我们要感谢 Bourdoncle 和修士的科钦成像设施 (科钦研究所, 巴黎) 的意见和协助显微镜。这项工作得到了法国甲国家组织癌症、CARPEM (个性化医学癌症研究)、法国基金会和欧洲联盟2020地平线研究和创新方案赠款资助的部分支持。协议 No 667980 (克拉)。

人的肿瘤得到了法国道德委员会的同意和协助公共-Hôpitaux de 巴黎 (AP-HP) 在应用与法国法律的第 i. 1121-1 条。在纳入该研究之前, 患者获得了书面的知情同意。
1. 获得人肺肿瘤
<ol>
	<li>从一期非小细胞肺癌患者中获得新鲜的肺部肿瘤标本, 他们接受了完全外科手术切除的肺部肿瘤4。</li>
	<li>将无固定的新鲜肿瘤运送到15毫升圆锥管中, 含 ice-cold 罗斯威尔公园纪念研究所 (RPMI 1640) 培养基。在冰上预留30分钟直到步骤2.7。 注意: 当肿瘤在下午收到时, 将其放置在4° c 过夜, 并处理第二天。在这种情况下, 补充 RPMI-1640 培养基与5% 胎牛血清 (FBS)。</li>
</ol>
2. 人肺肿瘤的琼脂糖嵌入和 vibratome 切片
注: 执行所有步骤, 直到2.10 在无菌条件下。
<ol>
	<li>在不含钙和镁的磷酸缓冲盐 (PBS) 20 毫升无菌中溶解1克熔点点琼脂糖, 准备5% 琼脂糖溶液嵌入。微波这个解决方案, 直到琼脂糖完全溶解。允许琼脂糖冷却在37° c, 放置在一个孵化器在37° c 5 分钟的解决方案。</li>
	<li>在组织培养罩, 制备完整的 RPMI-1640 培养基 (不含酚红), 含有10% 胎牛血清 (FBS), 4 毫米谷氨酰胺和1x 青霉素和链霉素。</li>
	<li>添加1.1 毫升完整的 RPMI-1640 培养基每井到一个6孔细胞培养板, 并放置一个30毫米细胞培养插入每一个井。把盘子放在37° c 的孵卵器里30分钟直到步骤2.12。</li>
	<li>放置灭菌不锈钢平垫圈 (内径为5毫米, 外径为10毫米, 厚度为 1 mm), 在 35 mm 的塑料盘中填充 RPMI 完全介质。垫圈将进一步用于集中抗体的 vibratome 切片。</li>
	<li>将肿瘤活检放在一个10厘米的塑料盘子里, 用锋利的刀片将肿瘤切开, 切成大约5x5 毫米长的小立方体形状的碎片。</li>
	<li>从孵化箱取出琼脂糖, 将溶液倒入35毫米的塑料碟中。</li>
	<li>用一副钳子将肿瘤的碎片小心地转移到纸巾上, 以消除过量的培养基。将碎片插入琼脂糖, 并将其放置在塑料盘的底部。在冰上留下琼脂糖凝胶5分钟固化。</li>
	<li>一旦琼脂糖凝固, 倒置的塑料碟和使用刮刀释放整个凝胶。</li>
	<li>使用锋利的刀片修剪周围的肿瘤片段的过量琼脂糖, 留下3-5 毫米的凝胶周围的组织。</li>
	<li>将每个琼脂糖块贴在 vibratome 的试样盘上, 用一滴无毒的丁基氰组织胶。用无菌 ice-cold PBS 填充 vibratome 缓冲托盘, 并将试样盘安装在托盘中。</li>
	<li>将琼脂糖嵌入组织切成350µm 厚的切片。调整 vibratome 设置在慢速范围 (0.2-0.3 毫米/秒) 和振动频率在一个中等范围 (1.5 毫米)。</li>
	<li>使用细钳, 仔细收集的肿瘤切片, 因为他们被切割, 并放置到细胞培养插入一个6井板 (步骤 2.3)。在每个插入上传输1切片。处理切片时要非常小心, 因为它们很容易损坏。</li>
	<li>用细钳将不锈钢垫圈放置在每个单独的切片上。确保垫圈在肿瘤组织周围的琼脂糖上有很好的定位。</li>
	<li>在37° c 的 5% CO2中, 将培养基保持在恒温箱中10分钟, 进行免疫。</li>
</ol>
3. 居民 CD8 T 细胞和肿瘤细胞的免疫
<ol>
