这项研究得到了 Adelson 医学研究基金会 （AMRF）、黑色素瘤研究联盟 （MRA） 和 U54CA224068 的支持。 图 1、 图 4 和 图 5 是使用 BioRender.com 创建的。

本研究符合有关人体组织采样的所有机构指南。获得患者的知情同意，并对可识别的样本数据进行匿名化处理。在手术室收集样本，置于 RPMI、生理盐水或 PBS 溶液中，并在用于研究之前通过病理科确认癌变。除非另有说明，否则所有步骤均应在 4 °C 或冰上进行。处理人体组织时，在生物安全罩内工作。有关本协议中使用的所有材料、试剂和仪器的详细信息，请参阅 材料表 。
1. 获取样本
<ol><li>在一管培养基中获取实体瘤样品并将其置于冰上。<ol><li>指示手术室工作人员将样品装在含有足够 RPMI、生理盐水或 PBS 的试管中运输，以完全浸没组织以避免样品脱水。 注意：确保样品未干燥很重要，因为这会降低活力。</li>
	</ol></li>
	<li>将所有离心机冷却至 4 °C，并将热混合器加热至 37 °C。</li>
	<li>取出准备好的人或小鼠肿瘤解离酶（材料表），在 37 °C 热混合器上解冻，解冻后移至冰中。</li>
	<li>取回 20 mL 等分试样的 RPMI。</li>
	<li>记录相关的样本信息（例如，患者 ID、去标识符、样本类型）。</li>
	<li>将试管倒置数次，然后将内容物倒入培养皿中，以确保转移所有样品材料，将样品转移到 60 mm 组织培养皿（置于冰上）中。如果样品在到达时未以培养基形式交付，请在培养皿中加入新鲜的 RPMI 培养基。<ol><li>如果样品碎片化，将含有样品的试管在 450 × g、4 °C 下旋转 5 分钟，弃去上清液，将样品重悬于 420 μL RPMI 中，然后移至第 2 部分。</li>
	</ol></li>
</ol>2. 机械和酶消化
<ol><li>将 420 μL 培养基从 60 mm 样品皿中移液到 1.5 mL 微量离心管中。尝试包含媒体中悬浮的任何样本片段。</li>
	<li>使用镊子将样品组织从 60 mm 培养皿转移到含有培养基的 1.5 mL 试管中。用管内的干净剪刀切开样品，使样品块的最大直径为 2-3 毫米。</li>
	<li>向样品中添加根据制造商规格（材料表）制备的消化酶，体积如下：人样品 42 μL 酶 H（非特异性蛋白酶），小鼠样品酶 D（非特异性蛋白酶），21 μL 酶 R（非特异性蛋白酶）和 5 μL 酶 A（核酸酶）。</li>
	<li>从培养皿中再添加 420 μL 培养基，或最多添加到试管上的 1 mL 标记处。培养基含量不超过 420 μL。</li>
	<li>将管子涡旋 5 秒，然后将其放入垂直放置在其侧面的热混合器中（图 1B）。在 37 °C、450 rpm 下孵育 15 分钟。 注：在孵育过程中，取出 GEM 生成所需的所有 10x Genomics 材料，因为这些材料需要 30 分钟才能平衡至室温</li>
</ol>3. 样本过滤
<ol><li>将 50 μm 比色皿过滤器放在新的 15 mL 锥形管顶部，然后将 1 mL 新鲜 RPMI 培养基通过滤池放入试管中来灌注过滤器。</li>
	<li>消解后，使用 1,000 μL 低吸附宽移液器吸头将样品转移至 15 mL 试管中。用 1 mL 新鲜培养基清洗 1.5 mL 试管，然后将培养基转移到过滤器中，以确保所有样品材料都通过过滤器。</li>
	<li>获取 1 mL 塑料注射器，仅取出柱塞（丢弃注射器的其余部分）。使用注射器柱塞的背面，以扭转运动研磨并推动样品在过滤器上。</li>
