Name: non-destructive evaluation of regional cell density within tumor aggregates following drug treatment
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Description: Watch this Scientific Journal Video about Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment at JoVE.com

这项研究得到了美国国立卫生研究院R01 BRG CA207725（MB / DTC）和NIH R01 CA233188（MB）的支持。我们要感谢AMC药房为这些实验提供的曲妥珠单抗。

AU565（HER2+）和MDA-MB-231乳腺癌细胞系用于本研究（见 材料表）。
1. 制备肿瘤聚集体
<ol><li>使用罗斯威尔公园纪念研究所（RPMI）1640基础培养基（+）在L-谷氨酰胺中制备AU565（HER2 +）乳腺癌细胞生长培养基，并补充10%（v / v）胎牛血清和1%青霉素/链霉素（见 材料表）。</li>
	<li>使用Dulbecco的改良鹰培养基（DMEM）制备MDA-MB-231三阴性乳腺癌细?...

<ol>
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意义 多细胞肿瘤球体（MCTS）是用于研究肿瘤进展和药物筛选1，2，3的强大3D体外模型。推进这些相对简单的聚合模型的实用性在很大程度上依赖于其关键特征的表征，例如形态学和细胞密度，已知这些特征会影响肿瘤模型的进展和治疗反应。然而，它们所需的尺寸在评估这些特征时带来了挑战，特别是对...

研究人员已经在很大程度上转向台式3D培养体外系统来研究肿瘤进展的一些关键特征。这项研究的大部分是由多细胞肿瘤球状体（MCTS）和更复杂的类器官1，2的重新出现引起的。虽然这些模型是无血管的，但它们为概括体内发生的生理和病理过程提供了强大的工具3，4，5。特别是，中型模型（直径300-500μm）可以模拟关键的肿瘤特征，例如3D结构，病理生理梯度和由于核心缺氧引起的转移信号传导。有据可查的是，这些模型显示了血管化体内肿瘤中的特征性同心层，即增殖细胞的外层，衰老/静止细胞的过渡层以及核心缺氧的细胞3，6，7，8，9.通过表征这些层内的细胞行为，在发育过程中和对药物的反应，可以从这些模型中获得独特的见解。然而，开发梯度所需的MCTS尺寸，使它们如此强大的体外模型所必需的，极大地限制了用于无损评估的工具。事实上，对MTCS进行非破坏性分析的最大挑战之一是量化细胞规模的细节。明场和相差显微镜通常用于无损评估3D MCTS的生长和发育。然而，这些模态仅限于2D投影，缺乏可视化这些模型的关键3D结构的能力10，11，12，13。关于细胞毒性和细胞增殖的信息通常通过荧光成像（即，光片显微镜、共聚焦显微镜）或离体免疫组织染色14、15、16收集。虽然这些方法提供了有关组织结构、细胞密度和细胞功能的有价值的高分辨率信息，但它们通常需要样品制备，例如光学清除、固定/染色或嵌入，以防止纵向分析。
光学相干断层扫描（OCT）是一种非破坏性结构成像方式，有可能克服上述一些挑战。它具有蜂窝分辨率和足够宽的视野（高达10 mm x 10 mm），能够可视化整个多细胞聚集体17，18，19。重要的是，由于所用光的可见性质，这种技术是完全非破坏性的，并且没有标签17。此外，样品可以在不需要样品制备的情况下 原位 成像，这样就可以直接从培养箱中取出样品，用OCT快速扫描（扫描持续时间约为5-10分钟），然后返回培养箱，从而实现纵向表征。最近出现了许多试图使用OCT来分析肿瘤球体行为的研究。在最令人兴奋的演示之一中，Huang等人使用OCT来无损检测大型肿瘤球体模型中的坏死核心，并指出活细胞和死细胞区域在光学衰减方面具有明显的差异，可用于无标记的生存能力监测20。类似地，Hari等人对用OCT成像的人类结肠癌（HCT116）球体进行了折射率（RI）测量，以研究样品21中缺氧的存在。他们的测量不足以进行直接推断，尽管他们确实在与坏死核心的部位相关（尽管不是大小）的位置观察到较低的RI，后来 通过 共聚焦显微镜识别出来。Abd El-Sadek等人使用OCT来可视化和量化乳腺癌肿瘤模型22的区域组织生存能力。他们报告了两种基于OCT的可视化组织动力学的方法，并显示这些指标的差异与显微镜识别的活/死细胞区域之间存在中等相关性。
我们发表的工作使用OCT建立在先前的文献的基础上，建立了一种定量的，非破坏性的方法，以在开发过程中测量MCTSs乳腺癌模型中的3D形态和细胞计数10，23。使用Imaris 3D渲染图像分析软件来计算OCT体积扫描中成像的细胞大小的物体（即斑点）的数量，在MCTS中无损测量细胞计数，这些计数在统计学上与在聚合解离时 通过 血细胞计数器确定的细胞计数相似。然而，由于OCT的结构性质，细胞因坏死而死亡后仍然存在的细胞膜可能被错误地计为活细胞。此外，这种表征被扩展到无损跟踪单个聚集体内的细胞活力，并受到有希望成功的药物治疗方案10。重要的是，值得注意的是，我们的OCT-Imaris方法报告了类似的细胞活力，并且在解离时对这些样品进行了基准测试。这种非破坏性和无标记的细胞方法使细胞能够在3D构建体和密集聚集体中纵向计数，而不会牺牲构建/聚集体结构。
本工作报告了一种改进的方法，通过利用OCT-Imaris测量3D聚集体形态和细胞数量的能力，直接量化密集聚集体中的区域细胞密度。这种方法学上的进步提供了MCTS模型的特征同心层内细胞空间分布和增殖的更详细的图片。这种局部密度测量不是简单地计算总体平均细胞密度，而是可以揭示细胞密度梯度，例如与压实相关的梯度。这种区域评估也适用于用化疗药物治疗的聚集体，以评估区域药物反应，通过局部细胞密度的变化来衡量。OCT和高级成像分析方法的这种组合提供了区域细胞活力的定量，可用于根据哪些区域经历细胞密度降低来探索药物渗透。这是第一份非破坏性地量化区域细胞密度和活力以响应致密细胞组织内药物并纵向测量的报告。这种对整个MCTS的三维细胞密度和空间分布的表征可能有助于优化癌症治疗中的药物递送，并提高对癌症模型进展的理解。

