should be handled according to health and safety guidelines. Inhibitors are prepared as stock solutions as recommended by the manufacturer. As an example we used the migrastatic inhibitor MI-192 to demonstrate the use of such inhibitors. We have tested a range of migrastatic inhibitors in this way with comparable results.</td>
		</tr>
		<tr>
			<td>Mountant</td>
			<td>various</td>
			<td>various</td>
			<td>for microscopic imaging</td>
		</tr>
		<tr>
			<td>NaOH (1 M)</td>
			<td>various&#160;</td>
			<td>various</td>
			<td>NaOH can be either purchased at the required molarity or prepared from solid form. The prepared solution should be filter sterilized using a syringe filter system. One M NaOH is corrosive and care should be taken during solution preparation.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>various</td>
			<td>various</td>
			<td>for fixing spheroids and cells; make up at 4%, caution health hazard, ensure that health and safety regulations are adhered to for collagen solution for invasion assay</td>
		</tr>
		<tr>
			<td>Pastettes (graduated pipette, 3ml)</td>
			<td>various</td>
			<td>various</td>
			<td>for invasion assay, solution removal</td>
		</tr>
		<tr>
			<td>PBS, sterile for tissue culture</td>
			<td>Sigma</td>
			<td>D1408-500ML&#160;&#160;</td>
			<td>needed for cell culture of glioma cell lines and washes for staining</td>
		</tr>
		<tr>
			<td>Pen/strep (antibiotics)</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>needed for cell culture of glioma cell lines</td>
		</tr>
		<tr>
			<td>Primary antibody, secondary antibody, DAPI, Phalloidin</td>
			<td>various</td>
			<td>various</td>
			<td>there are many manufacturers for these reagents, for secondary labelled antibodies we recommend Alexa Fluor (Molecular Probes). Here we used for primary antibodies mouse anti-acetylated tubulin antibody (1/100, Abcam). For secondary antibodies we used 1/500 anti-mouse Alexa Fluor 488 conjugated antibody, Molecular Probes. For nuclear stain we used DAPI (many manufacturers) and the actin stain Phalloidin (many manufacturers) both used at recommended dilution of 1/500.</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>needed for collagen solution for glioma invasion assay at 5X concentration</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td>needed for collagen solution for glioma invasion assay at 5X concentration</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td>for trypsinisation</td>
		</tr>
		<tr>
			<td>Ultra low attachment plates</td>
			<td>Sigma/Nunc</td>
			<td>CLS7007-24EA</td>
			<td>for glioma invasion assay; a low adherent plate is required, with 96-well plates preferred to allow for large-scale screening of compounds under investigation. There are several low adherence plates commercially available; it is advisable to test a variety of plates for optimum spheroid generation. In our experience Costar Ultra Low Cluster with lid, round bottom, works best for the generation of spheroids from glioma cancer cells in terms of 100% spheroid formation and reproducibility. These plates were also successfully used for the generation of glioma spheroids from patient-derived material, bladder and ovarian cancer cells in our laboratory. In addition, stem or progenitor neurospheres can be used in these plates to facilitate the generation of standardized neurosphere-spheroids</td>
		</tr>
		<tr>
			<td>Stripettes (serological pipettes, sterile, 5ml and 10 ml)</td>
			<td>various e.g. Costar</td>
			<td>CLS4488-50; CLS4487-50</td>
			<td>for tissue culture and collagen preparation</td>
		</tr>
		<tr>
			<td>various multichannel (50 - 250 uL) and single channel pipettes (10 uL, 50 uL, 200 uL 1 mL)</td>
			<td>various</td>
			<td>various</td>
			<td>for cell and spheroid handling</td>
		</tr>
		<tr>
			<td>Widefield microscopy</td>
			<td>various&#160;</td>
			<td>various</td>
			<td>for observation of spheroid generation and spheroid imaging; here wide-field fluorescence images were captured using an EVOS FL cell imaging system (Thermo Fisher Scientific)</td>
		</tr>
		<tr>
			<td>Zeiss LSM 880 CLSM equipped with a Plan Apochromat 63x 1.4 NA oil objective</td>
			<td>Zeiss</td>
			<td>quote from manufacturer</td>
			<td>Confocal images were captured using a Zeiss LSM 880 CLSM equipped with a Plan Apochromat 63x 1.4 NA oil objective. Diode 405nm, 458/488/514 nm argon multiline and HeNe 594nm lasers were used to excite Phalloidin 594, Alexa Fluor 488, and DAPI respectively. For each image a single representative optical section were captured, with all settings, both pre- and post-image capture, maintained for comparative purposes. All images were subsequently processed using the associated Zen imaging software and Adobe Photoshop.</td>
		</tr>
	</tbody>
</table>

characterization of the effects of migrastatic inhibitors on 3d tumor spheroid invasion by high-resolution confocal microscopy