	<li>用 5 mL 培养基和装有样品的培养皿中的任何其他剩余培养基（第 1 部分）清洗过滤器，以收集可能已从组织中分离的任何细胞。</li>
	<li>将 30 μm 细胞过滤器放在新的 15 mL 锥形管顶部，用 1 mL 培养基灌注过滤器，然后将含有解离样品的管中的内容物转移至过滤器。 注：此步骤将去除在使用 10x Genomics 微流控芯片运行样品时可能堵塞或导致润湿失败的大碎屑。</li>
</ol>4. 红细胞裂解
<ol><li>将样品在 450 × g、4 °C 下离心 5 分钟。吸出培养基，注意不要干扰沉淀。</li>
	<li>重悬于 ACK 裂解缓冲液中以去除红细胞 （RBC） 并记录使用的体积。体积将取决于颗粒大小和颗粒中 RBC 的程度。 注：当添加足够的 ACK 裂解缓冲液时，重悬的 ACK 样品溶液应为无色，因为这将清除样品中的红细胞。</li>
	<li>将样品管在 450 × g、4 °C 下离心 5 分钟，然后吸出上清液。</li>
	<li>根据需要重复步骤 4.2-4.3，直到离心后样品沉淀没有可见的红色（图 2）。记录此步骤中使用的任何额外体积的 ACK 裂解缓冲液。</li>
	<li>将样品沉淀重悬于新鲜培养基中，以准备计数。</li>
</ol>5. 细胞计数
<ol><li>将 10 μL 台盼蓝放入 1.5 mL 试管中。轻轻混合解离的细胞，取 10 μL 样品，与台盼蓝混合 （1：1）。在血细胞计数器的每一侧加载 10 μL 并计数细胞。</li>
	<li>记录细胞浓度、总活细胞计数、活力百分比和计数后的最终培养基体积。</li>
	<li>确保细胞计数在 700 至 1,200 个细胞/μL（7 × 10、5-1.2 × 10、6/mL）之间，以实现最佳的 10 倍基因组学运行。<ol><li>如果样品浓度过高，请用培养基稀释并重新计数。</li>
		<li>如果样品太稀释，将样品在 450 × g、4 °C 下离心 5 分钟，然后重悬于较小体积的培养基中。</li>
		<li>如果细胞活力低于 80%，则执行活死程序以去除死细胞。</li>
		<li>如果单元格少于 100 万个，请参阅第 6 节。</li>
		<li>如果细胞数量超过 100 万个且活力> 10%，请参见第 6 节。</li>
		<li>如果细胞数量超过 100 万个且活力< 10%，请参阅第 7 节。</li>
		<li>如果细胞活力高于 80%，请将处理过的细胞放在冰上，并立即根据制造商的说明进行 GEM 生成。</li>
	</ol></li>
</ol>6. 去除死细胞 - 试剂盒 1
注：使用死细胞去除试剂时，请在生物安全通风橱内工作，因为这些试剂很容易被污染。
<ol><li>将样品在 450 × g、4 °C 下离心 5 分钟。</li>
	<li>在 15 mL 锥形管中制备分离培养基，并在室温下保存：9.8 mL PBS、10 μL CaCl2 和 200 μL 热灭活 FCS（添加到生物安全罩中）。</li>
	<li>离心后，从沉淀中吸出培养基，并重悬于 1 mL 分离培养基中。</li>
	<li>将试管再次在 450 × g、4 °C 下离心 5 分钟。吸出上清液并将沉淀重悬于 100 μL 分离培养基中。</li>
	<li>将 100 μL 分离培养基中的样品转移到 0.2 mL PCR 联管的一个孔中。</li>
	<li>在生物安全通风橱中，向含有 100 μL 样品的分离培养基孔中加入 10 μL Annexin V 混合物和 10 μL 生物素选择混合物。移液管混合溶液，使其在室温下静置 4 分钟。</li>
