JMN 承认国家卫生研究院 (T32GM088129) 和国立牙科 & 颅颌面部研究研究所 (F31DE026682) 的财政支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。这个项目也得到了在贝勒医学院的细胞和细胞分类核心的支持, 从 NIH (P30 AI036211, P30 CA125123, S10 RR024574) 的资助和乔尔 m. Sederstrom 的专家协助。

这里描述的所有方法都已被贝勒医学院的机构动物护理和使用委员会 (IACUC) 批准。
1. 肿瘤的收获和消化
注: 收割所需时间为 3-5 分钟/肿瘤, 加工所需时间为1小时 + 2-3 分/肿瘤。
<ol><li>弄死小鼠通过二氧化碳吸入和喷雾每只老鼠下来与70% 乙醇在收割前防止头发污染。手术切除小鼠皮下肿瘤, 确保肿瘤没有任何污染组织 (如毛发、皮肤、腹膜等). 将每个肿瘤放置在1.8 毫升的基 RPMI-1640 培养基中, 而不需要胎牛血清 (血清) 在24井板中。为了更大的收成, 在冰上保持盘子的低温以保持细胞的活力。 注: 如果需要不育, 所有的肿瘤解剖将需要在一个生物安全柜内使用无菌外科工具 (如剪刀, 钳等), 以及任何进一步的步骤。最长直径在 0.5-1 厘米之间的肿瘤是最佳的, 更大或更小的肿瘤将需要试剂的缩放。</li>
	<li>用剪刀把肿瘤切成少于1毫米的3块。</li>
	<li>通过溶酶 i 10 毫克/毫升, 胶原酶 IV 在 2500 u/毫升, 和 DNase I 在 200 U/毫升在基 RPMI-1640 培养基 (没有血清), 制备10x 消化鸡尾酒。 注: 消化鸡尾酒可以提前一天准备, 储存在-20 摄氏度;但是, 不推荐长时间贮存, 因为酶一旦溶解就会失去活性。</li>
	<li>添加200µL 的10x 离解鸡尾酒含有胶原酶 i, 胶原酶 IV, 和 DNase i 到每个井与1毫米3件。</li>
	<li>允许样品消化1小时在37°c, 而轻微晃动 60-100 rpm 使用轨道平板振动筛。 注: 在酶消化过程中, 温度升高到37摄氏度可能会导致免疫细胞活化。</li>
	<li>1小时后, 加入1毫升的 RPMI-1640 培养基, 补充5% 的血清和2毫米 EDTA, 以抵消反应。</li>
	<li>将肿瘤消化和剩余的肿瘤片注入40µm 细胞过滤器, 安装在50毫升离心管的顶端。 注: 切断1毫升吸管尖端的尖端, 以便更容易地将肿瘤消化转移到滤网。</li>
	<li>使用10毫升注射器柱塞通过过滤器机械地将肿瘤碎片分解。定期冲洗过滤器与2毫升补充 RPMI-1640 培养基 (含5% 的血清) 和重复, 直到细胞过滤器是明确的肿瘤。 注: 大约 4-10 毫升的总介质应该足以充分冲洗滤网的肿瘤。 注: 将样品放在冰上, 直到完成所有样品的步骤, 以保持细胞的生存能力。</li>
	<li>使用补充的 RPMI-1640 介质, 将每50毫升离心管调整到同一体积。 注: 容积调整对离心机平衡至关重要。跳过这一步可能会导致离心机损坏或人身伤害。</li>
	<li>离心机细胞悬浮液 (5 分钟, 805 x g, 4 °c), 丢弃上清液, 并在2毫升补充 RPMI-1640 介质中重新悬浮。 注: 细胞骨料可能形成后, 离心, 是难以并用重悬。使用5毫升血清吸管, 并迅速混合, 以打破它之前, 继续。</li>
</ol>2. 免疫和肿瘤细胞分数的分离
注: 此步骤所需的时间约为1小时。
<ol><li>将3毫升的密度梯度介质添加到15毫升离心管的底部。为每个肿瘤准备和贴上足够的管子。</li>
	<li>将2毫升的肿瘤细胞悬浮在密度梯度介质的顶部, 吹打缓慢地向下侧管以防止两层混合。</li>
