Name: enrichment and characterization of the tumor immune and non-immune microenvironments in established subcutaneous murine tumors
Uploaded: 2018-06-07T14:45:00.000+00:00
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Description: 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

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。

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

Universidad San Sebastian

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

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

科研

癌症研究

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

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

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

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

医学

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 ...

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

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

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

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

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

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

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

在携带人类肿瘤的人源化小鼠模型中测试癌症免疫疗法

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

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

摘要

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此视频中的章节

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

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

肿瘤移植用于评估小鼠肿瘤浸润CD8 ⁺ T细胞的动力学

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价

在携带人类肿瘤的人源化小鼠模型中测试癌症免疫疗法

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

从原发肿瘤的免疫细胞的分离

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

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

摘要

探索更多视频

此视频中的章节

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

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

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

在携带人类肿瘤的人源化小鼠模型中测试癌症免疫疗法

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

从原发肿瘤的免疫细胞的分离

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

肿瘤移植用于评估小鼠肿瘤浸润CD8 ⁺ T细胞的动力学

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价