我们要感谢Lee Niswander博士和Rada Massarwa博士对这里描述的切片培养共聚焦成像方案的技术专长和贡献。进一步感谢Kalen Dionne博士提供了关于优化脑肿瘤组织切片和培养参数的专业知识。

在开始收集患者组织样本之前，必须根据批准的机构评估委员会（IRB）方案从每个患者获得知情同意书。该协议的作者收到了科罗拉多大学医院和Inova Fairfax医院批准的IRB协议下所述的工作的同意。从这些切片培养物收集的数据不用于指导患者护理决定。 预切片准备<ol><li>在计划肿瘤切除和组织收集前（或在2周内利用以前产生的培养基）前一天准备"组织加工"培养基和"切片培养维持"培养基。向500mL高葡萄糖DMEM中加入5mL青霉素 - 链霉素溶液（10,000U / mL）和5mL 1M HEPES以产生组织处理介质。 </li><li>使用神经元培养基（ 如 Neurobasal）准备250 mL切片培养维持培养基胡桃酚红。用10mM HEPES，1xB-27补充剂，400μML-谷氨酰胺，600μML-丙氨酰-L-谷氨酰胺二肽，60U / mL青霉素，60μg/ mL链霉素和6U / mL制霉菌素补充该培养基。 </li><li>将所有培养基储存在4°C不超过2周。 </li></ol> 2.手术日:组织获取<ol><li>在组织采集当天，使用高压灭菌的玻璃器皿和层流罩中的无菌技术，在组织处理介质中制备50-100mL的低熔点琼脂糖的1％和2％（wt / vol％）溶液。需要两种浓度的琼脂糖来适应不可预测的肿瘤组织一致性变化。 </li><li>使用微波加热琼脂糖悬浮液，直到观察到温和的沸腾。将琼脂糖溶液置于37°C水浴中，保持液态，直至使用。 </li><li>在6孔板的每个孔中放置1mL切片培养物维持培养基。 </li><li>使用无菌镊子将空PTFE培养物插入每个孔中。将板放置在保持在37℃的5％CO 2气氛的加湿，水夹套的组织培养箱中。 </li><li>在层流罩内使用无菌巴斯德吸管，在肿瘤组织获取之前，将95％O 2 /5％CO 2气体的混合物在含有冰冷的组织加工介质的烧瓶中起泡约15至30分钟。在加工过程中，使用超氧化介质可以最大限度地减少组织中的缺氧。 </li><li>准备标有50 mL锥形管（足够分析需要数量的单个肿瘤区域），包含20 mL等量的超氧化冰冷加工介质，以便在手术室和切片设备之间运输组织。 </li><li>结合神经外科医生，对肿瘤区域进行术前计划，以进行样本采集。选择具有对比度增强的肿瘤区域，如患者的临床想象所示g（T1后钆磁共振成像（MRI）序列）。以前的经验表明，该区域产生可行的肿瘤组织，而不是坏死组织。 注意:尝试从周围（周围）白质生成切片;然而，背景自体荧光和肿瘤细胞密度降低限制了这些切片的实验效用。 </li><li>在肿瘤切除开始时获得肿瘤组织。避免收集暴露于广泛的双极烧灼的肿瘤组织，这可能会损害继发于热损伤的组织活力。与片状切除术中获得的组织相比，在片状肿瘤切除术期间获得的组织样品显示增强的活力。这一观察结果可能涉及由于手术切除和延长获取时间而导致的血流不均匀肿瘤组织剥夺。 </li><li>如果需要，将肿瘤组织标记并存储在单独的锥形管中以分离样品来自不同的肿瘤区域。 （ 即表面与深部肿瘤组织）。如果术中手术导航可用，可以记录精确的组织位置。 </li></ol> 3.切片培养准备注意:本协议要求使用新鲜未固定的人体组织。所有样品均被认为是传染性的，应根据普遍的血源性病原体方案进行处理。任何时候都应穿戴适当的个人防护装备。在使用前，镊子和解剖刀应暴露于15分钟的紫外线。在使用过程中，用70％乙醇（EtOH）间歇性喷洒工具，使液体在使用前蒸发。切片过程以半无菌的方式使用具有过滤空气的水平层流罩进行。 <ol><li>将肿瘤组织块放置在具有冰冷加工介质的培养皿中。我们要清洗肿瘤组织并尽量减少附着的红细胞ea移液管轻轻地交换和丢弃培养皿中的培养基三次。 </li><li>使用手术刀将肿瘤块切成矩形盒形。将组织块修剪成约3mm×3mm×10mm。通过切割或用细镊子轻柔牵引小心地清除任何连接的血管。 </li><li>将5 - 7 mL 37°C琼脂糖吸取到小型立方体（〜2 cm 3 ）塑料包埋模具中。使用前用无菌温度计确认琼脂糖溶液的温度不超过37℃。 </li><li>让琼脂糖在冰床上坐1分钟左右。 </li><li>将2 - 4个肿瘤组织"条带"放入琼脂糖中，长轴垂直取向。 注意:组织条在凝固前会倾向于"沉淀"在琼脂糖中。为了避免这种并发症，请将组织条暂时保持在垂直方向，直到琼脂糖进一步固化。 </li><li>将含有琼脂糖模板的组织放在冰床上2 - 5 min以方便固化。 </li><li>从冰中取出霉菌，用手术刀切割形状的两侧，轻轻取出琼脂糖块。避免在块上施加过大的力，这可能会破坏琼脂糖。 </li><li>使用大量的氰基丙烯酸酯胶将琼脂糖块固定在vibratome标本板上。让胶水设定约1 - 2分钟。 </li><li>用冰冷的加工介质填充vibratome储存器以浸没固定在样品板上的琼脂糖块。 </li><li>在切片过程中，将95％O 2 /5％CO 2气体混合物通过修剪的无菌塑料移液器进入振动储器。 </li><li>将vibratome切片厚度设置为300 - 350μm。 </li><li>根据组织一致性调整刀片前进速度和刀片振幅。 "更硬"GBM组织需要较慢的刀片提前速度和更高的振幅。精确的刀片速度和幅度设置将根据vibratome规格而有所不同。 <br/&gt;注意:切片时通常会出现几个问题。如果组织条从琼脂糖块上移开，尝试将组织包埋在较高浓度的琼脂糖中。如果肿瘤切片比想要的（机器组）厚，或者是不对称的，则增加振动和振幅降低的速度。为了确保肿瘤组织不被刀片固定器卡住或"拖动"，因为切片发生，当刀片移动时，请小心地将刀片夹持器和切片之间定位。 </li><li>使用不锈钢微球，将组织切片转移到具有冰冷处理介质的培养皿中。 </li><li>从培养箱中获得含有PTFE插入物和平衡的切片培养基的6孔板（如第2部分步骤4中所制备）。 </li><li>板组织切片使用不锈钢微球。通过使用小的细毛刷画来最小化直接接触和操纵组织以产生"流体波"，以将切片从刮刀上轻轻地推出nd到培养物插入物上。 注意:最小化在组织培养插件顶部引入的加工介质的量。如果过量的介质转移导致切片浮动，请使用无菌巴斯德吸管移除介质。 </li><li>将含有肿瘤切片的6孔板返回到保持在37℃，5％CO 2气氛的培养箱中。 注意:整个嵌入和切片方案应在手术室肿瘤组织获取90分钟内完成。 </li></ol>切片文化维护<ol><li> 12-24小时后，用无菌镊子抓住每个插入物边缘转移切片培养物，并转移到含有新鲜切片培养维持培养基的平板上。确保在转移插入物之前将等分于每个孔中的切片培养物维持培养基在培养箱中平衡至少15分钟。 </li><li>用新鲜平衡的切片将插入物移入新的6孔板每48小时播放一次媒体。 </li><li>如果需要，使用移液管等分试样切片培养基，以便立即用于生物化学测定（ 即 ELISA）或在-80℃下冷冻以备将来使用。 </li></ol> 5. 通过绿色荧光蛋白表达逆转录病毒的肿瘤细胞标记注意:用于分析肿瘤细胞迁移的延时显微术需要在切片培养物内稳定的长期荧光标记细胞。建议使用逆转录病毒，因为它选择性地感染分裂细胞，从而丰富肿瘤细胞群体内的荧光标记，而不是切片内存在的小胶质细胞或其他细胞类型。感染的标准化表明，10 4 CFU /μL的病毒滴度导致足够的绿色荧光蛋白表达用于跟踪和分析细胞迁移。增加病毒滴度，使用非选择性病毒（ 即腺病毒，慢病毒）或ot她标记所有细胞的手段可能排除了在迁移过程中识别清除细胞边界，从而使分析复杂化。可以根据需要利用替代荧光标记物进行优化。 <ol><li>无论是通过标准协议5，14或从可商购的来源获得用于肿瘤切片的感染逆转录病毒。通过将适当的体积添加到未补充的神经元培养基中以达到10 4 CFU /μL的病毒滴度来稀释病毒上清液。 </li><li>培养7 - 10天之后，感染感染的肿瘤切片培养物为5 - 10μL病毒（10 4 CFU /μL）。将病毒轻轻滴到每个组织切片的表面上。如果切片漂浮在插入物的表面上，则减少添加的上清液体积。将含有切片培养物的板返回培养箱。 </li><li>在24小时后开始评估标记肿瘤细胞的切片<html>病毒感染（见第6节）。该时间延迟将取决于病毒掺入和荧光基因表达动力学。对于这里使用的病毒构建体，需要72小时用标准荧光显微镜观察强壮的荧光信号。 注意:很少，切片显示病毒标记细胞的主要外围分布，这可能使共聚焦成像复杂化。为了避免这种并发症，尝试减少切片上的切片厚度和厚度变化。如果切片培养物在中心附近具有较厚的区域，则这可能会阻止充分的营养渗透，从而限制了活跃分裂肿瘤细胞的群体。筛选这种并发症的定性测定是向切片培养基中加入四唑染料试剂（ 即 MTT）。添加试剂后切片不变蓝色的区域表明缺乏代谢活动，损害切片的健康。 </li></ol>le"&gt; 6。