我们感谢德国癌症中心 （DKFZ） 的单细胞开放实验室 （scOpenLab） 的有益讨论。这项研究得到了德国癌症援助组织马克斯-埃德计划赠款编号70111964（S.T.）的支持。

新鲜冷冻胶质瘤样本来自德国海德堡国家肿瘤疾病中心（NCT）组织库。海德堡医学院机构审查委员会批准了患者材料的使用，并获得了研究中包括的所有患者的知情同意。
1. 实验准备
<ol><li>在冰上或在 4 °C 下执行所有步骤。</li>
	<li>预冷管、盘子、剃须刀刀片、杜恩克和害虫至 4 °C。</li>
	<li>提前准备所有缓冲。这些缓冲器在室温下是稳定的。建议进行无菌过滤，尤其是蔗糖。股票准备从科塞斯等人19日修改。请参阅表 1-7。</li>
	<li>从液氮或 -80 °C 冰柜存储中取出样品，并保持干燥的冰层，直到第 2.1 步。</li>
</ol>2. 组织解剖和分离
<ol><li>将新鲜冷冻组织样本（10-60毫克）转移到预冷的培养皿中。薄荷/切碎新鲜冷冻组织，用剃须刀刀片切成冰上的小块。</li>
	<li>在预冷的 1.5 mL 管中加入 500 μL 的冰镇核裂解缓冲器。将组织片放在含有核裂解缓冲器的1.5mL管中，并转移到杜塞尔（材料表）。 注:有两个 Dounce 同质化害虫:初始样品减少的"松散"或"A"害虫和完全样本均质化的"紧"或"B"害虫。</li>
	<li>将组织片与"松散"的害虫缠成约20次，直到摩擦减少。如果存在碎屑，则样品可以通过 100 mm 滤网过滤预先清除。</li>
	<li>Dounce 与 "紧" 害虫 20 笔触， 以实现完整的组织均质化。</li>
	<li>将同质性（约 500 μL）转移到预冷的 2 mL 管中。添加 1 mL 的冰镇裂解缓冲器。轻轻混合，在冰上孵育5分钟。轻轻地与宽孔移液器尖端混合，在孵化过程中重复 1-2 次。</li>
	<li>使用 30 μm 滤网过滤整个同质性，收集到 15 mL 猎鹰管中，然后传输回新的预冷 2 mL 管中。单个过滤器通常足以满足整个同质性。</li>
	<li>在光显微镜下检查，以验证大碎片的清除和核膜的完整性。核需要圆，核膜不应该扭曲。如果存在碎片，可以重新过滤原子核。</li>
	<li>在长凳顶部离心机上，将核离心在 500 x g 下 5 分钟，在 4 °C 下离心。取出超高纳坦，留下 +50 μL，颗粒中含有核。轻轻地将颗粒重新吸回另一个 1 mL 的核裂解缓冲器中，并在冰上孵育 5 分钟。</li>
	<li>在 4°C 下将核离心在 500 x g 下 5 分钟。 去除超高颗粒而不打扰颗粒，加入500 mL的1倍同质化缓冲（HB）（表4），孵育5分钟，无需再悬浮。然后，将核重新悬回另一个 1.0 mL 的 1x HB 中。</li>
	<li>在 4 °C 下将核离心在 500 x g 下 5 分钟。 取出超高纳坦，轻轻将 1x HB 的 200 μL 内的核重新插入新的 2.0 mL 管中。</li>
</ol>3. 梯度离心
<ol><li>加入200微升50%碘化醇溶液（表5），最终浓度为25%碘化醇。混合好10次，移液器设置在300ml。</li>
	<li>在 25% 混合物下加入 300 μL 的 29% 碘化醇溶液 （表 6）。使用 P1000 精细尖端以避免层的混合物。</li>
	<li>在 29% 混合物下加入 300 μl 的 35% 碘化醇溶液 （表 7）。使用 P1000 精细尖端以避免层的混合物。 注意:此步骤要求在管道过程中逐步去除移液器尖端，以避免过多的体积位移。</li>
	<li>将样品放在摆动的桶离心机中，在 4°C 下以 3，500 x g 的速度旋转 20 分钟，同时关闭制动器。</li>
	<li>轻轻取出样品，不摇晃，在光线下观察。在第二层和第三层之间应可见 95% 纯核的清晰白色波段（图 1）。</li>
</ol>4. 核分离
<ol><li>以 29%-35% 的相间水平吸气顶层，直到白核带。</li>
	<li>以 200 mL 的体积收集核带，转移到新鲜管中，并用 20μm 滤芯过滤器（材料表）进行过滤。 注:核不需要在过滤前重新悬浮。</li>
	<li>在光显微镜下检查，以验证大碎片的清除和核膜的完整性。核需要圆，核膜不应该扭曲。</li>
	<li>计数核使用特里潘蓝色染色在六细胞计和阿利库特核为 snrna - seq / snatac - seq 。</li>
</ol>5. 单核RNA和ATAC seq
<ol><li>立即使用单细胞基因表达和单细胞ATAC试剂盒（材料表）处理核。 注:10 倍基因组系统的核样本浓度为 snRNA-seq 的 1500-3000 个核，snNA-seq 的核样本浓度为 3500-7000 个核/微升。使用 1x PBS 可以稀释核。</li>
	<li>在基因组核心设施对生成的库进行排序。</li>
	<li>对数据进行质量控制分析。如果核含有独特的分子识别器 （UMI） >1000，则包含 500 >基因数量和线粒体基因的百分比<5%，并且均值在 UMI 和基因的三个标准偏差范围内，则包括核进行进一步分析。</li>
</ol>