	<li>小心地放置在离心机的分层管和旋转20分钟在 805 x g, 20 °c, 没有刹车。 注意: 验证适当的平衡并确保在离心过程中不使用刹车是非常重要的。此过程的任何中断都会导致图层混合, 并且完整的样本恢复将会很困难。</li>
	<li>将肿瘤浸润的白细胞 (直至) 层和顶层培养基, 用转移吸管小心转移到新的15毫升管上。丢弃所有剩余的密度梯度介质, 并在2毫升的补充 RPMI-1640 中并用重悬在管底部的肿瘤颗粒。 注: 离心后从上到下将有四个可见层, 分别对应于: 培养基、TILs、密度梯度介质和肿瘤细胞颗粒。有关各个层的示例, 请参见图 2A 。</li>
	<li>在分离的管子 (5 分钟, 805 x g, 4 °c) 中, 离心两个直到和肿瘤标本, 并丢弃上清。</li>
	<li>并用重悬在200µL 和肿瘤颗粒在2毫升补充 RPMI-1640 培养基中, 直到样品。保持冰块以保持生存能力。</li>
</ol>3. 电镀和染色
注: 电镀所需时间为三十年代/肿瘤;表面染色所需时间为1小时;细胞内染色所需时间为40分钟;流式细胞术分析所需的时间为 1-4 min/肿瘤。
<ol><li>为每个免疫染色板准备并标记一个96井的 U 型底板, 并为细胞计数增加一个盘子。 注: 通常, 总共准备了三个板块: 淋巴细胞聚焦板、髓系聚焦板和细胞计数板。</li>
	<li>整除将单细胞悬浮液放入每一个染色板, 并将一组容积放入计数板中。 注: 计数板用于计算添加到每个染色面板的单元格总数, 从而允许准确的细胞数量计数。96井板取样和容积分析能力的流式细胞仪可以提供每个样本中每个µL 的单元数, 因此计算添加到每个染色面板中的单元格数。计数板可以获得第二天后固定过夜4摄氏度, 冲洗与外地资产管制署缓冲 (磷酸盐缓冲盐水 (PBS) 与2% 的血清), 并 resuspending 在一套量的外地资产管制署缓冲区。</li>
	<li>用200µL 的 Dulbecco 磷酸盐缓冲盐水 (DPBS) 以离心 (5 分钟, 805 x g, 4 摄氏度) 清洗两次细胞, 并在每次冲洗之间丢弃上清, 以除去任何自由的血清。</li>
	<li>添加50µL 的 Fc 块, 混合样品使用多通道吸管, 并孵化20分钟4摄氏度。</li>
	<li>添加50µL 的2x 浓缩细胞外靶向抗体和固定生存能力染色混合物, 混合使用多通道吸管, 并孵化30分钟在黑暗中的 RT 或4°c。 注: 抗体的确切染色条件取决于制造商的指示。所有染色稀释必须在 DPBS, 没有添加的血清, 以防止饱和的可修复的活性染料。请参阅本手稿中使用的染色面板的最佳稀释的材料/设备表。抗体和可修复的生存能力染色解决方案是在2x 浓度, 以提供最终1x 染色浓度100µL 的总染色量时添加到 Fc 块。</li>
	<li>用离心 (5 分钟, 805 x g, 4 °c) 将样品清洗两次, 并在每次冲洗之间丢弃上清液。</li>
	<li>并用重悬在200µL 1x 固定/通透溶液和孵化过夜4摄氏度保护免受光照。 注意: 这个实验可以在一夜之间暂停, 在这一点上: 保持样品在4摄氏度的保护免受光照。</li>
	<li>第二天, 离心板 (5 分钟, 805 x g, 4 °c), 并丢弃上清。</li>
	<li>冲洗一次与200µL 1x 通透缓冲器, 离心机 (5 分钟, 805 x g, 4 °c), 并丢弃上清。</li>
	<li>添加100µL 1x 浓缩细胞内抗体染色板, 在1x 通透缓冲和孵化30分钟在黑暗中的 RT 或4°c (这取决于染色建议从制造商)。</li>
	<li>冲洗一次与1x 通透缓冲 (5 分钟, 805 x g, 4 °c), 放弃上清, 和漂洗第二次与资产管制署缓冲 (PBS 与2% 血清)。</li>
	<li>并用重悬300µL (PBS 2% 胎) 的样品, 将样本转移到流式细胞术管, 进行流式细胞仪分析。</li>
</ol>