	<li>在无酚红的 RPMI-1640 培养基中稀释抗体 (最终浓度10µg/毫升) 和核染料 (最终浓度1µg/毫升)。 注: 使用荧光结合的 anti-CD8 (小鼠 igg, 克隆 SK1) 和抗 EpCAM (上皮细胞黏附分子) (小鼠 igg HEA-125) 抗体标签 CD8 T 淋巴细胞和肿瘤上皮细胞, 分别。使用荧光结合的 anti-CD90/Thy1 (小鼠 IgG, 克隆 5E10) 抗体标记成纤维细胞和活化内皮细胞。使用 4 ', 6-diamidino-2-吲 (DAPI) 标记细胞核的细胞与一个受损的等离子膜, 以评估组织的可行性。</li>
	<li>从孵化箱中取出培养皿, 用吸管抽吸不锈钢垫圈内的任何液体。不要删除1.1 毫升 RPMI-1640 介质放置在细胞培养插入 (步骤 2.3)。</li>
	<li>在不接触切片和使用吸管尖端的情况下, 在每个肿瘤切片上加40µL 的溶液中含有抗体。</li>
	<li>孵育板在37° c 为15分钟, 允许抗体染色。</li>
	<li>用细钳, 取下垫圈, 取出切片, 十年代在无酚红的 RPMI-1640 介质中浸出。将切片放回单元格区域性插入, 并在每个切片上添加一滴酚无红色 RPMI-1640 介质。保持在37° c 的板10分钟进行成像。</li>
</ol>
4. 人肺肿瘤切片中居民 CD8 T 细胞迁移的影像学分析
注: 本协议中使用的共焦显微镜是一个装有25x 水浸泡物镜的直立旋转圆盘 (25 x/0.95)。
<ol>
	<li>准备显微镜设置<ol>
		<li>设置显微镜热室的温度在37° c 前几个小时开始成像会议。</li>
		<li>建立一个系统, 以不断灌注肿瘤切片与含氧 (5% CO2, 95% O2) 苯酚无红色 RPMI 培养基 (图 1)。使用蠕动泵将灌注介质流进成像室, 并将溶液抽吸到废物收集瓶中。</li>
		<li>运行蠕动泵, 让氧溶液通过灌注管。</li>
	</ol>
	</li>
	<li>用细钳, 取出6井板上的切片, 并将其转移到35毫米的含酚无红 RPMI 培养基的塑料碟中。固定切片与19.7 毫米直径的切片锚与2毫米间隔线程。将培养皿放在显微镜的成像阶段。连接灌注系统的入口和出口提示。打开灌注系统, 将介质流量设置为0.8 毫升/分钟。</li>
	<li>将浸水目标降低到切片。聚焦在片的顶部, 明亮的光场。</li></ol>使用适当的萤光灯, 选择一个包含 CD8 T 细胞的感兴趣区域, 肿瘤胰岛 (阳性 EpCAM) 和基质区 (阳性 CD90)。通过评估切口表面的 DAPI 染色和更深的组织, 检查切片内细胞的活性。 注意: 一个健康的切片只包含少数的 DAPI 阳性细胞在几微米的切割表面。
	<li>使用以下操作设置图像处理会话。<ol>
		<li>根据3荧光荧光信号的强度, 将曝光时间设置在50到800毫秒之间, 激光强度介于20到40% 之间。</li>
		<li>在肿瘤切片中选择 z 堆叠厚度到图像。 注: 在 z 维度中, 10-12 架横跨60-70 µm 的光学平面被捕获。</li>
		<li>在第一个标记为 CD8 T 单元格的下面, 选取大约10µm 的起始位置。</li>
		<li>定义每个 z 堆栈图像之间的时间间隔, 介于10到三十年代之间, 总记录时间介于10到30分钟之间。</li>
	</ol>
	</li>
	<li>按参考4中的描述分析驻留 CD8 T 细胞迁移。<ol>
		<li>将数据导入跟踪软件后, 在该字段中为每个 CD8 T 单元格创建点。根据成像荧光细胞调整细胞直径和检测阈值。</li>
		<li>检查轨道, 删除任何不相关的轨道, 删除它们。通过连接和断开不同轨道和同一轨道的不同时间点来纠正轨道。将数据 (跟踪速度、跟踪位移长度) 导出到电子表格软件。</li>
	</ol>
	</li>