癌症研究中的药物发现和开发越来越多地基于3D格式的药物屏幕。针对癌细胞的迁移和侵入性潜力，以及由此而转移的疾病传播的新型抑制剂正在被发现，并被视为高侵入性癌症（如胶质瘤）的补充疗法。因此，在添加药物后生成数据，以便对3D环境中的细胞进行详细分析。此处描述的方法，将球体入侵检测与高分辨率图像捕获和由共聚焦激光扫描显微镜 （CLSM） 进行数据分析相结合，可详细描述潜在抗迁移抑制剂的影响MI-192对胶质瘤细胞。球状物从细胞系生成，用于低附着96孔板中的入侵测定，然后准备进行CLSM分析。由于易用性和可重复性，描述的工作流程比其他常用的球形生成技术更可取。与传统广域方法相比，共聚焦显微镜获得的图像分辨率增强，可以识别和分析以下三维环境中迁移细胞的不同形态变化。用止血药物MI-192治疗。

三维球形技术正在促进对药物-肿瘤相互作用的理解，因为它更具有癌症特定环境的代表性。球体的生成可以通过多种方式实现;该协议使用了低粘附性 96 孔板。在测试了来自不同制造商的几种产品后，选择此处使用的板材，因为它们在成功的球形生产和均匀性方面始终表现最佳。替换步骤，其中生长培养物被胶原蛋白基质替换，是协议的关键点;必须非常小心，以消除大部...

Watch this Scientific Journal Video about Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy at JoVE.com

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

使用组蛋白脱乙酰雅酶（HDAC）抑制剂的三维（3D）入侵测定中，压伤抑制剂对胶质瘤癌细胞迁移的影响以高分辨率共聚焦显微镜为特征。

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 ...