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作者没有什么可透露的。

时间由多种复杂的细胞成分和分子组成。最近的证据表明, 准确的描述这个环境可以提供更好的理解治疗成功或失败, 甚至可以帮助确定机制的治疗耐药性。例如, 增加各种免疫抑制细胞 (如MDSCs、T 调节细胞等) 的瘤内水平, 可以减轻效应的免疫应答, 并消除免疫治疗效应。另一方面, 效应免疫种群 (如肿瘤特异性 CD8+ t 细胞、自然杀伤细胞、t 辅助细胞) 的瘤内存在增加, 可以成为?...

抑制时间最近被认为是癌症的一个标志, 众所周知, 在肿瘤的发展、进展和保护方面发挥重要作用, 并减轻他们对免疫治疗1的敏感性。时间由大量的细胞子集和表型组成, 所有这些都为肿瘤的免疫状态提供了重要的洞察力。这些免疫亚群可以进一步分层成淋巴细胞或髓系细胞的起源, 这共同构成了大多数先天和适应性免疫反应2,3。最近在癌症免疫治疗领域的进展表明, 免疫治疗策略 (即免疫检查点抑制剂, 嵌合体抗原受体 T 细胞等) 有可能导致持久性癌症回归然而, 它们在4、5的实体肿瘤癌症中仍然相对无效。许多团体已经显示了时间可以在治疗成功6,7的关键障碍, 因此, 仍然需要准确地评估新的免疫疗法在临床前的设置专门关注其能够调节或克服时间8。
目前的工作特点的时间通常利用显微镜或流式细胞术, 以及抗体标签策略, 以确定免疫细胞子集和其特点9,10。这两种策略提供独特的不同信息, 因为显微镜允许细胞子集的空间欣赏和流式细胞术提供高吞吐量和更广泛的细胞变化量化。尽管最近有改进的荧光复合免疫组化优化光谱成像系统, 现在可以支持多达7参数, 有限的面板大小使广泛的免疫分析困难, 因此这种技术往往为更集中的分析预留。因此, 流式细胞术仍然是最广泛使用的免疫分析技术之一。尽管其在时间表征中的广泛应用, 但用于加工和染色的方法相当多变。通常的协议使用肿瘤游离酶 (即胶原酶, DNase等) 和手工离解方法, 以实现单细胞悬浮, 其次是抗体染色和分析7。尽管每种方法都有好处, 但许多群体显示了通过这些技术11可以诱发的广泛的变异性。这使得跨研究比较的肿瘤微环境分析非常困难, 即使在评估相同的小鼠肿瘤模型。此外, 这些方法提供了有限的潜力, 以评估肿瘤细胞和肿瘤免疫渗透成分独立, 因为两个组成部分是散置后消化。样本数量, 然后限制多面板染色和分析, 这成为一个主要的问题时, 试图描述罕见的免疫子集 (即肿瘤特异性 T 细胞)12。最近的技术, 如大规模细胞术, 或飞行时间的细胞术 (CyTOF), 允许高维细胞子集的表型分析与一些系统支持面板设计超过42独立参数13。尽管 CyTOF 技术在免疫分析方面具有巨大的威力, 但由于费用、分析专业知识和对设备的访问, 它仍然受到限制。此外, 许多 CyTOF 协议建议纯化免疫子集, 以提高信噪比14, 因此我们建议, 我们的浓缩方法可以用于 CyTOF 分析的上游, 以提高数据质量。
在此, 我们描述了一种肿瘤微环境消化和分析方法, 纳入肿瘤免疫渗透分离。这种方法的目的是允许独立高通量的肿瘤免疫浸润和肿瘤细胞分数的分析, 以更广泛地描述肿瘤微环境。利用该方法, 我们进一步论证了进行全肿瘤分析的重要性, 认为皮下实体瘤模型具有显著的空间免疫异质性。总的来说, 这种方法可以更准确和一致地比较样本, 因为它丰富的免疫细胞亚群在肿瘤和允许独立的分析肿瘤细胞和免疫分数的肿瘤。