Time-Lapse Single Photon Laser Scanning Confocal Imaging of Tumor Cell Migration 注意:成功转导后，证实培养物的健康，细胞可以在对照条件下成像，然后在处理条件下进行相同的成像时间。使用该方案，细胞在每种条件下成功成像并追踪12小时。然而，更短或更长的成像周期和环境操纵也可能是有益的。 <ol><li> 加载显微镜 <ol><li>在成像之前，将1 mL新鲜切片培养基置于玻璃底盘中。 </li><li>允许玻璃底盘中的培养基在培养箱中平衡15分钟。 注意:此时，可将可溶性标记试剂，如荧光共轭凝集素（ 即用于小胶质细胞标记的Isolectin IB 4 ）或配体缀合的量子点添加到切片培养基中用于鉴定细胞成像期间的唾液亚群。 </li><li>使用层流罩中的无菌镊子将要成像的插入物转移到玻璃底盘。将菜肴运送到显微镜舞台。 </li><li>在密封的显微镜分级培养箱中，在37℃和5％CO 2气氛下保持切片培养物。如果有可能的话，使用无菌的H 2 O加湿培养箱，以防止过度的介质蒸发（对于较长的成像实验尤其重要）。 </li><li>利用具有长工作距离10X空气物镜的共聚焦显微镜和用于单光子和/或多光子的激光激发。 注意:确保显微镜物镜提供足够的工作距离。由于来自台式培养箱，玻璃底盘和组织培养插入物的附加高度的结果，长工作距离目标是至关重要的。 </li><li>固定玻璃底板，取下塑料培养皿盖，盖上气 -渗透膜。 </li></ol></li><li> 图像采集 <ol><li>使用显微镜目视检查切片，并在切片边缘和中心之间找到具有足够密度的荧光标记肿瘤细胞的合适场。由于成像期间组织移位的敏感性增加，避免了切片边缘的成像场。可以采用组织切片竖琴来限制这种变化。 </li><li>在多维分析模式下使用成像软件设置第一个（底部）和最后一个（顶部）Z-堆叠边界，使得要成像的所有位置都包含可见的荧光细胞信号。通过150 - 200μm的切片成像，以10μm的恒定Z步距为跟踪细胞路径提供了足够的分辨率（可根据个别成像需要进行调整）。 </li><li>如果显微镜具有电动化阶段，请确保每个Z型叠层采集都有足够的时间。将采集之间的时间间隔设置为相等每个位置的扫描时间x图像位置数（ 即成像肿瘤区域）。 注意:为了同时激发绿色和红色荧光团，请使用双线激光线扫描程序，同时激活488 nm和633 nm。选择的激光激发波长将根据表达的各种荧光蛋白而变化。 </li><li>在成像的肿瘤区域之间保持均匀的激光功率和共焦孔设置。利用最低的激光功率设置，以清楚地划分肿瘤细胞体和过程以限制光毒性。具体的成像参数单位（ 即功率和共焦孔设置）将根据显微镜和激光源规格而变化。 </li><li>通过在朝向目标前进的焦平面中添加2-3个"缓冲"Z堆叠步骤来补偿潜在的介质蒸发。这有效地防止组织离开Z堆叠采集范围离子在垂直平面上。 </li></ol></li></ol>图像后处理和肿瘤细胞追踪注意:许多共焦成像系统配备了专有的图像处理软件。下面讨论的处理步骤包括可以跨软件平台执行的通用协议。将给出开源平台NIH ImageJ和MTrackJ 15的具体说明。 <ol><li>打开一个Z-stack文件。在ImageJ中，单击"图像→堆栈→Z项目"。选择第一个和最后一个Z-stack包含，并选择"Max Intensity"作为投影类型。结果是称为最大强度投影（MIP）的渲染。从每个成像区域捕获的每组Z-stack图像创建一个MIP。 </li><li>连接来自每个区域的MIP以创建时间序列。点击"图像→堆叠→图像到堆叠"。 </li><li>手动识别位置通过选择视觉上近似的肿瘤细胞体的中心点来形成细胞体"质心"。这是通过使用MTrackJ（NIH ImageJ的开放源代码插件）的"添加"轨道功能点击细胞体来实现的。 </li><li>单击以划分一系列图像的每个帧中的单元格体位置。这为每个单元格创建一个独特的"轨迹"。顺序地，标记下一个单元格的单元格本体位置。重复此过程，直到所有单元迁移路径被跟踪。 </li><li>一旦在给定的肿瘤微区域中跟踪细胞群体，使用MTrackJ的"测量"功能导出所有细胞轨迹坐标。将文件保存为.xls格式，以便可以使用电子表格或用户生成的软件分析原始数据。 </li><li>使用沿单元格迁移轨迹记录的每个点记录的坐标，执行定量分析，包括迁移速度的推导，方向性和其他迁移指标，如Parker 等[ 16]所述 。 注意:所有计算的距离和速度都是实际值的低估。这是通过产生最大强度投影将三维图像（和迁移路径）转换为二维数据所固有的。 </li></ol>