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	<tbody>
		<tr>
			<td>96 well plates</td>
			<td>Greiner Bio-One&#160;</td>
			<td>650970</td>
			<td>CellStar Cell-Repellent Surface, https://shop.gbo.com/en/usa/products/bioscience/cell-culture-products/cellstar-cell-repellent-surface/</td>
		</tr>
		<tr>
			<td>0.25% trypsin, 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>AU565 breast cancer cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI software</td>
			<td>open-source</td>
			<td>(Fiji Is Just) ImageJ v2.1/1.5.3j</td>
			<td>Downloaded from https://imagej.net/software/fiji/</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher Scientific</td>
			<td>0267151B</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris image analysis software</td>
			<td>Bitplane</td>
			<td></td>
			<td>Current version 9.8</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Lonza</td>
			<td>17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354263</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231 breast cancer cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Z1 AxioVision</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin streptomycin</td>
			<td>Corning</td>
			<td>30-0002CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral Domain Optical Coherence Tomography</td>
			<td>ThorLabs</td>
			<td>TEL220C1</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 cell culture flasks</td>
			<td>Greiner Bio-One&#160;</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Trastuzumab</td>
			<td></td>
			<td></td>
			<td>Remnant clinical samples of Trastuzumab were used in this study, generously gifted by the Albany Medical College Pharmacy.&#160;</td>
		</tr>
	</tbody>
</table>

non-destructive evaluation of regional cell density within tumor aggregates following drug treatment

多细胞肿瘤球状体（MCTS）模型已被证明对癌症进展和药物发现的 体外 研究越来越有用。这些相对简单的无血管构建体模仿 体内 肿瘤的关键方面，例如3D结构和病理生理梯度。MCTSS模型可以提供对球体发育期间和药物响应的癌细胞行为的见解;然而，它们所需的尺寸极大地限制了用于非破坏性评估的工具。探索光学相干断层扫描结构成像和Imaris 3D分析软件，以快速，无损和无标记地测量MCTS中的区域细胞密度。该方法用于评估 4 天成熟期的 MCTS，以及临床上相关的抗 HER2 药物曲妥珠单抗的 5 天延长治疗期间。简而言之，AU565 HER2 +乳腺癌MCTS 是通过 液体覆盖物创建的，无论是否添加Matrigel（基底膜基质），以探索不同形态的聚集体（分别为较厚的盘状2.5D聚集体或扁平2D聚集体）。在成熟的MCTS中表征了外部区域，过渡区域和内核内的细胞密度，揭示了与外层相比，核心区域具有更高细胞密度的细胞密度梯度。基质添加重新分配了细胞密度并增强了这种梯度，降低了外区密度并增加了核心中的细胞压实度。在逐渐深的100μm区域内进行药物治疗后（0小时，24小时，5天）的细胞密度定量，以评估药物反应的潜在区域差异。到最后一个时间点，几乎所有的细胞死亡似乎都被限制在每个聚集体的外200μm，而聚集体中更深处的细胞似乎基本上不受影响，这表明药物反应的区域差异可能是由于药物渗透的限制。目前的方案提供了一种独特的技术来无损量化致密细胞组织内的区域细胞密度并对其进行纵向测量。