该协议证明，我们现在可以在3D癌症模型中详细确定哑压抑制剂的效果。主要优点是采用一种易于和一对一的方法，可以采用这种方法。该技术允许在3D环境中评估癌症研究中的哑压药物，允许在肿瘤环境相关环境中测试药物。这种方法可用于癌症细胞毒性药物的测试，或者，例如，评估辐射对癌症增殖和细胞迁移的影响。用普通的96孔板练习介质去除步骤和胶原蛋白替代是有用的。此外，在共合显微镜上进行培训也是必不可少的。这种技术的视觉演示对于研究人员了解球体所需的微妙处理及其在共和显微镜中的使用至关重要。在实验的第一天，有标准培养培养，在这种情况下，U251，准备适当的细胞系。在组织培养罩中，在培养中加入0.5毫升的尝试性脑试增素，使癌细胞增平。将癌细胞计数在血细胞计上，稀释至每毫升10至第三细胞的浓度为5倍。将细胞悬浮液留在标记清楚的无菌万能管中。通过温和的反转重新暂停细胞，以避免细胞笨拙。使用移液器将细胞培养的200微升添加到96井板的每个井中。在每个空井中加入 200 个 1X PBS 微升，以避免蒸发。在37摄氏度的培养箱中孵化细胞。24小时后，通过亮场显微镜检查细胞。细胞系，如胶质瘤癌细胞系，在24小时内在井底形成球状体。再孵育球体48小时，使3D细胞架构形成。第三天，放置胶原蛋白，5倍培养基，一摩尔氢氧化钠，和一个20毫升的冰管。小心和缓慢地将10.4毫升的冷胶原蛋白加入冷冻培养管。避免气泡。然后，轻轻加入1.52毫升的冷无菌5X培养培养。避免气泡。使用前，轻轻加入72微升的冷无菌单摩尔氢氧化钠，并将溶液留在冰上。通过移液轻轻混合，避免气泡。有效的混合会导致介质中颜色从红色更改为橙色红色。将混合物留在冰上直到使用。接下来，要从第一天准备的 96 井板中去除 190 微升的上流液，请使用移液器，以向井的一侧倾斜。小心不要打扰在井底形成的球体。轻轻将100微升的胶原蛋白混合物加入到每一个井中，在井的一侧下移液。避免气泡和任何球体扰动。在室温下将剩余的胶原蛋白混合物留在20毫升管中，以评估聚合。在培养箱中孵育板至少10分钟，使胶原蛋白聚合。当剩余的胶原蛋白变成半固体和海绵状，球体准备用抑制剂治疗。将浓度为2倍的抑制剂加入培养培养的5毫升。移液器 100 微升的介质轻轻地向下每一个井的侧面，以避免球类扰动。在零小时、24小时、48小时和72小时的时间，通过光场显微镜观察和成像每个球体，以评估药物活动。然后，将盘子返回孵化器。现在，将板放在组织培养罩中，用 100 微升 1X PBS 轻轻更换上流液。注意不要打扰球体，避免接触胶原蛋白。再重复此洗涤步骤三次。拆下每个井中的最终洗涤液，并在 1X PBS 中用 100 微升 4%甲醛进行更换。将 96 井板放在实验室工作台上，用铝箔盖住，并在室温下离开 24 小时。第二天，小心地去除甲醛，并更换为1X PBS。再重复三次。然后，用 100 微升新鲜准备好的 Triton X-100 溶液更换 1X PBS 洗涤液。在室温下孵育30分钟。同时，在50毫升管中用50毫升1X PBS和05%脱脂奶粉准备阻断溶液，并彻底混合。30 分钟后，取出 Triton X-100，用 1X PBS 洗涤三次。在每个井中加入100微升的阻解溶液，孵育15分钟。接下来，在阻断缓冲液中稀释抗小鼠IgG乙酰化管状蛋白抗体，比例为1比100。将原抗体阻断缓冲液混合物在15，682倍g下离心5分钟。小心地去除每个井中的阻尼溶液，并从管中加入25至50微升的抗体阻断缓冲液。在黑暗中在室温下孵育一小时。然后，从每个井中去除抗体溶液，用100微升1X PBS洗涤三次。除任何额外的荧光污渍外，在推荐浓度下稀释阻断缓冲液中的二次抗体。再次，在15，682倍g下离心二次抗体溶液5分钟。从每个井中去除阻解溶液，并加入25至50微升的二级抗体上流剂。在黑暗中孵育1个半小时，在室温下。去除二次抗体染料溶液，用1X PBS洗涤三次，每井100微升。用塑料 200 微升移液器将单个胶原蛋白插头小心地举到玻璃滑梯的中心。加入一滴合适的安装剂，以覆盖胶原蛋白塞。将盖玻片放在样品顶部，并在室温下在黑暗中存储幻灯片。三维球类技术正在增进对药物-肿瘤相互作用的理解，因为它更代表癌症特异性环境。在这项研究中，发现了潜在的抗或亲迁移效应。这在 KNS42 中尤为明显，在控制或处理过的球体中似乎没有迁移。在10微摩尔的最高抑制剂浓度下观察到细胞死亡。核分裂和迁移细胞在U251细胞中很明显。U251细胞球体有尖峰辐射远离原来的球体，增加细胞四舍五入细胞明显与增加的抑制剂浓度。在KNS42中观察到微管坍塌和核碎片。具有单细胞尖峰的表状突起表示 KNS42 迁移。在最低的抑制剂浓度，这种表型是明显的，但随着抑制剂浓度的增加而丢失。将所有试剂留在冰上直到准备就绪至关重要。否则，胶原蛋白开始聚合之前，它已被添加到球体。有空间量化由共和显微镜生成的图像，以评估，例如，哑球药物对细胞形态的影响。它首次使我们能够确认米格拉静态抑制剂MI-192对微管乙酰化水平的影响，并在细胞迁移方面进一步探讨这一点。在固定步骤处理甲醛时，应格外小心。通常的健康和安全准则适用。

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

一种确定环境富集对小鼠结肠肿瘤模型结肠菌群生物多样性影响的方法

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

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 ...

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 ...

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 ...

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 ...