<ol>
	<li>Beadle, C., 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=The+role+of+myosin+II+in+glioma+invasion+of+the+brain.">The role of myosin II in glioma invasion of the brain.</a> Mol Biol Cell. 19, 3357-3368 (2008).</li><li>Farin, 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=Transplanted+glioma+cells+migrate+and+proliferate+on+host+brain+vasculature:+a+dynamic+analysis.">Transplanted glioma cells migrate and proliferate on host brain vasculature: a dynamic analysis.</a> Glia. 53, 799-808 (2006).</li><li>Panopoulos, A., Howell, M., Fotedar, R., Margolis, 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=Glioblastoma+motility+occurs+in+the+absence+of+actin+polymer.">Glioblastoma motility occurs in the absence of actin polymer.</a> Mol Biol Cell. 22, 2212-2220 (2011).</li><li>Ivkovic, S., 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=Direct+inhibition+of+myosin+II+effectively+blocks+glioma+invasion+in+the+presence+of+multiple+motogens.">Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens.</a> Mol Biol Cell. 23, 533-542 (2012).</li><li>Assanah, 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=Glial+progenitors+in+adult+white+matter+are+driven+to+form+malignant+gliomas+by+platelet-derived+growth+factor-expressing+retroviruses.">Glial progenitors in adult white matter are driven to form malignant gliomas by platelet-derived growth factor-expressing retroviruses.</a> J Neurosci. 26, 6781-6790 (2006).</li><li>Chaichana, K. 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=Preservation+of+glial+cytoarchitecture+from+ex+vivo+human+tumor+and+non-tumor+cerebral+cortical+explants:+A+human+model+to+study+neurological+diseases.">Preservation of glial cytoarchitecture from ex vivo human tumor and non-tumor cerebral cortical explants: A human model to study neurological diseases.</a> J Neurosci Methods. 164, 261-270 (2007).</li><li>Grube, S., 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=Overexpression+of+fatty+acid+synthase+in+human+gliomas+correlates+with+the+WHO+tumor+grade+and+inhibition+with+Orlistat+reduces+cell+viability+and+triggers+apoptosis.">Overexpression of fatty acid synthase in human gliomas correlates with the WHO tumor grade and inhibition with Orlistat reduces cell viability and triggers apoptosis.</a> J Neurooncol. 118, 277-287 (2014).</li><li>Hovinga, K. E., 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=Inhibition+of+notch+signaling+in+glioblastoma+targets+cancer+stem+cells+via+an+endothelial+cell+intermediate.">Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate.</a> Stem Cells. 28, 1019-1029 (2010).</li><li>Merz, F., 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=Organotypic+slice+cultures+of+human+glioblastoma+reveal+different+susceptibilities+to+treatments.">Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.</a> Neurooncol. 15, 670-681 (2013).</li><li>Xu, J., 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=Vorinostat+modulates+cell+cycle+regulatory+proteins+in+glioma+cells+and+human+glioma+slice+cultures.">Vorinostat modulates cell cycle regulatory proteins in glioma cells and human glioma slice cultures.</a> J Neurooncol. 105, 241-251 (2011).</li><li>Verhaak, R. G., 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=Integrated+genomic+analysis+identifies+clinically+relevant+subtypes+of+glioblastoma+characterized+by+abnormalities.">Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities.</a> in PDGFRA, IDH1, EGFR, and NF1. Cancer cell. 17, 98-110 (2010).</li><li>Gill, B. J., 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=MRI-localized+biopsies+reveal+subtype-specific+differences+in+molecular+and+cellular+composition+at+the+margins+of+glioblastoma.">MRI-localized biopsies reveal subtype-specific differences in molecular and cellular composition at the margins of glioblastoma.</a> Proc Natl Acad Sci USA. 111, 12550-12555 (2014).</li><li>Snuderl, 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=Mosaic+amplification+of+multiple+receptor+tyrosine+kinase+genes+in+glioblastoma.">Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma.</a> Cancer cell. 20, 810-817 (2011).</li><li>Kakita, A., Goldman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+and+dynamics+of+SVZ+cell+migration+in+the+postnatal+forebrain:+monitoring+living+progenitors+in+slice+preparations.">Patterns and dynamics of SVZ cell migration in the postnatal forebrain: monitoring living progenitors in slice preparations.</a> Neuron. 23, 461-472 (1999).</li><li>Meijering, E., Dzyubachyk, O., Smal, I., van Cappellen, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+in+cell+and+developmental+biology.">Tracking in cell and developmental biology.</a> Sem Cell Dev Biol. 20, 894-902 (2009).</li><li>Parker, J. J., 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=Gefitinib+selectively+inhibits+tumor+cell+migration+in+EGFR-amplified+human+glioblastoma.">Gefitinib selectively inhibits tumor cell migration in EGFR-amplified human glioblastoma.</a> Neurooncol. 15, 1048-1057 (2013).</li><li>Brat, D. J., 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=Pseudopalisades+in+glioblastoma+are+hypoxic,+express+extracellular+matrix+proteases,+and+are+formed+by+an+actively+migrating+cell+population.">Pseudopalisades in glioblastoma are hypoxic, express extracellular matrix proteases, and are formed by an actively migrating cell population.</a> Cancer Res. 64, 920-927 (2004).</li><li>Shweiki, D., Itin, A., Soffer, D., Keshet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+induced+by+hypoxia+may+mediate+hypoxia-initiated+angiogenesis.">Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.</a> Nature. 359, 843-845 (1992).</li><li>Shamir, E. R., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+culture:+experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: experimental models of mammalian biology and disease.</a> Nat Rev Mol Cell Biol. 15, 647-664 (2014).</li><li>Charles, N. A., Holland, E. C., Gilbertson, R., Glass, R., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+tumor+microenvironment.">The brain tumor microenvironment.</a> Glia. 60, 502-514 (2012).</li><li>Di Cristofori, 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=The+vacuolar+H++ATPase+is+a+novel+therapeutic+target+for+glioblastoma.">The vacuolar H+ ATPase is a novel therapeutic target for glioblastoma.</a> Oncotarget. 6, 17514-17513 (2015).</li><li>Vaira, 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=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> Proc Natl Acad Sci USA. , 8352-8356 (2010).</li><li>Gerlach, M. 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=Slice+cultures+from+head+and+neck+squamous+cell+carcinoma:+a+novel+test+system+for+drug+susceptibility+and+mechanisms+of+resistance.">Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.</a> Br J Cancer. 110, 479-488 (2014).</li><li>Holliday, D. 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=The+practicalities+of+using+tissue+slices+as+preclinical+organotypic+breast+cancer+models.">The practicalities of using tissue slices as preclinical organotypic breast cancer models.</a> J Clin Pathol. 66, 253-255 (2013).</li><li>Maund, S. L., Nolley, R., Peehl, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+comprehensive+characterization+of+a+faithful+tissue+culture+model+of+the+benign+and+malignant+human+prostate.">Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate.</a> Lab Invest. 94, 208-221 (2014).</li></ol>

