这项工作得到部分支持, 由科氏研究所支助 (核心) 赠款 P30-CA 14051 从国家癌症研究所, 由国家卫生补助研究所 (CA206157-22 和 GM118066) 和生硬的大理石癌症研究基金安洁莉卡阿蒙。安洁莉卡还是霍华德休斯医学研究所和格伦生物医学研究基金会的研究员。在美国意大利癌症基金会 (AICF) 的支持下, 由玛丽居里行动和意大利癌症研究协会 (AIRC) 的癌症研究奖学金, 以及五年期癌症研究奖学金。他得到了霍华德休斯医学研究所的一份奖学金的支持。

文化条件
RPE-1 hTERT 细胞系培养 Dulbecco 的改良鹰培养基 (材料表) 补充10% 胎牛血清, 2 毫米 l-谷氨酰胺, 100 U/毫升青霉素/链霉素。细胞在37°c 孵化, 5% CO2在一个湿润的环境中。下面描述的协议是使用10厘米的菜肴开发的, 所有的技术细节都是指这些菜肴。如果使用不同的菜肴, 相应地向上或向下缩放。
1. RPE-1 细胞同步
<ol><li>使用新鲜解冻的细胞, 没有经历超过15通道的所有实验。在1天, 解冻和免除 ~ 2.5-5.0 x 105 RPE-1 hTERT 细胞成10厘米的组织培养皿。添加8毫升标准生长培养基和孵化过夜。使用标准单元格计数器 (如 hemocytometer) 确定单元格编号。 注: 细胞密度是指 RPE-1 细胞的生长条件。生长条件可能在不同的细胞系之间变化;考虑相应地调整它们。</li>
	<li>在2天, 通过吸定期生长培养基和增加8毫升培养基, 其中含有5毫米胸苷, 同步 G1/S 边界的细胞。 注: 胸苷浓度和治疗长度可能不同的细胞线。最好进行试验, 以确定最佳条件的细胞线 (s) 进行测试。</li>
	<li>在3天, 24 小时后添加胸苷培养基 (步骤 1.2), 释放从胸苷块的吸气培养基和洗涤细胞三倍的 1x PBS。添加8毫升的定期生长培养基, 并返回板到孵化器。</li>
</ol>2. 通过干扰有丝分裂激酶 Mps1 的活性产生异倍体细胞
<ol><li>在3天, 6 小时后, 从胸苷块释放 (步骤 1.3; 这是约 3-6 小时前细胞进入有丝分裂), 吸入培养基, 一次性冲洗 1x PBS, 并替换为中含有 500 nM reversine (Mps1 抑制剂)。 注: RPE-1 细胞进入有丝分裂 9-12 小时后, 胸苷洗出10,11。然而, 细胞周期的动力学变化在不同的细胞系之间。因此, 通过建立的方法进行细胞周期分析 (例如), 通过测量 DNA 含量通过流动力学分析) 来确定这些动力学在先导实验中是至关重要的。此外, Mps1 抑制剂的最佳工作浓度可能会因细胞系而异。以前使用 RPE1 细胞的实验成功地使用了 reversine 的工作浓度为 500 nM10,11,12。最好进行试验, 以确定最佳浓度的细胞线进行测试。</li>
	<li>在4天, 12 小时后 reversine 治疗 (约6小时后细胞有丝分裂), 吸入 reversine 培养基和洗涤细胞三倍的 1x PBS。将8毫升的定期生长培养基添加到板上, 并将细胞恢复到孵化器中。</li>
</ol>3. 异倍体循环细胞的去除和 ArCK 种群的富集
<ol><li>在6天, 细胞进入第一次有丝分裂后约72小时 (步骤 2.2; 这是大约 66 h 在 reversine 洗涤以后, 对应于 2-3 细胞周期), 抽吸媒介, 洗涤一次与 1x PBS 并且替换与包含300毫微米 nocodazole 的媒介。 注: 最近的工作表明, 窝藏异倍体核型的 RPE-1 细胞 genomically 不稳定, 并在第一次错误有丝分裂10 后, 在 2-3 细胞周期内拘捕其增殖。然而, 这种动力学可能会在不同的细胞线之间变化。因此, 进行实验以确定异倍体循环细胞清除的适当时机是至关重要的。</li>
	<li>在7天, 12 小时后 nocodazole 治疗, 吸入介质, 并添加3毫升 1x PBS 的板。通过敲击盘子的侧面, 摆脱有丝分裂细胞。在每个水龙头之间旋转盘子, 以允许甚至切除有丝分裂细胞。</li>