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			<td height="41" style="height:41px;width:216px;">6 to 12 week old male C57BL/6 mice</td>
			<td style="width:113px;">Jackson Laboratory&#160;</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Mice used in our tumor studies</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">MEER Murine Syngeneic Cancer Cell line</td>
			<td style="width:113px;">Acquired from collaborator&#160;</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">E6/E7 HPV antigen expressing murine tonsillar epithelial cancer cell line&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% isopropyl alcohol&#160;</td>
			<td style="width:113px;">EMD Milipore&#160;</td>
			<td style="width:119px;">PX1840</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Murine surgical tool kit</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:119px;">MOUSEKIT</td>
			<td style="width:167px;">Primarily only need scissors, tweezers, and a scalpel.&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">24-well plate&#160;</td>
			<td style="width:113px;">Denville Scientific Inc.</td>
			<td style="width:119px;">T1024</td>
			<td style="width:167px;">Or any 24-well flat bottom plate.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI-1640 media</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">R0883</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase I&#160;</td>
			<td style="width:113px;">EMD Milipore</td>
			<td style="width:119px;">234153</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Collagenase IV</td>
			<td style="width:113px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;">LS004189</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase I&#160;</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">11284932001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Low Speed Orbital Shaker</td>
			<td style="width:113px;">BioExpress (supplier: Genemate)&#160;</td>
			<td style="width:119px;">S-3200-LS</td>
			<td style="width:167px;">Or any orbital shaker.&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Thermoregulating incubator</td>
			<td style="width:113px;">Fisher Scientific</td>
			<td style="width:119px;">13-255-27</td>
			<td style="width:167px;">Or any other thermo-regulated incubator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8192</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:113px;">AMRESCO</td>
			<td style="width:119px;">E177</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">1 mL Pipette and tips</td>
			<td style="width:113px;">Eppendorf</td>
			<td style="width:119px;">13-690-032</td>
			<td style="width:167px;">Or any other pipette and tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40 um Cell Strainers</td>
			<td style="width:113px;">Fisher Scientific</td>
			<td style="width:119px;">22363547</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">50 mL Centrifuge Tubes</td>
			<td style="width:113px;">Denville Scientific Inc.</td>
			<td style="width:119px;">C1062-P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">15 mL Conical Tubes</td>
			<td style="width:113px;">Denville Scientific Inc.</td>
			<td style="width:119px;">C1012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">10 mL Luer-Lok Syringe without needles</td>
			<td style="width:113px;">BD</td>
			<td style="width:119px;">309604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Lymphoprep Density Gradient Medium</td>
			<td style="width:113px;">STEMCELL Technologies</td>
			<td style="width:119px;">7811</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Transfer Pipettes</td>
			<td style="width:113px;">Denville Scientific Inc.</td>
			<td style="width:119px;">P7212</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">96-well U-bottom plates</td>
			<td style="width:113px;">Denville Scientific Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPBS</td>
			<td style="width:113px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D8573</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">FoxP3 Transcription Factor Staining Buffer Set&#160;</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">00-5523-00</td>
			<td style="width:167px;">Fix/Permeabilization Buffer and Permeabilization Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermoregulated Centrifuge</td>
			<td style="width:113px;">Eppendorf&#160;</td>
			<td style="width:119px;">5810 R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Attune NxT Flow Cytometer</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">For 96-well cell volumetric counting&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:216px;">Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)&#160;Clone 2.4G2&#160;</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">553141</td>
			<td style="width:167px;">Fc Block</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">L34962</td>
			<td style="width:167px;">Fixable viability stain</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">CD45 Monoclonal Antibody (30-F11), APC-eFluor 780</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">47-0451-80</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">TCR beta Monoclonal Antibody (H57-597), APC</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">17-5961-82</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">CD8-&#945; Antibody (KT15), PE</td>
			<td style="width:113px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-53473&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">CD4 Monoclonal Antibody (GK1.5), PE-Cyanine5</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">15-0041-81</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), Alexa Fluor 700</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">56-5321-82</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">CD11c Monoclonal Antibody (N418), PE-Cyanine5</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">15-0114-82</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">F4/80 Monoclonal Antibody (BM8), eFluor 450</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">48-4801-80</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">CD11b Monoclonal Antibody (M1/70), APC</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">17-0112-81</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Ly-6G (Gr-1) Monoclonal Antibody (RB6-8C5), PE</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">12-5931-82</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25-5982-80</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">CD273 (B7-DC) Monoclonal Antibody (122), PerCP-eFluor 710</td>
			<td style="width:113px;">ThermoFisher Scientific</td>
			<td style="width:119px;">46-9972-82</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Ki-67 Monoclonal Antibody (B56), BV711</td>
			<td style="width:113px;">BD Biosciences</td>
			<td style="width:119px;">563755</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