在先前的出版物中，建立了一种使用OCT10对细胞聚集体内全局细胞密度进行非破坏性测量的方法。在本文中，该技术被扩展为评估发育中的细胞聚集体的区域细胞密度。图1显示了该扩展的示意图，其中可以通过观察小的（直径为100μm）的球形塞子（用图1B，C中的黄色圆圈表示）来评估细胞密度。MDA-MB-231肿瘤球体最初通过?...

Watch this Scientific Journal Video about Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment at JoVE.com

Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

本方案开发了一种基于图像的技术，用于在3D肿瘤聚集体中快速，无破坏性和无标记的区域细胞密度和活力测量。研究结果揭示了细胞密度梯度，在发育中的聚集体中，核心区域的细胞密度高于外层，并且在用曲妥珠单抗处理的HER2 +聚集体中主要是外周细胞死亡。

药物治疗后肿瘤聚集体内区域细胞密度的无损评估

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These ...

该方案以非破坏性方式提供有关肿瘤模型进展的信息。它还通过单一集合体中的整个药物治疗方案而不是依赖于年龄匹配样本来提供治疗反应的指示。在聚集体中获取细胞密度和活力的传统方法需要样品固定和切片，而我们的方法则以非破坏性方式提供数据。因此，随着时间的推移，可以在单个聚合中跟踪更改。该技术有望提高我们对聚合模型的离散区域内药物反应的理解，并对这些模型在体内反应和药物筛选应用中的反映程度产生影响。首先，在标准条件下制备所需细胞系的70%至90%汇合细胞培养物。按照标准胰蛋白酶消化方法从培养瓶中分离细胞单层，并将细胞悬浮液加入离心管中。将10微升细胞悬浮液加入血细胞计数器并通过显微镜计数以确定悬浮液中的细胞数量。通过离心沉淀细胞，并将细胞重悬于培养基中，所需浓度为每毫升2.5倍10至第五细胞。对于用基底膜基质制备的悬浮液，从零下20摄氏度的储存中取出基质小瓶，并放入冰箱中解冻过夜。准备一个装有生长培养基的容器，冷藏10分钟以冷却。使用冷冻移液器吸头将基质加入到冷冻培养基中，使得该溶液的最终浓度为5%，将50微升该培养基加入到圆底，非粘附的96孔板的每个孔中，使得在加入细胞悬浮液后，这些孔中基质的最终浓度将为2.5%。对于没有基质制备的悬浮液，向每个孔中加入50微升的普通生长培养基。将50微升细胞悬浮液分配到每个孔中。接种后立即在室温下将板以123G离心10分钟，以确保在每个孔的底部收集细胞沉淀。利用 OCT 系统进行结构成像。将 A 扫描速率设置为 5.5 千赫兹，以进行高分辨率图像采集。将液体介质中样品的折射率设置为 1.33。在屏幕右侧的图像参数窗口中，通过输入 X、Y 和 Z 值来设置视场，以便样本包含在此感兴趣区域中。单击3D采集模式，然后单击记录以收集样品的3D体积扫描。要创建卷重建，请打开 Imaris 并导航到其竞技场内转换后的 TIF 文件。转到编辑，然后单击“图像属性”并将 OCT 图像中的体素大小输入到相应的 XYZ 框中。接下来，单击“确定”。单击对象树上方的“添加新曲面”。在树下方的菜单中，单击跳过自动创建并手动编辑。在显示调整窗口中，手动滑动红色和黑色箭头以增强样品与背景之间的对比度，并改善样品的可视化效果。将切片位置调整到样品一条边缘处的切片。使用转义键将鼠标从导航模式更改为选择模式，然后单击“绘制”。手动跟踪显示信号的区域的轮廓。通过在输入框中输入下一个位置来推进切片位置。下一个位置应小于或等于样品中比前一个位置进一步的100个切片。手动跟踪显示信号的区域。通过样品的厚度重复此步骤，直到到达样品的另一边缘。然后单击“创建曲面”和左侧菜单将这些切片拼接在一起。最后，单击“编辑”，然后单击“掩码选择”，然后单击“确定”以完成卷重建。要获取样品的总细胞密度，请选择添加新斑点。在算法设置菜单中，取消选择所有框。单击蓝色箭头以移动到源通道屏幕。