	<li>为确保完全去除有丝分裂细胞, 重复进行多轮的抖动过程 (建议 3-5 次), 在每轮之间吸出和增加3毫升 1x PBS。</li>
	<li>摇动后, 轻轻地吸入附着在盖子或盘子边缘的液体。简要地, 用10X 放大倍数检查显微镜下的板。确保少数有丝分裂细胞被观察到。如果在10X 目标下发现五多个有丝分裂细胞, 重复另一轮的震荡。 注意: 有丝分裂细胞会出现圆形, 并可能在摇晃后部分分离。在将细胞暴露到下一轮 nocodazole 治疗之前, 确保完全清除有丝分裂细胞是至关重要的。如果不这样做, 可能导致有丝分裂细胞死亡和/或导致二、四倍体杂交细胞的产生。</li>
	<li>最后一次摇动后, 用5毫升的 1x PBS 洗涤细胞。将含有 330 nM nocodazole 的8毫升培养基加入板中, 孵育12小时。</li>
	<li>重复 nocodazole 治疗/抖动 (步骤 3.1-3.5) 每12小时, 直到没有发现有丝分裂细胞后, 孵化。通常, 建议4-5 轮治疗/震动。 注意: 这里提供的信息是指 RPE-1 细胞。不同细胞系间的治疗/震荡次数可能有所不同。为了避免长时间和不必要的暴露于 nocodazole, 在试点试验中, 必须仔细确定有效和彻底清除异倍体循环细胞所需的子弹数量。</li>
	<li>经过 4-5 轮的 nocodazole 治疗/抖动 (步骤 3.1-3.6), 增加10毫升的定期生长培养基和孵化。允许细胞在未来的操作或化验使用前休息 12-24 小时。 注: 在最终的震荡后, 板上剩余的细胞是被逮捕的细胞周期, 如最近的10所示, 港湾复杂的核型。这些细胞被称为 ArCK (用复杂的核型) 细胞被逮捕。在隔离的一周内对 ArCK 的人群进行化验是可取的。</li>
	<li>或者, 要评估被拘捕的细胞与复杂的核型的百分比, 收集和计数的细胞从每次抖动使用标准的细胞计数器。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57215/57215fig1.jpg"> 图 1: 用于生成和隔离具有复杂核型 (ArCK) 和代表性图像的异倍体单元格的方法的总体示意图。(A) ArCK 单元生成和隔离协议示意图。(B)在治疗的每个阶段 RPE-1 hTERT 细胞的代表性图像。最后一个面板 (用红色表示) 代表了独立的 ArCK 单元格。缩放栏100µm.<a href="//cloudflare.jove.com/files/ftp_upload/57215/57215fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
4. ArCK 人口的特征
<ol><li>通过执行以下化验, 确认成功地隔离 ArCK 人口。<ol>
<li>驴β-乳糖酶染色 (衰老的标志) 使用一个商业上可用的套件 (见材料表)。</li>
		<li>通过使用商业上可用的工具包 (见材料表) 测量促炎性细胞因子 (如 CCL2) 的分泌。</li>
		<li>确认 p53, p21, p16 的细胞周期抑制剂的增加水平。 注意: 为了正确控制这些化验, ArCK 的人群应与 euploid 和异倍体循环细胞进行比较。前者为低通道, 野生型 RPE-1 hTERT 细胞呈指数增长。后者可通过诱导 RPE-1 hTERT 中的染色体错误分离和在第一次异常有丝分裂后收获细胞72小时获得。为此, 请按照上文所述的步骤1.1 中的协议进行, 并在第一个 nocodazole 处理之前收获细胞 (步骤 3.1)。</li>
	</ol>
</li>
	<li>执行单细胞测序, 以评估被拘捕细胞的核型。 注意: 单细胞测序是确定单个细胞级别13上的染色体和亚染色体变化的一种成熟的方法。有关如何执行单单元排序的详细过程, 请在10的其他位置找到。</li>
</ol>