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未声明任何利益冲突。

本文介绍了一种直接、快速、稳健的人肺肿瘤切片技术, 并利用共焦显微镜对保存在肿瘤环境中的 CD8 T 细胞的动态行为进行了评价。这个协议是一个以前的朱庇特文章的细化, 描述了对小鼠的淋巴结切片2监测的镀 T 细胞。
荧光染料的漂白必须特别考虑第一代染料, 如荧光素异硫氰酸酯和藻。我们发现在双光子显微镜下, 直接耦合的抗体有明显的漂白。与此相反, 最小的漂白被发现与共聚焦显微镜, 特别是使用明亮的荧光染料最近开发。
抗体是相对较大的分子, 因此它们渗透到组织中可能是困难的。这一步可以提高孵化时间。此外, 使用更小的染色试剂, 如直接耦合的抗体片段, 是一种很好的替代品。
本协议中描述的所有抗体都已经过验证, 并且已知可以产生明亮的着色4。因此, 不需要同种控件。然而, 如果对这种方法的修改使用其他抗体是需要的, 那么使用同种控制抗体是极力推荐的。
这一实验系统有一定的局限性。切割过程将损害组织, 这可能会影响 CD8 T 细胞的行为, 特别是在切割表面附近。此外, 一些可溶性因素 (如趋化因子) 有助于控制 T 细胞的迁移, 可以失去。绕过这些问题的一种方法是, 通过监测位于组织的大概更健康区域的表面上几十微米的 T 细胞。已发表的实验表明, 在组织内深部的 t 细胞的迁移比在切口表面附近的 t 细胞更活跃.4。我们已经用共焦显微镜进行了研究, 但有可能是双光子显微镜将增加空间分辨率的深度。
这种方法依赖于非中性分子的荧光耦合抗体的使用。全尺寸抗体易受不同方式影响 T 细胞的正常功能。首先, anti-CD8 抗体可以改变 T 细胞迁移通过触发细胞内信号级联能够影响细胞骨架的淋巴细胞。其次, anti-CD8 抗体可以调节 CD8 和 MHC I 类分子之间的相互作用, 这是抗原识别过程中的一个重要步骤。第三, 完整的抗体可以绑定到 Fc 受体表达的许多髓细胞分布在肿瘤, 因此容易增加非特异性信号。我们没有迹象表明这一问题影响了我们的数据, 可能是因为居民髓细胞的 Fc 受体与文化中的细胞不同, 在肿瘤环境中饱和了免疫球蛋白。事实上, 在标记过程中添加血清, 一个已知的阻断游离 Fc 受体的过程, 没有改变免疫染色。尽管如此, 没有 fc 区的抗体片段, 以及在其 fc 区域和骆驼抗体片段中突变的抗体,8代表了其他可替代的策略来跟踪带有荧光示踪的驻留细胞。
在过去的几十年中, 几个模型系统已经开发和使用的 real-time 成像的 T 淋巴细胞。这些包括体外的 T 细胞的准备, 以前纯化和培养抗原呈递细胞。这些准备工作在确定抗原识别和反应机制方面取得了显著进展。然而, 它们缺乏组织环境的复杂性。其他的准备工作已经建立, 更紧密地模仿当地组织的结构。例如, 三维胶原凝胶基质, 其中纯化 t 细胞已被植入, 以及 reaggregated 组织片段的培养, 已被用来监测的动态行为的 t 淋巴细胞的荧光成像技术9.这些系统目前的优势, 建筑接近本土组织, 但他们缺乏其他重要的细胞和可溶性因素。在小鼠, 双光子活体显微镜已被用来跟踪 T 细胞在真正的生理环境的一个完整的器官10。然而, 这种方法在技术上很难建立。此外, 小鼠模型显示出显著的差异与人类的生理。
本议定书所描述的技术是一个复杂的多肿瘤环境, 是独特的定位, 作为一个重要的联系之间的共培养研究和动物模型。
本文所描述的协议也可用于跟踪其他细胞的运动, 而不是 CD8 T 淋巴细胞的抗体生成。此外, 该技术可以适应其他人和小鼠的肿瘤和组织。例如, 我们已经能够实时图像的动态 B 细胞标记的 anti-CD19 抗体在新鲜的人扁桃体。
本协议中所描述的技术允许对组织中的 T 细胞运动进行控制的治疗分子进行筛选。文化系统的可及性允许应用一些药理试剂并测试其对 T 细胞迁移的影响。现在人们已经承认, 在生长的肿瘤, CD8 T 细胞表现出低迁移和减少接触与肿瘤细胞11。重要的是, t 细胞在肿瘤细胞区的稀少存在与不良结局相关, 被认为是对癌症免疫治疗的抵抗机制之一。在肿瘤中限制 T 细胞迁移的多重障碍已经被确定, 包括一个稠密的细胞外基质。在这方面, 鉴定能够恢复 T 细胞运动和与恶性细胞相互作用的分子是癌症免疫治疗的一个重要步骤。在先前的研究中, 我们发现胶原酶的部分降解, 对人肺切片有很好的应用, 可以改善 T 细胞与肿瘤细胞的接触。
最后, 将人类肿瘤切片保留在文化中长达数天的可能性7,12,13在 T 细胞和免疫治疗方面提供了许多有希望的应用。然而, 模型系统在长期培养过程中的稳定性是一个重要的参数, 需要进行全面的评估。
获得新鲜和健康的组织是生产优质切片的关键因素, CD8 细胞、肿瘤细胞和基质元素免疫良好。在4° c 前保持肿瘤活检是至关重要的。
用细钳处理切片是该协议中的另一个关键步骤。这应该是非常谨慎的执行, 因为琼脂糖可以很容易损坏。琼脂糖的切割会损害不锈钢垫圈所造成的密封, 从而导致含有溶液的抗体泄漏。