作者没有什么可以披露的。

来自人类癌症组织的有机切片培养物为临床前的翻译实验提供了有吸引力和利用率不足的平台。了解肿瘤细胞在天然肿瘤微环境中的迁移，增殖和细胞死亡的人群行为缺乏。重要的是，在细胞行为水平上以动态，时间分辨的方式研究肿瘤对治疗的反应可以揭示治疗抵抗的新机制。人类肿瘤切片培养提供人类疾病过程与当前离体和体内建模技术之间的联系19 。最近，我们在这里验证作为描述的方法来研究GBM的迁移，在与EGFR放大和信令16细胞迁移行为第一次可测量间肿瘤变化报告的技术。这项研究还利用切片培养模型来测试蛋糕EGFR抑制剂，吉非替尼作为GBM 16的潜在抗侵入疗法的特异性有效性。 上面已经讨论了组织切片，逆转录病毒感染，成像和图像分析范例中的几个常见的缺陷。然而，逆转录病毒感染方案值得进一步关注。鉴于患者对患者的变化，可能会挑战每个切片内滴定病毒标记的肿瘤细胞的密度。如果细胞数量不足，则由病毒构建体标记，每天切片加入5-10μL等份的逆转录病毒上清液，直至达到所需标准肿瘤细胞的浓度。在初级切片培养物中，复制细胞的百分比通常低于转化的细胞系，因此限制了在任何时间点允许逆转录病毒掺入的细胞亚群。或者，如果太多的细胞被标记，尿液分界细胞迁移路径，用神经元介质稀释病毒上清液以达到较低的有效滴度。肿瘤相关小胶质细胞掺入表达逆转录病毒的荧光蛋白，频率约为所有标记细胞的1％。我们能够通过使用小神经胶质结合凝集素进行后期成像数据分析，目视分离这些细胞以进行单独分析（ 图5 ）。 肿瘤微环境包括营养供体，细胞相互作用和细胞外基质等方面都在GBM 20的发病机制中发挥作用。直接人类GBM切片培养物消除了在小动物模型或播散细胞培养物中传代的需要，同时提供了人类肿瘤微环境的重要概括。此外，切片培养提供了跨样品的营养物质的均匀接触，同时保持细胞和细胞与ECM的相互作用。通过减少var在细胞获取已知发生在肿瘤内的营养物质的情况下，我们建议观察到的培养物差异揭示了在群体水平上的肿瘤细胞行为（ 即迁移）之间的内在差异。然而，解释从人类肿瘤产生的切片培养物收集的数据由于固有的肿瘤内和肿瘤内异质性而复杂化。至关重要的是，需要进一步的研究来表征在人体肿瘤切片培养物离体维持期间可能发生的潜在的遗传和表观遗传学变化。 与I期/ II期临床试验平行使用人类肿瘤切片培养是将切片参数与患者临床结果相关联的有希望的策略。在切片培养可用于个体化肿瘤治疗之前，需要验证这些潜在的预测/预后参数。我们的工作以及其他工作证明了生物标志物验证的可行性SUP类="外部参照"&gt; 21，以及在切片培养物从GBM 9，16的治疗剂的快速离体测试。使用肺22 ，结肠22 ，头颈部23 ，乳房24和前列腺癌25组织的类似人切片培养技术表明，该方法可广泛应用于人类癌症。