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作者没有什么可透露的。

这种新的方法生成和丰富的被逮捕的细胞与复杂的核型 (ArCK) 允许研究的细胞, 有多个染色体的增益或损失, 并停止分裂。该方法的设置是为了方便快速、可靠地隔离 ArCK 细胞。
这项试验中最关键的一步是严格控制药物治疗的时间线, 最重要的是清除异倍体循环细胞。为了获得最佳结果, nocodazole 治疗和抖动的时机特别重要, 以确保循环细胞从板块中移除, 而它们仍然是圆形和有丝分裂, 防止有丝分裂细胞死亡或滑入 G1 的可能性,可能造成二、四倍体杂交人口。不建议在12小时 nocodazole 孵化时, 抖动的时间偏离两个以上的时间。
未来对 ArCK 细胞的研究有可能有助于更深入地了解复杂的核型对细胞生理学的影响。特别是, 最近的一项研究表明, 免疫系统的细胞能够与 ArCK 细胞的免疫清除功能进行交互并触发10。这里描述的方法为 ArCK 细胞的进一步表征提供了一个良好的出发点, 包括澄清未变形细胞免疫清除的分子机制和致癌转化的研究可以绕过此监视机制, 这是字段14、15中的关键问题。

染色体分离过程中的错误导致体检, 这种情况的特征是染色体数不是单倍体补充的倍数1,2,3,4。异倍体核型触发复制重音产生进一步的基因组不稳定5, 6, 7, 8, 9, 10, 增加核型复杂性, 并最终导致细胞周期逮捕的细胞亚群10。这里提出的方法的目的是生成和分离这样的亚群从循环细胞。通过使用廉价的组织培养技术, 该协议促进了细胞周期被捕获的复杂核型异倍体细胞的分离和鉴定。这些细胞被称为 ArCK (以复杂的核型) 细胞及其 euploid 循环对应物作为对照。
该议定书是第一个为此目的建立, 并允许隔离和进一步研究 ArCK 线包括, 但不限于, 其诱发衰老和衰老相关分泌表型 (SASP), 他们的亲炎特征, 以及它们与免疫细胞接触的能力。这里提出的方法已经在未变形, 永生化的人类细胞, 但尚未在癌症线测试。由于抑制一个或多个通路, 某些转化细胞可能对细胞周期停止敏感;因此, 应在其他单元格行中执行进一步的验证。

<table border="0" cellpadding="0" cellspacing="0" width="953">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMEM (Dulbecco&#8217;s Modified Eagle Medium)&#160;</td>
			<td style="width:311px;">Thermofisher</td>
			<td style="width:319px;">11995-073</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymidine</td>
			<td>Sigma Aldrich</td>
			<td>T1859</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reversine</td>
			<td>Cayman Chemical Company</td>
			<td>10004412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nocodazole</td>
			<td>Sigma Aldrich</td>
			<td>M1410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Anti-p53 antibody</td>
			<td style="width:311px;">Santa Cruz</td>
			<td style="width:319px;">sc-126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Anti-p21 antibody</td>
			<td style="width:311px;">Santa Cruz</td>
			<td style="width:319px;">sc-6246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Anti-p16 antibody</td>
			<td style="width:311px;">BD</td>
			<td style="width:319px;">554079</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Senescence b-Galactosidase Staining Kit</td>
			<td style="width:311px;">Cell Signaling Technology&#160;</td>
			<td style="width:319px;">9860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteome Profiler Human Cytokine Array Kit&#160;</td>
			<td style="width:311px;">R&#38;D Systems</td>
			<td style="width:319px;">ARY005B</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and isolation of cell cycle-arrested cells with complex karyotypes