CD8 T 细胞在抗癌的斗争中起着关键作用。然而, 许多抑制机制阻止 T 淋巴细胞杀死恶性细胞。在过去的几年中, 一些有希望的策略, 释放刹车的 T 细胞已经开发和使用的诊所5。保留相当注意的两种方法是免疫检查点靶向抗体, 如 anti-PD-1 和过继 T 细胞疗法。然而, 尽管最近取得了成功, 但只有一部分患者受益于这些疗法6。一个主要的挑战是找出抗肿瘤免疫疗法的机制, 以便开发更有效的治疗方法。另一项挑战是开发模型系统, 使新的治疗方法的特点和测试, 其效力和安全性。 到目前为止, 大多数的这些化验都是基于体外细胞培养系统, 它不太能模仿肿瘤组织的复杂性。
前体组织切片是一种很有前途的技术, 因为它保留了原始的组织微环境7。多年来, 我们建立了这种方法来跟踪各种组织中 T 淋巴细胞的动态变化。我们的结果表明, 荧光标记 T 细胞添加到小鼠的淋巴结切片迁移到组织, 并响应其适当的微提示1,2。 同样, 在人肺肿瘤切片上的 T 淋巴细胞被迅速吸收到肿瘤基质中3。利用这一技术, 我们确定了细胞外基质作为控制人类肿瘤 T 细胞分布和迁移的一个重要因素3,4。由于体外纯化和镀膜 t 细胞的行为并不一定反映了居民瘤 t 淋巴细胞的行为, 我们最近改进了这种方法 4。
在这里, 我们描述了一个协议, 以跟踪居民 T 淋巴细胞的切片由新鲜人的肺部肿瘤。不同的步骤包括制备人肺肿瘤切片 (包括琼脂糖嵌入和 vibratome 切片的嵌入组织), 在新鲜的肿瘤切片内源性 T 细胞的荧光抗体染色, 并监测这些细胞与共聚焦显微镜。