胶质母细胞瘤（GBM）的实验室研究受到缺乏忠实地重现人类疾病所需病理特征（即肿瘤细胞迁移和侵袭）的模型的阻碍。 2D和3D 的体外侵袭测定的比较研究，以及3D啮齿动物切片培养物模型已经发现在这两个上下文机理上不同的细胞迁移的程序，从2D系统发现的译可能限制与人类疾病1，2，3。这里描述的器官型肿瘤切片培养和成像范例允许研究从手术切除获得的离体人肿瘤组织的切片内的肿瘤细胞迁移。因此，手术切除的肿瘤组织的切片培养物与延时共聚焦显微镜结合提供了研究本地的肿瘤细胞迁移的平台微环境无组织溶解或培养传代。 有一种使用由人类肿瘤异种移植物，逆转录病毒诱导的肿瘤和细胞产生的叠加来研究肿瘤侵袭1，2，3，4，5 GBM的啮齿动物脑切片培养物模型的大量文献。最近，几个小组已经直接从人GBM组织6，7，8，9，10中描述的器官切片培养物的产生。然而，关于切片技术和培养基的公开方案之间存在明显差异。此外，使用器官切片培养物已经集中在包括细胞信号变化的静态实验终点ng，增殖和死亡。本文所述的方案通过采用时间推移激光扫描共聚焦显微镜的动态肿瘤细胞行为的时间分辨观察来扩展先前的切片培养范例。的帧间11和瘤内12最近发现，在人GBM 13遗传变异强调联这种异质性与肿瘤细胞的行为和其对肿瘤对治疗的反应的影响的重要性。在这里，我们报告了使用来自人类癌症组织的直接切片培养物的简化和可重复的方案，以近似实时地观察肿瘤细胞迁移。

<table><tbody><tr><td>DMEM High Glucose&amp;nbsp;</td><td>Invitrogen (Gibco)</td><td>11960-044</td><td></td></tr><tr><td>Neurobasal-A Medium, minus phenol red</td><td>Invitrogen (Gibco)</td><td>12349-015</td><td></td></tr><tr><td>B-27 Supplement (50x), serum free</td><td>Invitrogen (Gibco)</td><td>17504-044</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Invitrogen (Gibco)</td><td>15140-122</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Invitrogen (Gibco)</td><td>35050-061</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Invitrogen (Gibco)</td><td>25030-081</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Invitrogen (Gibco)</td><td>15630-080</td><td></td></tr><tr><td>Nystatin Suspension</td><td>Sigma-Aldrich</td><td>N1638-20ML</td><td>10,000 unit/mL in DPBS, aseptically processed, BioReagent, suitable for cell culture</td></tr><tr><td>UltraPure Low Melting Point Agarose</td><td>Invitrogen (Gibco)</td><td>16520-050</td><td>Melts at 65.5 &amp;#176;C, Remains fluid at 37 &amp;#176;C, and sets rapidly below 25 &amp;#176;C.</td></tr><tr><td>Isolectin GS-IB4 from &lt;em&gt;Griffonia simplicifolia&lt;/em&gt;, Alexa Fluor 647 Conjugate</td><td>Thermo Fisher (Molecular Probes)</td><td>I32450</td><td>Used in media to label Microglia/Macrophages</td></tr><tr><td>pRetroX-IRES-ZsGreen1 Vector</td><td>Clonetech</td><td>632520</td><td></td></tr><tr><td>Retro-X Concentrator&amp;nbsp;</td><td>Clonetech</td><td>31455</td><td>Binding resin for non-ultracentrifugation concentration of viral supernatants</td></tr><tr><td>pVSG-G Vector</td><td>Clonetech</td><td>631530</td><td>part of the Retro-X Universal Retroviral Expression System</td></tr><tr><td>GP2-293 Viral packaging cells</td><td>Clonetech</td><td>631530</td><td>part of the Retro-X Universal Retroviral Expression System</td></tr><tr><td>Cyanoacrylate Glue (Super Glue)</td><td>Sigma-Aldrich</td><td>Z105899</td><td>Medium-viscosity</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Peel-A-Way Embedding Mold (Square - S22)</td><td>Polysciences, Inc.</td><td>18646A-1</td><td>Molds for tumor sample embedding</td></tr><tr><td>Stainless Steel Micro Spatulas</td><td>Fisher Scientific</td><td>S50823</td><td>Bend instrument 45 degrees at the neck of the spoon blade</td></tr><tr><td>Curved Fisherbrand Dissecting Fine-Pointed Forceps</td><td>Fisher Scientific&amp;nbsp;</td><td>08-875</td><td></td></tr><tr><td>Single Edge Razor Blade (American Safety Razors)</td><td>Fisher Scientific</td><td>17-989-001</td><td>Blade edge is 0.009&quot; thick. Crimped blunt-edge cover is removed before loading onto vibratome.</td></tr><tr><td>Leica VT1000 S Vibratome</td><td>Leica Biosystems</td><td>VT1000 S</td><td></td></tr><tr><td>Hydrophilic PTFE cell culture insert&amp;nbsp;</td><td>EMD Millipore</td><td>PICM0RG50</td><td>30 mm, hydrophilic PTFE, 0.4 &amp;micro;m pore size</td></tr><tr><td>35 mm Glass Bottom Dishes&amp;nbsp;</td><td>MatTek</td><td>P35G-1.5-20-C Sleeve</td><td>20 mm glass diameter. Coverslip glass thickness 1.5 mm</td></tr><tr><td>LSM 510 Confocal Micoscope</td><td>Zeiss</td><td>LSM 510</td><td>10x Air Objective (c-Apochromat NA 0.45)</td></tr><tr><td>PECON Stagetop Incubator</td><td>PeCON Germany</td><td>(Discontinued)</td><td>Incubator PM 2000 RBT is a comprable product designed for use with Zeiss Microscopes.</td></tr></tbody></table>

a human glioblastoma organotypic slice culture model for study of tumor cell migration and patient-specific effects of anti-invasive drugs