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作者没有什么可透露的。

肿瘤内异质性领域正处于一个激动人心的阶段，正在开发新的分析和平台来挑战和扩展现有的知识。肿瘤内异质性是导致胶质瘤28中疾病进展和抵抗当前治疗方式的关键因素。最近关于脑肿瘤的研究集中在这一重要方面，使用单细胞转录和表观基因组测定，以更好地描述细胞异质性在同一肿瘤29，30，31，32。<s...

扩散性低级胶质瘤（LGG）是成人中最常见的原发性脑肿瘤，是脑半球中经常出现的渗透性肿瘤。LGG表现出肿瘤间和肿瘤内异质性，这不仅是由肿瘤人群驱动的，而且是由复杂参与肿瘤发育和进展的非恶性细胞驱动的，1、2、3、4、5。
在过去十年中，在胶质瘤领域收集的基因组数据大量增加。这些数据主要来自散装肿瘤测序研究，对分子定性以及目前脑肿瘤5、6、7、8、9、10、11的分类做出了巨大贡献。然而，尽管这些研究揭示了与胶质瘤相关的广泛的分子景观，但在治疗干预方面仍然缺乏进展，令人失望。脑肿瘤治疗耐药性的障碍之一是肿瘤内异质性。为了解决这个问题，各种研究一直集中在基因组学，转录学，蛋白体学和表观遗传异质性存在于肿瘤的单细胞水平12，13，14，15，16，17。
虽然在过去几年中，单细胞领域取得了显著的技术进步，但主要限制因素之一是提供分离细胞和进行这些实验所需的新鲜标本。为了克服这一限制，已经进行了几次成功的尝试，例如从冷冻组织中，使用核而不是细胞18，19进行snRNA-seq和snATAC-seq。这些方法大多依赖于荧光激活的细胞分拣 （FACS） 或过滤策略。单细胞和单核方法都有其优点和缺点。单细胞方法保持线粒体记录，虽然可能信息丰富，但也可能减少转录体覆盖，因为他们的高丰度。单核分离方法消除了线粒体分数的高百分比，从而使核记录20的更深入的覆盖。
近年来，有各种商业平台被用来检测单细胞基因组学数据，包括RNA-seq和ATAC-seq。其中最突出的平台是10倍基因组铬平台，用于单细胞基因表达和单细胞ATAC分析。由于该平台在微流体室的帮助下工作，任何碎屑或聚合物都可能堵塞系统，导致数据、试剂和有价值的临床样本丢失。因此，单细胞研究的成功在很大程度上取决于单细胞/细胞核的准确分离。
我们将在这里展示的协议是DroNc-seq和Omni-ATAC-seq协议的稍微修改的组合，并利用类似的方法进行最近的研究，利用snRNA-seq来了解人脑中的神经紊乱和神经元细胞类型18，19，21，22，23，24。该协议采用冷冻样品的酶/机械分离组合，然后进行过滤和梯度离心，并允许从新鲜冷冻胶质瘤组织快速准确地分离单核。我们已经成功地利用这个协议从脑肿瘤标本的相同核制剂中生成snRNA-seq和snATAC-seq数据。

