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

<ol>
	<li>Liu, C., Yang, M., Zhang, D., Chen, M., Zhu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+cancer+immunotherapy:+Current+progress+and+prospects.">Clinical cancer immunotherapy: Current progress and prospects.</a> Front Immunol. 13, 961805 (2022).</li><li>Baghban, R., 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=Tumor+microenvironment+complexity+and+therapeutic+implications+at+a+glance.">Tumor microenvironment complexity and therapeutic implications at a glance.</a> Cell Commun Signal. 18 (1), 59 (2020).</li><li>Sun, 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=Single-cell+RNA+sequencing+in+cancer:+Applications,+advances,+and+emerging+challenges.">Single-cell RNA sequencing in cancer: Applications, advances, and emerging challenges.</a> Mol Ther Oncolytics. 21, 183-206 (2021).</li><li>Huang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+single-cell+RNA+sequencing+and+its+applications+in+cancer+research.">Advances in single-cell RNA sequencing and its applications in cancer research.</a> J Hematol Oncol. 16 (1), 98 (2023).</li><li>Ottaiano, 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=From+chaos+to+opportunity:+decoding+cancer+heterogeneity+for+enhanced+treatment+strategies.">From chaos to opportunity: decoding cancer heterogeneity for enhanced treatment strategies.</a> Biology. 12 (9), 1183 (2023).</li><li>Hwang, B., Lee, J. H., Bang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+technologies+and+bioinformatics+pipelines.">Single-cell RNA sequencing technologies and bioinformatics pipelines.</a> Exp Mol Med. 50 (8), 1-14 (2018).</li><li>Erfanian, N., 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=Deep+learning+applications+in+single-cell+genomics+and+transcriptomics+data+analysis.">Deep learning applications in single-cell genomics and transcriptomics data analysis.</a> Biomed Pharmacother. 165, 115077 (2023).</li><li>Jovic, D., 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=Single-cell+RNA+sequencing+technologies+and+applications:+A+brief+overview.">Single-cell RNA sequencing technologies and applications: A brief overview.</a> Clin Transl Med. 12 (3), e694 (2022).</li><li>Burja, B., 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=An+optimized+tissue+dissociation+protocol+for+single-cell+RNA+sequencing+analysis+of+fresh+and+cultured+human+skin+biopsies.">An optimized tissue dissociation protocol for single-cell RNA sequencing analysis of fresh and cultured human skin biopsies.</a> Front Cell Dev Biol. 10, 872688 (2022).</li><li>Denisenko, 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=Systematic+assessment+of+tissue+dissociation+and+storage+biases+in+single-cell+and+single-nucleus+RNA-seq+workflows.">Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows.</a> Genome Biol. 21 (1), 130 (2020).</li><li>Sade-Feldman, 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=Defining+T+cell+states+associated+with+response+to+checkpoint+immunotherapy+in+melanoma.">Defining T cell states associated with response to checkpoint immunotherapy in melanoma.</a> Cell. 176 (1-2), 404 (2019).</li><li>Pelka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+organized+multicellular+immune+hubs+in+human+colorectal+cancer.">Spatially organized multicellular immune hubs in human colorectal cancer.</a> Cell. 184 (18), 4734-4752.e20 (2021).</li><li>Bill, R., 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=CXCL9:SPP1+macrophage+polarity+identifies+a+network+of+cellular+programs+that+control+human+cancers.">CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.</a> Science. 381 (6657), 515-524 (2023).</li><li>Al'Khafaji, A. 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=High-throughput+RNA+isoform+sequencing+using+programmed+cDNA+concatenation.">High-throughput RNA isoform sequencing using programmed cDNA concatenation.</a> Nat Biotechnol. 42 (4), 582-586 (2024).</li><li>Xing, Y., 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+effect+of+cell+isolation+methods+on+the+human+transcriptome+profiling+and+microbial+transcripts+of+peripheral+blood.">The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood.</a> Mol Biol Rep. 48 (4), 3059-3068 (2021).</li><li>Ma, F., Hernandez, G. E., Romay, M., Iruela-Arispe, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+to+study+vascular+diversity+and+function.">Single-cell RNA sequencing to study vascular diversity and function.</a> Curr Opin Hematol. 28 (3), 221-229 (2021).</li><li>Fan, X. -. 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=Impact+of+cold+ischemic+time+and+freeze-thaw+cycles+on+RNA,+DNA+and+protein+quality+in+colorectal+cancer+tissues+biobanking.">Impact of cold ischemic time and freeze-thaw cycles on RNA, DNA and protein quality in colorectal cancer tissues biobanking.</a> J Cancer. 10 (20), 4978-4988 (2019).</li><li>LoCoco, P. 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=Reliable+approaches+to+extract+high-integrity+RNA+from+skin+and+other+pertinent+tissues+used+in+pain+research.">Reliable approaches to extract high-integrity RNA from skin and other pertinent tissues used in pain research.</a> Pain Rep. 5 (2), e818 (2020).</li><li>Montanari, 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=Automated-mechanical+procedure+compared+to+gentle+enzymatic+tissue+dissociation+in+cell+function+studies.">Automated-mechanical procedure compared to gentle enzymatic tissue dissociation in cell function studies.</a> Biomolecules. 12 (5), 701 (2022).</li><li>Yosefov-Levi, O., Tornovsky, S., Parnas, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+tissue+dissection+to+isolate+viable+single+cells.">Pancreatic tissue dissection to isolate viable single cells.</a> J Vis Exp. (195), (2023).</li><li>Janssen, P., 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+effect+of+background+noise+and+its+removal+on+the+analysis+of+single-cell+expression+data.">The effect of background noise and its removal on the analysis of single-cell expression data.</a> Genome Biol. 24 (1), 140 (2023).</li><li>Adam, M., Potter, A. S., Potter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychrophilic+proteases+dramatically+reduce+single+cell+RNA-seq+artifacts:+A+molecular+atlas+of+kidney+development.">Psychrophilic proteases dramatically reduce single cell RNA-seq artifacts: A molecular atlas of kidney development.</a> Development. 144 (19), 3625-3632 (2017).</li><li>Sutermaster, B. A., Darling, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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">
	<tbody>
		<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。