尽管手术，化学治疗和放射治疗，胶质母细胞瘤（GBM）仍然具有极差的临床预后。进入周围脑实质的肿瘤侵袭代表着持久的治疗挑战。为了开发GBM的抗迁移疗法，为控制实验提供生理学相关背景的模型系统至关重要。在这里，我们提出了一种用于在手术切除期间获得的人类GBM组织产生切片培养物的方案。这些培养物允许离体实验，而不通过动物异种移植物或单细胞培养物传代。此外，我们描述了使用延时激光扫描共聚焦显微镜与细胞跟踪，以定量研究肿瘤细胞的迁移行为和与治疗相关的反应。手术组织采集后90分钟内可重复产生切片。逆转录病毒介导的荧光细胞随后在培养的两周内完成蜂鸣，共聚焦成像和肿瘤细胞迁移分析。我们已经成功地使用这些切片培养物来发现人类GBM中与迁移行为增加相关的遗传因素。此外，我们已经验证了模型检测针对抗迁移疗法的患者特异性变异的能力。展望未来，人类GBM切片培养是快速离体评估治疗剂对肿瘤敏感性的有吸引力的平台，以推进个性化神经肿瘤治疗。

<html>我们的小组成功地从50多位接受GBM初始切除术的患者中获得了切片培养。这种切片生成，培养，逆转录病毒标记，成像和迁移分析协议已经精简到可重复的工作流程中（ 图1 ）。重要的是，这些器官型GBM切片与整个培养物中的起始肿瘤组织一致，包括在培养物中维持长达15天的病理标志物和小胶质细胞（ 图2 ）。此外，我们利用该系统对微环境变化进行肿瘤反应的功能测定。作为生理完整性的指标，我们检查了GBM切片培养物如何通过测量血管内皮生长因子（VEGF）的产生来回应缺氧（1％O 2 ），这是在体内GBM微环境中大量发生的过程17 ，"18。我们证明，通过将切片培养物置于缺氧条件下，切片具有快速的生理反应，诱导VEGF释放到培养基中（ 图3 ）。 为了评估肿瘤细胞迁移的定性和定量方面，我们利用延时图像生成详细的迁移图。这些图划分了在肿瘤微区域（1 mm 2 ）范围内跟踪的所有GBM细胞，为肿瘤群体的动态迁移行为提供了静态可视化。计算每个细胞的迁移速度和方向性（细胞位移/总距离行进）的定量测​​量，以便对肿瘤区域，肿瘤样本和对治疗反应的迁移参数变化进行研究（ 图4 ）。 细胞标记和成像方案des这里描述还提供足够的空间和时间分辨率来评估通过天然肿瘤微环境迁移期间细胞形态的变化。我们观察到在切片培养物中混合形态上不同的运动性肿瘤细胞和小胶质细胞的存在（ 图5A ）。肿瘤细胞运动的特征在于"搜索和爆发"过程，其涉及从静电细胞重复突出和退缩丝状伪足，随后是短时间的有效运动。大约每10分钟成像切片区域也提供足够的时间分辨率来记录经历细胞分裂的肿瘤细胞的延时图像。这些分裂细胞从迁移暂停，完成有丝分裂，并且子细胞在3小时的时间内无延迟地重新启动迁移（ 图5D ）。相比之下，小胶质细胞以更高和更一致的速度迁移，具有比相邻肿瘤细胞更低的方向性，表明他们相对低效的迁移（ 图5C，5E-G ）。这些观察对于了解生物学潜在的患者或细胞特异性治疗反应可能是重要的。 最后，我们使用该方案来显示患者 - 患者在群体水平的细胞迁移参数变异性，包括表皮生长因子受体（ EGFR ）基因组扩增与肿瘤细胞增殖迁移潜力的相关性16 。此外，肿瘤切片的时间间隔显微镜之前，并与抗侵入的药物，吉非替尼治疗后，证明迁移显著减少，这是特定于EGFR扩增的肿瘤切片16。 <img alt="图1" src="/files/ftp_upload/53557/53557fig1.jpg" /> 图1: </st荣&gt; 人类GBM器官切片文化感染，成像和细胞迁移分析工作流程。肿瘤组织通过术中导航设备定位于特定区域。切片一周后，将ZsGreen表达的逆转录病毒加入到切片培养物中以标记有丝分裂活性的肿瘤细胞。感染后3天，为共焦成像制备切片。 3D成像数据被后处理成2D图像用于细胞迁移路径跟踪，肿瘤细胞迁移图的产生以及肿瘤细胞迁移参数的计算。该图的部分原始出版于Parker 等 ，2013年16月，经牛津大学出版社许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53557/53557fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图2. 人体GBM器官切片在整个 体内 培养中 保留组织学特征 。 （ A ）T1对比增强MRI序列用于定位和记录组织采集区域（箭头）。 （ B ）来自A 组突变区域获得的组织产生的切片培养物的培养第8天初始供体组织（OR）和切片的H＆E染色。（ C ）保持GBM的微环境病理和细胞特征， 体内贯穿整个文化。 （I，II）在低（顶）和高（下）放大倍数下，CD68（小胶质细胞/巨噬细胞标志物）的免疫组织化学显示15天培养后切片中的小胶质细胞持久性。切片培养第4天的H＆E染色证实了假性坏死坏死的维持在低（iii）和高（iv）放大倍数下，GBM的逻辑标志。刻度棒=200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53557/53557fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/53557/53557fig3.jpg" /> 图3: 人类GBM切片培养物分泌VEGF对低氧的响应。 （ A ）实验使用含有相同肿瘤区域产生的3个相似大小的肿瘤切片的个体培养插入物。将切片维持在正常氧气中12小时，然后维持两个12小时的缺氧间隔，每个间隔之前加入新培养基。 （ B ）从两个代表性肿瘤产生的切片培养物中通过ELISA测量的VEGF分泌进入介质（平均值±标准偏差），显着增加缺氧比常氧（p &lt;0.05）。 （ C ）来自4种不同肿瘤的切片培养物的合并分析显示，与常氧相比，连续12小时缺氧间隔后VEGF分泌增加（p &lt;0.05）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53557/53557fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/53557/53557fig4.jpg" /> 图4: 肿瘤细胞路径轨迹数据允许定量确定细胞速度和方向性。 （ A ）方向性为1的肿瘤细胞沿着直线向量表现出完美的效率，而具有较低方向性的那些细胞参与低效的曲折路径，如该原理图16所示 。经牛津大学许可转载按。 （ B ）代表性路径轨迹数据（"低"分辨率〜55分钟细胞跟踪间隔）的分析证明了速度和方向性的细胞变异性。 （ C ）绘制每个细胞轨迹的方向性与迁移速度，以可视化跨细胞群体的迁移行为（每个点代表单个细胞）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53557/53557fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/53557/53557fig5.jpg" /> 图5: GBM切片培养物中的小胶质细胞由相对于肿瘤细胞的高迁移速度和低方向性表征。 （ A ）复制选择性表达用Isolectin-I标记的ZsGreen逆转录病毒和小胶质细胞的肿瘤细胞<html> B 4 -647共轭体存在于代表性的切片培养物的代表性肿瘤微区域中。 （ BC ）同一肿瘤微区域中单独追踪的GBM（ B ）和小胶质细胞（ C ）的路径显示不一致的迁移行为。刻度棒=200μm。 （ D ）积极迁移的肿瘤细胞暂停，缩回其过程（箭头），经历细胞分裂，并且两个子细胞沿相反方向迁移（箭头）。刻度棒=50μm。 （ E和F ）与同一区域的肿瘤细胞相比，小胶质细胞显示迁移速度增加（p &lt;0.0001）和降低的方向性（p &lt;0.0001）。 （G）基于速度和方向性的肿瘤和小胶质细胞的分布显示了两种细胞群体的独特的迁移表型。et ="_ blank"&gt; 请点击此处查看此图的较大版本。