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Homogenizer</td>
			<td>Active motif</td>
			<td>40401</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Thermo Scientific</td>
			<td>R1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol (aka Optiprep)</td>
			<td>Stem cell technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>MACs Smart Strainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-463</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(Ac)2</td>
			<td>Sigma</td>
			<td>63052-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abcam</td>
			<td>ab142227</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Kit: Nuclei EZ Prep</td>
			<td>Sigma</td>
			<td>NUC101-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Separation Filters (20 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell ATAC</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell Gene Expression</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (1000 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (200 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2069GPK</td>
			<td></td>
		</tr>
	</tbody>
</table>

nuclei isolation from fresh frozen brain tumors for single-nucleus rna-seq and atac-seq

成人扩散胶质瘤表现出肿瘤间和肿瘤内异质性。直到最近，大多数大规模的分子分析工作都集中在导致脑肿瘤分子分类的批量方法上。在过去五年中，单细胞测序方法突出了胶质瘤的几个重要特征。这些研究大多利用新鲜的脑肿瘤标本，利用流细胞学或抗体分离方法分离单细胞。今后，使用来自生物库的新鲜冷冻组织样本将为单细胞应用提供更大的灵活性。此外，随着单细胞场的进步，下一个挑战将是从单个细胞或相同的样本制备中生成多细胞数据，以更好地解开肿瘤的复杂性。因此，简单而灵活的协议，允许数据生成的各种方法，如单核RNA测序（snRNA-seq）和单核检测的转位酶-可访问色度素与高通量测序（snATAC-seq）将是重要的领域。
单细胞领域的最新进展，加上10倍基因组学平台等可访问的微流体仪器，促进了单细胞在研究实验室中的应用。为了研究脑肿瘤异质性，我们开发了一个增强的方案，用于将单核从新鲜冷冻胶质瘤中分离出来。此协议合并了现有的单细胞协议，并结合了同质化步骤，然后是过滤和缓冲介导梯度离心。生成的样本是纯单核悬浮，可用于从相同的核制剂生成单核基因表达和色度素可访问数据。

单核基因组学是一个不断发展的领域，数据和协议有限。影响单核检测结果的一个关键因素是纯核和完整核的分离。我们结合了两个已发布的协议（DroNc-seq 和 Omni-ATAC-seq 协议），在相对较短的时间内将高质量和纯核从新鲜冷冻胶质瘤组织块中分离出来，从而保持成绩单的稳定性（图 1）。
使用各种过滤步骤以及使用碘糖醇/蔗糖梯度的梯度离心，可以隔?...

Watch this Scientific Journal Video about Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq at JoVE.com

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

肿瘤内异质性是肿瘤的内在特征，包括胶质瘤。我们开发了一个简单而高效的协议，利用缓冲和梯度离心的组合，将单核从新鲜冷冻胶质瘤组织中分离出来，用于单核RNA和ATAC测序研究。

单核RNA-seq和ATAC-seq的新鲜冷冻脑肿瘤的核隔离

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus

Research

JoVE Journal

Cancer Research

11.9K 次观看.  University Hospital Heidelberg. 肿瘤内异质性是肿瘤的内在特征，包括胶质瘤。我们开发了一个简单而高效的协议，利用缓冲和梯度离心的组合，将单核从新鲜冷冻胶质瘤组织中分离出来，用于单核RNA和ATAC测序研究。

视频: Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

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 ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

从妇科和乳腺癌肿瘤获得癌症干细胞球

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

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 ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

快速筛选工作流程，利用患者衍生胶质瘤干细胞确定GBM的潜在组合疗法

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

用于癌症表观遗传学研究的ATAC-Seq优化

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

来自新鲜组织的胰腺癌来源的肿瘤类器官和成纤维细胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

作者聚焦:从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...