<table>
	<tbody>
		<tr>
			<td>Upright confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td>The use of a spinning disk confocal is recommended</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td></td>
			<td>This sofware is used for visualization of images and tracking of T cell migration</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>World Precision Instruments</td>
			<td>14142</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mm Culture inserts</td>
			<td>Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) medium, GlutaMAX Supplement</td>
			<td>ThermoFisher</td>
			<td>61870010</td>
			<td>Complete RPMI-medium is made by adding 10 % heat-inactivated fetal calf serum and Penicillin/streptomycin</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>20012019</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170088</td>
			<td></td>
		</tr>
		<tr>
			<td>Low gelling temperature Agarose, type VII-A</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-toxic butyl Cyanoacrylate Glue, Vetbond</td>
			<td>3M</td>
			<td>1469</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice anchors, 19.7 diameter, 2 mm spacing threads</td>
			<td>Warner Instruments</td>
			<td>64-1415</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel washers, 5 mm of inner diameter</td>
			<td>Amazon</td>
			<td>B004K1FDGQ</td>
			<td></td>
		</tr>
		<tr>
			<td>BV605-conjugated anti-CD8 (clone SK1)</td>
			<td>BD Biosciences</td>
			<td>565289</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-EpCAM (clone HEA-125)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-113</td>
			<td></td>
		</tr>
		<tr>
			<td>BV510-conjugated anti-CD90 (clone 5E10)</td>
			<td>Biolegend</td>
			<td>328125</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo imaging of resident cd8 t lymphocytes in human lung tumor slices using confocal microscopy

CD8 T 细胞是对抗癌症的关键人物。为了使 CD8 T 细胞杀死肿瘤细胞, 他们需要进入肿瘤, 在肿瘤微环境中迁移, 并对肿瘤抗原作出适当反应。最近发展的改进成像方法, 如2光子显微镜, 和使用强大的小鼠肿瘤模型已经揭示了一些机制, 调节抗肿瘤 T 细胞的活动。虽然这些系统提供了宝贵的洞察力, 但它们并不总能预测人类的反应。在人类方面, 我们在这一领域的知识主要来自于对人类患者的固定肿瘤样本的描述, 以及体外研究。然而,体外模型缺乏复杂的三维肿瘤环境, 因此, 是体内T 细胞活动的不完全近似。由说明组织制成的新鲜切片代表了一个复杂的多肿瘤环境, 可以作为共研究和动物模型之间的重要联系。最初设置在小鼠淋巴结1 , 以前在朱庇特文章2中描述, 这种方法现在已被转化为人类肿瘤, 以检查两个镀膜的动力学3以及驻留 T 细胞4。
本文介绍了一种用于制备人肺肿瘤切片、免疫 CD8 t 和肿瘤细胞的方法, 以及利用共聚焦显微镜对肿瘤微环境中 CD8 t 淋巴细胞进行追踪的协议。该系统是唯一的放置在屏幕上的新的免疫治疗药物, 有利于 T 细胞迁移肿瘤。