Watch this Scientific Journal Video about A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs at JoVE.com

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

目前的胶质母细胞瘤（GBM）的离体模型并没有针对人肿瘤浸润的生理学相关研究进行优化。在这里，我们提出了新鲜人类GBM组织的器官切片培养物的产生和维持方案。提供延时显微术和定量细胞迁移分析技术的描述。

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

a-human-glioblastoma-organotypic-slice-culture-model-for-study-tumor

Nanyang Technological University

Research

JoVE Journal

Medicine

12.8K 次观看.  Stanford University Hospital, Stanford University School of Medicine. 目前的胶质母细胞瘤（GBM）的离体模型并没有针对人肿瘤浸润的生理学相关研究进行优化。在这里，我们提出了新鲜人类GBM组织的器官切片培养物的产生和维持方案。提供延时显微术和定量细胞迁移分析技术的描述。 

视频: A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

多端根结核细胞迁移的实时监测

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

科研

癌症研究

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 ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

测量组织肿瘤组织切片的实时药物反应

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

与器官型脑片和癌细胞的3D球体共培养系统

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

医学

<meta charset="utf8"></head><body>乐高：揭示脂质失调和药物靶点的人类 GBM 类器官模型

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

<meta charset="utf8"></head><body>实验室工程胶质母细胞瘤类器官培养和药物筛选

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

实验室工程胶质母细胞瘤类器官培养和药物筛选

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

摘要

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多端根结核细胞迁移的实时监测

胶质母细胞瘤的3D球形模型

测量组织肿瘤组织切片的实时药物反应

一种研究并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

与器官型脑片和癌细胞的3D球体共培养系统

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

摘要

探索更多视频

此视频中的章节

多端根结核细胞迁移的实时监测

胶质母细胞瘤的3D球形模型

测量组织肿瘤组织切片的实时药物反应

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

与器官型脑片和癌细胞的3D球体共培养系统

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

一种研究并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型