enrichment and characterization of the tumor immune and non-immune microenvironments in established subcutaneous murine tumors

肿瘤免疫微环境 (时间) 最近被认为是一个关键的调解者的治疗反应, 在实体肿瘤, 特别是免疫疗法。最近在免疫治疗方面的最新进展突出了需要重现的方法, 准确和彻底的特点肿瘤及其相关的免疫渗透。肿瘤酶消化和流细胞分析可以广泛地描述多种免疫细胞亚群和表型;然而, 分析的深度往往受到限制的荧光限制的面板设计和需要获得大量的肿瘤样本, 以观察罕见的免疫人群的兴趣。因此, 我们已经开发出一种有效的高通量的方法来分离和丰富肿瘤免疫浸润的非免疫肿瘤成分。所描述的肿瘤消化和离心密度分离技术允许分离的肿瘤和肿瘤免疫浸润分数的特点, 并保持细胞活力, 从而提供了一个广泛的描述肿瘤免疫状态。该方法用于表征实体肿瘤中广泛的空间免疫异质性, 进一步证明了对肿瘤免疫分析技术的一致性要求。总体上, 该方法为皮下固体小鼠肿瘤的免疫学特性提供了一种有效、适应性强的技术;因此, 这个工具可以用来更好地表征肿瘤免疫功能和在临床前评价新的免疫治疗策略。

我们的研究结果表明, 从非免疫性肿瘤成分分离的显著优势, 如议定书所解释的。此外, 使用所描述的方法, 我们证明了明显的免疫异质性的建立实体肿瘤。
许多肿瘤离解技术的一个重要问题是样本生存能力的丧失, 最常见的原因是严酷的消化条件 (即高温、营养供给不足等)。使用描述的消化方法, 有或没有密度梯?...

Watch this Scientific Journal Video about Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors at JoVE.com

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

在这里, 我们描述了一个方法, 以分离和丰富的组成部分, 肿瘤免疫和非免疫微环境的建立皮下肿瘤。该技术允许对肿瘤免疫浸润和非免疫肿瘤分数进行单独分析, 从而可以对肿瘤免疫微环境进行综合表征。

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

enrichment-characterization-tumor-immune-non-immune-microenvironments

Research

JoVE Journal

Cancer Research

9.5K 次观看.  Baylor College of Medicine. 在这里, 我们描述了一个方法, 以分离和丰富的组成部分, 肿瘤免疫和非免疫微环境的建立皮下肿瘤。该技术允许对肿瘤免疫浸润和非免疫肿瘤分数进行单独分析, 从而可以对肿瘤免疫微环境进行综合表征。

视频: Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

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

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 tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

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

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

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 ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

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 ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

一种确定环境富集对小鼠结肠肿瘤模型结肠菌群生物多样性影响的方法

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

缺血再灌注损伤中转移性肝肿瘤的模样

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

小肠神经内分泌肿瘤类球体的建立与特征

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

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 ...