	<li>4 分钟后，涡旋葡聚糖包被的磁性颗粒 30 秒。向样品中加入 20 μL 磁珠和 60 μL 分离培养基（总体积为 200 μL）。如果活力低于 40%，则将磁珠的量放大至 40 μL，同时调整分离培养基的体积（最多 40 μL），保持总体积为 200 μL。移液混合，让溶液在室温下静置 3 分钟。</li>
	<li>将试管放在 10x Genomics 磁分离器（高设置）上，在室温下放置 5 分钟。</li>
	<li>在不干扰磁珠的情况下将液体从联管转移到新的 1.5 mL 低结合微量离心管中。不要从磁铁上取下条管。</li>
	<li>将含有样品的 1.5 mL 试管在 450 × g、4 °C 下离心 5 分钟。吸出上清液，并根据沉淀大小和细胞计数将沉淀重悬于适当体积的 RPMI 培养基中，如第 5 节所述。如果活力仍低于 80%，则重复死细胞去除程序（图 3 和 图 4）。 注：请勿将细胞重悬于分离培养基中以用于下游 GEM 生成，因为氯化钙会干扰包埋过程和逆转录 （RT） 步骤。</li>
	<li>如果细胞活力高于 80%，请将处理过的细胞放在冰上，并根据制造商的说明立即进行 GEM 生成。</li>
</ol>7. 去除死细胞 - 套件 2
注：使用试剂盒试剂时，请在生物安全通风橱内工作。
<ol><li>将微珠和 20x 结合缓冲液原液放入生物安全罩内。</li>
	<li>将样品悬浮液在 450 × g、4 °C 下离心 5 分钟。</li>
	<li>在无菌条件下使用 1 mL 的 20x 结合缓冲液原液，移液到 19 mL 不含 DNAse 和 RNAse 的蒸馏水中，制备 1x 缓冲溶液。</li>
	<li>离心完成后，吸出上清液，加入微珠，轻轻混合样品，并在室温下孵育 15 分钟。<ol><li>当细胞计数为 107 或更少时，添加 100 μL 微珠。</li>
		<li>对于高于 107 个总细胞计数的微珠，将微珠调整为每 107 个活细胞 100 μL 的最终浓度。</li>
	</ol></li>
	<li>在孵育过程中，获得一个 MACS 阳性选择柱、MACS multistand 和相应的磁分离器。确保分离器在多功能支架上调平，并且支架附近没有其他金属或磁性物体，因为磁力很强大，可能会影响死细胞去除效率。将色谱柱紧贴地放入分离器支架中，机翼朝外。</li>
	<li>样品孵育后，如果总重悬体积小于 500 μL，则添加 1x 结合缓冲液，直到总体积为 500 μL。</li>
	<li>将 15 mL 锥形管置于色谱柱下方，并用 3 mL 1x 结合缓冲液灌注色谱柱。</li>
	<li>将锥形管更换为用于细胞样品采集的新型 15 mL 锥形管，并将样品加入色谱柱中，使其完全流过色谱柱。</li>
	<li>用 4 x 3 mL 的 1x 结合缓冲液洗涤色谱柱，在上一次洗涤液完全流过色谱柱后，一次只添加一次洗涤液。</li>
	<li>第四次洗涤后，将含有分离细胞的锥形管在 450 × g、4 °C 下离心 5 分钟。丢弃色谱柱和剩余的 1x Binding Buffer。</li>
	<li>根据沉淀大小，将样品溶液重悬于适量的 RPMI 培养基中，并使用第 5 节中描述的方法对分离的细胞进行计数（图 3 和 图 5）。<ol><li>如果活力保持在 80% 以下，并且细胞总数为 106 或更少，请重复第 6 部分。</li>
		<li>如果细胞活力高于 80%，请将处理过的细胞保存在冰上，并立即根据制造商的说明进行 GEM 生成。</li>
	</ol></li>
</ol>