从显示的下拉菜单中，选择蒙版通道。在 XY 直径框中输入样品的平均细胞直径。确保选中背景减法。单击蓝色箭头可移动到分类点的屏幕。在菜单底部的图形中，单击黄色阈值的左边缘并将其拖动到图形的左边缘，以便所有对象都包含在黄色阴影阈值中。然后单击绿色箭头以完成点创建。通过单击“统计信息”获取标识的对象数。然后单击“总体”，最后单击“总点数”。单击创建的曲面，然后导航到曲面样式质量选项卡。将选区更改为中心点，并将像素宽度更改为小于 20 以获得最佳可见性。导航到“统计信息”。然后单击“详细”，然后单击“位置”并记录中心点的位置。从对象树上方的菜单中选择添加新参考框架。选中菜单中 X、Y 旁边的可见框和固定框。单击并拖动参考框图标的中心，使其与中心点对齐。取消选择“XY 可见”和“修复”框，然后为 XZ 选择它们。再次单击并拖动参考框图标的中心，使其与中心点对齐。最后，对 YZ 平面重复此操作，在这三个固定平面之间交替，直到参考系与中心点完全对齐。双击对象树中的参考框架，然后将其重命名为中心或类似名称。单击创建的点并导航到统计信息。然后单击详细，然后单击位置参考框架。单击位置 X 参考框，直到它从最高值到最低值排序。记录最大值以获取聚合的半径。执行手动计算以确定其他感兴趣的位置。为此，请在 X 中心值上添加 100 微米，然后使用中心点的 Y 和 Z 值定义沿中心轴的第一个位置。选择添加新点。然后选择“手动跳过自动创建编辑”。按住键盘上的 Shift 按钮，然后单击屏幕上的任意位置以放置新位置。输入第一个感兴趣位置的 XYZ 位置值。然后选择“添加新参考帧”并将其与点对齐。将 100 微米添加到每个连续的 X 位置。将最后一个参考系放置在距离样品外半径小于50微米的位置。单击创建的点并导航到统计信息。在菜单的右下角，单击“将所有统计信息导出到文件”，然后将数据保存到电子表格中。打开电子表格。导航到距原点参考框选项卡的距离。使用 mod 函数以数字方式将每个距离分配给其相应的参考系。过滤此列中的值以处理每个参考系中的距离。对于每个参考帧，使用 COUNTIF 函数计算距帧 50 微米范围内的物体数量。生成的值对应于该区域插头中的像元数。将此值除以100微米插头的体积，得到细胞密度。学生的T测试显示，对于MDA-MB-231球体模型，球体核心的细胞密度明显高于过渡层和外层。结果表明，该球状核在四天后被压实。在AU565和MDA-MB-231聚合模型中，基质的添加似乎不会影响体积或细胞计数，因此似乎对细胞增殖的影响可以忽略不计。相反，基质添加似乎重新分配了细胞密度，促进了显着的核心压实并降低了外层的细胞密度。这些发现为基质使细胞聚集在不同乳腺癌细胞系中的物理机制提供了有价值的见解。接下来观察用基质制备的AU565肿瘤球体，用曲妥珠单抗处理成熟的聚集体，并通过五天的药物治疗方案评估区域细胞密度。在整个骨料厚度中每100微米设置一次插头。随着时间的推移，在每个聚集体的内部500微米内观察到细胞密度的微小波动，表明细胞死亡最小。细胞死亡的可视化作为指示性药物反应，主要在肿瘤球体的外层，这与曲妥珠单抗的药物渗透问题一致。现在，我们可以在不牺牲结构的情况下，无损地测量聚集体中的细胞活力。这样可以评估奇异肿瘤聚集体中的纵向药物反应，以识别候选抗癌药物的时间和渗透质量。

Research

JoVE Journal

Cancer Research

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

科研

癌症研究

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

术中高强度聚焦超声装置治疗肝转移的可行性、安全性和准确性评价

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

腹腔灌洗液中低密度中性粒细胞分泌的中性粒细胞胞外陷阱介导肿瘤细胞生长与附着

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...