Although many advancements in cancer research have led to the development and FDA approval of agents targeting inhibitory receptors expressed on immune and tumor cells, known as checkpoint blockade inhibitors, therapy resistance and identifying mechanisms that drive patient response remain challenging1. The complex challenge of characterizing tumor heterogeneity on its molecular diversity and the intricate interplay between tumor cells and the immune microenvironment necessitates new approaches to dissect this complex ecosystem at the single-cell resolution. Understanding the molecular intricacies within tumors and their microenvironments is pivotal for advancing therapeutic strategies and deciphering the complex biology underlying cancer progression2. Single-cell RNA sequencing (scRNA-seq) has become increasingly popular because it provides high-resolution analysis of individual cells within complex tumor samples3,4.
Tumor heterogeneity, encompassing genetic, epigenetic, and phenotypic diversities, poses a challenge in uncovering the intricacies of cancer biology5. The conventional methods of bulk RNA sequencing tend to obscure the unique expression profiles and phenotypes of heterogeneous cell populations present within tumors, as these methods average signals across entire cell populations4. In contrast, scRNA-seq allows the dissection of individual cell types, revealing diverse gene expression, repression patterns, and cellular states otherwise overlooked4,6. The premise of scRNA-seq lies in its capacity to decode individual cells&#39; genetic and molecular signatures, holding immense promise in discovering new cancer therapeutics7.
By deciphering the gene expression profiles of tumor cells and the surrounding stromal and immune cells, researchers can identify novel therapeutic targets and develop precision genetic medicine strategies tailored to individual patients6. Furthermore, dissecting the immune repertoire within the tumor microenvironment provides crucial insights into the interplay between tumor cells and immune effectors, paving the way for immunotherapeutic interventions3. Despite advances in using scRNAseq on clinical samples, several challenges during the processing steps can harm the cell&#39;s RNA integrity, viability, and quality of the data generated. Moreover, processing tissue with low cell viability can increase ambient RNA levels in the droplets generated during the encapsulation of individual cells, resulting in cross-contamination and incorrect annotation of cell types, while long dissociation protocols performed at 37 &#176;C can lead to the upregulation of a stress response gene signature in multiple cell types8,9,10.
The stepwise procedure (Figure 1A) outlined in this protocol commences with rapid mechanical and enzymatic dissociation of tumors, followed by filtration and the removal of dead cells, depending on the viability of each tumor sample. At present, this procedure has been verified in melanoma11, colorectal12, head and neck squamous cell carcinoma13, pancreatic and lung (unpublished data) tumor samples. These techniques ensure the generation of high-quality viable cell suspensions crucial for downstream analyses14. Notably, removing red blood cells, devoid of synthesized RNA, becomes imperative to purify the sample15. Subsequent evaluation of cell counts and the elimination of dead cells serve as a prerequisite for successful 10x Genomics Gel bead-in Emulsion (GEM) generation, barcoding, and increase the recovery of transcripts and sequenced cells without jeopardizing the exclusion of sensitive populations (e.g., neutrophils and epithelial cells)16. These steps are fundamental in unlocking the potential of single-cell resolution analysis within tumor samples9,10, uncovering novel gene expression profiles and immune signatures necessary for driving innovative therapeutic interventions, and identifying new tumor vulnerabilities.

作者聚焦：探索成功针对肿瘤的免疫应答策略

解离人和小鼠肿瘤组织样品用于单细胞 RNA 测序

The scope of our research is to study the mechanisms of response or resistance in standard of care immunotherapies. Our lab is trying to understand what drives a successful immune response against tumors, and if we can identify these predictors of therapy to find targets that can overcome resistance. The biggest advancement in recent years has been the development and integration of single-cell RNA sequencing techniques.They enable us in real time to systematically study molecular features in individual cells in an unbiased manner, which is helpful in the dissection of these complex and evolving tumor ecosystems. While many preclinical models are being used to study the response mechanisms to immunotherapy, we still don't know the immunological determinants that are important in human immunity. Processing and analyzing patient samples in real time is crucial to our ability to investigate all cellular compartments within a given tumor.Using unbiased genomic and transcriptomic techniques, our lab discovered that defects in the antigen presentation machinery are associated with immunotherapy resistance in melanoma. More recently, using single-cell approaches, we discovered unique T cell states and myeloid cell polarities associated with patient outcomes in melanoma and head neck cancer, respectively. The biggest advantage of our protocol lies in its simplicity.It is a universal protocol adjusted for the dissociation of small and large tumor specimens from multiple types of cancers in a short period of time, ending in the generation of highly-viable, single-cell suspensions.

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...

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。

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

1.1K 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?...