用于单细胞 RNA 测序分析的肿瘤解离方案

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+high-yield,+high-throughput+cell+enrichment:+fluorescence+versus+magnetic+sorting.">Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting.</a> Sci Rep. 9 (1), 227 (2019).</li></ol>

M.S-F.接受了 Bristol Myers-Squibb、Istari Oncology 的资助，并且是 Galvazine Therapeutics 的顾问。

该方案描述了人或小鼠肿瘤样品解离成单细胞悬液，以使用 10x Genomics 微流控系统进行 scRNA 测序。在处理通常来自罕见癌症的组织或正在进行的临床试验或小鼠肿瘤长期实验的组织时，必须在所有步骤之间以最佳方式仔细保存样品。所有步骤都应在冰上或 4 °C 下快速进行，以保持天然细胞 RNA 谱并防止降解，因为在室温下长时间工作会影响转录组质量17,18</s...

尽管癌症研究的许多进步导致针对免疫和肿瘤细胞上表达的抑制性受体的药物（称为检查点阻断抑制剂）的开发并获得 FDA 批准，但治疗耐药性和确定驱动患者反应的机制仍然具有挑战性1。表征肿瘤分子多样性的异质性以及肿瘤细胞与免疫微环境之间错综复杂的相互作用的复杂挑战，需要新的方法来以单细胞分辨率剖析这个复杂的生态系统。了解肿瘤及其微环境中的分子复杂性对于推进治疗策略和破译癌症进展的复杂生物学至关重要2。单细胞 RNA 测序 （scRNA-seq） 越来越受欢迎，因为它可以对复杂肿瘤样本中的单个细胞进行高分辨率分析 3,4。
肿瘤异质性，包括遗传、表观遗传和表型多样性，对揭示癌症生物学的复杂性构成了挑战5。传统的大量 RNA 测序方法往往会掩盖肿瘤中存在的异质细胞群的独特表达谱和表型，因为这些方法会平均整个细胞群的信号4。相比之下，scRNA-seq 允许解剖单个细胞类型，揭示不同的基因表达、抑制模式和细胞状态，否则会被忽视 4,6。scRNA-seq 的前提是它能够解码单个细胞的遗传和分子特征，在发现新的癌症疗法方面具有巨大的前景7。
通过破译肿瘤细胞以及周围基质细胞和免疫细胞的基因表达谱，研究人员可以确定新的治疗靶点，并针对个体患者制定精准遗传医学策略6。此外，剖析肿瘤微环境中的免疫库为了解肿瘤细胞和免疫效应子之间的相互作用提供了重要的见解，为免疫治疗干预铺平了道路3。尽管在临床样本中使用 scRNAseq 取得了进展，但处理步骤中的一些挑战可能会损害细胞的 RNA 完整性、活力和所生成数据的质量。此外，处理细胞活力低的组织会增加单个细胞封装过程中产生的液滴中的环境 RNA 水平，从而导致交叉污染和细胞类型的错误注释，而在 37 °C 下进行的长期解离方案会导致多种细胞类型中应激反应基因特征的上调 8,9，10.
该方案中概述的逐步程序（图 1A）从肿瘤的快速机械和酶解离开始，然后过滤和去除死细胞，具体取决于每个肿瘤样本的活力。目前，该程序已在黑色素瘤11、结直肠12、头颈部鳞状细胞癌13、胰腺和肺（未发表数据）肿瘤样本中得到验证。这些技术可确保生成对下游分析至关重要的高质量活细胞悬液14。值得注意的是，去除不含合成 RNA 的红细胞对于纯化样品 15 变得势在必行15。随后的细胞计数评估和死细胞的消除是成功生成 10x Genomics 凝胶微珠溶出液 （GEM） 、条形码的前提，并提高转录本和测序细胞的回收率，而不会危及敏感细胞群（例如中性粒细胞和上皮细胞）的排除16。这些步骤对于释放肿瘤样本中单细胞分辨率分析的潜力 9,10、揭示推动创新治疗干预所需的新基因表达谱和免疫特征以及识别新的肿瘤脆弱性至关重要。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>1x PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Corning</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389213</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L low-retention wide pipette tips</td>
			<td>Rainin</td>
			<td>30389218</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389212</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Magnetic Separator</td>
			<td>10x Genomics</td>
			<td>120250</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL aspirating pipette</td>
			<td>Corning</td>
			<td>357558</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389226</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389225</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389240</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389239</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>60 x 15 mm Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco</td>
			<td>A10492-1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>3090659</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>551660</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Solution</td>
			<td>Sigma Aldrich</td>
			<td>2115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Ranger&#160;</td>
			<td>10x Genomics</td>
			<td>N/A</td>
			<td>https://www.10xgenomics.com/support/software/cell-ranger/latest</td>
		</tr>
		<tr>
			<td>Cell counting slides for TC20 cell counter</td>
			<td>Bio-Rad</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics 30&#956;m sterile disposable filters</td>
			<td>Sysmex</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics 50&#956;m sterile disposable filters</td>
			<td>Sysmex</td>
			<td>04-004-2327</td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell removal Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td>Store at -20 &#176;C; kit 2</td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Fine Tip</td>
			<td>VWR</td>
			<td>82027-386</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Microcentrifuge tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySepTM Dead Cell Removal 
			(Annexin V) Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17899</td>
			<td>Store at 4 &#176;C; Kit 1</td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5425R</td>
			<td>Eppendorf</td>
			<td>5406000640</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5910R</td>
			<td>Eppendorf</td>
			<td>2231000657</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Easypet 3 Electronic Pipette controller</td>
			<td>Eppendorf</td>
			<td>EP4430000018</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Thermomixer F1.5 Model 5384</td>
			<td>Eppendorf</td>
			<td>EP5384000012</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>Store at 4 &#176;C, use in a Biological hood</td>
		</tr>
		<tr>
			<td>German Stainless Scissors</td>
			<td>Fine Science Tools</td>
			<td>14568-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Dmi1 Microscope</td>
			<td>Leica Microsystems</td>
			<td>454793</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>Rainin</td>
			<td>17014382</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadro MACS Magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-051</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 (1x)</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>TempAssure 0.2 mL PCR 8-Tube strips</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit, Human</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-929</td>
			<td>Store at -20 &#176;C, prepare enzymes according to kit instructions: 
			Reconstitute lyophilized Enzyme H vial in 3 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.</td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit, Mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-730</td>
			<td>Store at -20 &#176;C, prepare enzymes according to kit instructions: 
			Reconstitute lyophilized Enzyme D vial in 3 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td>Store at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