染色体错误隔离导致体检, 这种情况下细胞的染色体数目不平衡。几行证据有力地表明, 体检引发基因组不稳定, 最终产生具有复杂核型的细胞, 从而阻止其增殖。对具有复杂核型的细胞进行分离和表征, 对于研究染色体数目不平衡对细胞生理学的影响至关重要。迄今为止, 还没有建立可靠隔离这种异倍体细胞的方法。本文提供了一种利用标准的、廉价的组织培养技术对复杂核型异倍体细胞进行富集和分析的协议。该协议可用于分析具有复杂核型的异倍体细胞的几种特征, 包括其诱导的衰老相关的分泌形态、炎症性质和与免疫细胞相互作用的能力。因为癌细胞常常会造成染色体数量的失衡, 所以破译体检如何影响正常细胞的细胞生理学是至关重要的, 最终目的是揭露其亲和抗癌变的作用。

这种方法利用一个体外组织培养系统来隔离异倍体细胞, 其复杂的核型可以阻止其增殖。这个种群被称为 ArCK (以复杂的核型) 细胞被拘捕。图 1A显示了实验方案。野生型循环 RPE-1 hTERT 细胞在 G1/S 边界与胸苷同步, 并与 Mps1 抑制剂进行治疗, 以诱导染色体错误分离。在诱导染色体错误隔离后, 将主轴毒物 nocodazole 添加到细胞中72小时。12小时 nocodazole 治疗后, 仍能增殖的异倍体细胞进入有丝分裂, 并由于干扰微管聚合而被困在这个细胞周期阶段。这些被困的细胞, 然后很容易删除的温和的震动。这一震动过程重复了 3-5 次, 以确保完全清除循环细胞。图 1B显示了在治疗的每个阶段 RPE-1 细胞的代表性图像。
与 euploid 和异倍体循环单元相比, ArCK 细胞显示的细胞周期抑制剂 (如 p53、p21 和 p16) 的水平增加,如图2A 所示。此外,图 2B显示 ArCK 细胞中的正β-乳糖酶染色, 与 euploid 控制细胞相比, 它是细胞衰老的一个成熟标记。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57215/57215fig2.jpg"> 图 2: 使用 ArCK 单元格的代表性结果.(A)在 euploid、异倍体循环和 ArCK 细胞中评估细胞周期抑制剂水平的西方印迹。p53、p21 和 p16 的含量由西方印迹分析确定。肌动蛋白作为装载控制。(B) β-乳糖酶染色法评估 ArCK 细胞的衰老程度。在 euploid 细胞和 ArCK 细胞中测定了衰老相关的β-乳糖酶 (β-加仑) 活性。缩放栏100µm.<a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
这种细胞周期骤停是异倍体细胞具有复杂核型的显著特征, 如从图3中的单细胞测序中的代表性染色体分析显示, 其中 ArCK 细胞显示多个随机染色体增益和损失。此发现与以前的报表10完全一致。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57215/57215fig3.jpg"> 图 3: ArCK 单元格的代表性单细胞测序.显示六个代表性 ArCK 细胞的核型的分割图。分割图在日志2尺度上显示所有染色体从1到 X 的复制号相对于 euploid 引用 (野生类型 RPE-1 是雌性原点的双倍细胞线)。染色体的增加是红色的, 染色体的损失在绿色突出显示。<a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

体检导致基因组不稳定, 最终产生细胞周期-被捕获的细胞与复杂的核型。本文提供了一种简单方便的分离异倍体细胞的方法。

复杂核型细胞周期捕获细胞的生成与分离

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

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Research

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Cancer Research

10.3K 次观看.  Massachusetts Institute of Technology. 体检导致基因组不稳定, 最终产生细胞周期-被捕获的细胞与复杂的核型。本文提供了一种简单方便的分离异倍体细胞的方法。

视频: Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

科研

癌症研究

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

细胞毒性细胞因子诱导的杀伤性T细胞的分离和扩张，用于癌症治疗

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

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 ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

癌症患者血浆样本中无细胞DNA检测

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

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

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

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

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

鼠心肺复合物的脱细胞化

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...