人肺肿瘤通常是高度渗透在 CD8 T 细胞, 在基质中优先发现3,4。通过遵循这个协议, 人们应该能想象出大量的 immunostained CD8 T 在人肺肿瘤切片中的显示。具有抗 EpCAM 和 anti-CD90 抗体的 co-labeling 应允许描绘肿瘤胰岛和基质区。纤维胶原蛋白在肿瘤基质中丰富, 在双光子显微镜下采用二次谐波生成, 可直观地可视化无外源性染色3,4。请注意, 一些人的肺部肿瘤目前没有明确区分肿瘤岛和基质。这种肿瘤应该被丢弃, 以及标本呈现大面积的细胞坏死鉴定的非特异性结合抗体和 DAPI 染色。与人类肺癌和卵巢癌的公布结果相一致, CD8 T 细胞在基质中较肿瘤胰岛更集中于4。在图 2中显示了与肿瘤细胞和 CD90 相关的 CD8 T 细胞分布的典型例子。
切片法可以分析不同肿瘤区的居民 T 细胞运动。比较不同区域 CD8 T 细胞运动的有用标准包括平均速度 (路径长度除以采样时间) 和位移长度 (轨道起点和终点之间的向量)。虽然肿瘤小岛稀少, CD8 T 细胞迁移更积极地在这个车厢与基质相比 (图 3)。平均, 单个 CD8 T 细胞的平均速度应接近4µm/分钟的肿瘤基质与高内和 inter-tumor 变化4。在肿瘤基质中, 细胞外基质纤维的密度和取向对其细胞的运动有强烈的影响3,4。与松散矩阵区域相比, 一个成功的实验将导致在稠密矩阵区域中出现较少的 T 细胞, 与已发布的结果3,4一致。
请注意, 有些人的肺部肿瘤表现出非常高水平的自发荧光排除了居民 T 细胞的成像。在成像会话开始时, 此类特性很快被观察到。Autofluorescent 肿瘤应丢弃。一个典型的例子, 常驻 CD8 T 细胞在一个 autofluorescent 肿瘤显示在图 4。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55709/55709fig1.jpg"> 
图 1: 灌注系统的照片.A) 苯酚无红 RPMI 溶液富含 5% C02, 95% O2。B) 然后用蠕动泵灌注溶液。C) RPMI 溶液通过入口进入培养皿。在出口处, 溶液由泵从燃烧室吸入到一个废弃容器中。请注意, 吸入尖端设置在一个水平, 以确定解决方案的高度。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55709/55709fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55709/55709fig2.jpg"> 
图 2: 人肺肿瘤中居民 CD8 T 细胞的分布.CD8 (绿色)、EpCAM (蓝色) 和 CD90 (红色) 的人肺肿瘤切片的代表性图像。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55709/55709fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55709/55709fig3.jpg"> 
图 3: 人肺肿瘤中居民 CD8 T 细胞的运动.人肺癌切片基质 (红) 和肿瘤细胞 (蓝) 区个体居民 CD8 T 细胞的运动轨迹。用共聚焦显微镜对该切片进行了染色, 并对20分钟进行了成像。在双光子显微镜下利用二次谐波 (倍) 在末端检测纤维胶原。根据 CD8 T 细胞位移长度, color-coded 轨道。此图像已从4中修改。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55709/55709fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55709/55709fig4.jpg"> 
图 4: autofluorescent 人肺部肿瘤的典型图像.肿瘤切片被染色为 CD8 (绿色) 和 EpCAM (蓝色)。倍信号是红色的。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55709/55709fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy at JoVE.com

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

本协议描述了一种图像驻留肿瘤浸润 CD8 T 细胞标记的人肺肿瘤切片荧光耦合抗体的方法。这项技术允许 real-time 分析 CD8 T 细胞迁移使用共聚焦显微镜。

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 ...

ex-vivo-imaging-resident-cd8-t-lymphocytes-human-lung-tumor-slices

Nanyang Technological University

Research

JoVE Journal

Cancer Research

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

11.1K 次观看.  Université Paris Descartes. 本协议描述了一种图像驻留肿瘤浸润 CD8 T 细胞标记的人肺肿瘤切片荧光耦合抗体的方法。这项技术允许 real-time 分析 CD8 T 细胞迁移使用共聚焦显微镜。

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

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

科研

癌症研究

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

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

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 ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

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 ...

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

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

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 ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...