dissociation of human and mouse tumor tissue samples for single-cell rna sequencing

人类肿瘤样本包含有关其微环境和免疫库的大量信息。将人体组织样品有效解离成活细胞悬液是单细胞 RNA 测序 （scRNAseq） 流程所需的起始量。与大量 RNA 测序方法不同，scRNAseq 使我们能够在单细胞水平推断肿瘤标本中的转录异质性。近年来，采用这种方法带来了许多发现，例如识别免疫和肿瘤细胞状态以及与免疫疗法和其他类型治疗的临床反应相关的程序。此外，应用于解离组织的单细胞技术可用于使用 DNA 条形码抗体 （CITEseq） 识别可接近的染色质区域 T 和 B 细胞受体库以及蛋白质的表达。
使用这些技术时，解离样品的活力和质量是关键变量，因为这些会极大地影响单细胞与环境 RNA 的交叉污染、数据质量和解释。此外，长解离方案可导致敏感细胞群的消除和应激反应基因特征的上调。为了克服这些限制，我们设计了一种快速通用解离方案，该方案已在多种类型的人和小鼠肿瘤上得到验证。该过程从机械和酶解离开始，然后是过滤、红血裂解和活死富集，适用于细胞起始量低的样品（例如，针芯活检）。该方案可确保清洁且有活力的单细胞悬液，这对于成功生成凝胶微珠内乳液 （GEM）、条形码和测序至关重要。

获得悬浮在 RPMI 中的黑色素瘤活检并立即置于冰上。将样品转移至含有 420 μL RPMI 和消化酶的 1.5 mL 微量离心管中，用剪刀切成小块，随后在 37 °C 垂直放置的热混合器中进行酶消化 15 分钟（图 1）。消化后，将样品通过 50 μm 无菌一次性过滤器过滤，并用新鲜的 RPMI 洗涤过滤器以提高细胞产量（图 1）。洗涤后留下的组织剩余部分被丢弃，因为这些是不...

作者聚焦：探索成功针对肿瘤的免疫应答策略

该方案概述了快速解离人和小鼠肿瘤样品以进行单细胞 RNA 测序的程序。

解离人和小鼠肿瘤组织样品用于单细胞 RNA 测序

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into ...

dissociation-human-mouse-tumor-tissue-samples-for-single-cell-rna

Research

JoVE Journal

Cancer Research

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing

1.3K 次观看.  Massachusetts General Hospital. 我们的研究范围是研究标准护理免疫疗法的反应或耐药机制。我们的实验室正在努力了解是什么推动了针对肿瘤的成功免疫反应，以及我们是否能够确定这些治疗的预测因素以找到可以克服耐药性的靶点。近年来最大的进步是单细胞 RNA 测序技术的开发和整合。它们使我们能够实时、无偏见地系统地研究单个细胞中的分子特征，这有助于剖析这?...

视频: 作者聚焦：探索成功针对肿瘤的免疫应答策略

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

科研

癌症研究

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 ...

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

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

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 ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

用于低肿瘤含量组织中肿瘤富集的自动解剖方案

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA 测序的三种微分表达分析方法：利马、边缘、DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

组织中蛋白质和RNA表达的空间分析：一种微调虚拟显微切割的方法

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

分离细胞核用于实体瘤标本中的多组测序

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

作者聚焦：在具有挑战性的肿瘤微环境中增强用于多组测序的细胞核分离

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...