Name: in vivo immunogenicity screening of tumor-derived extracellular vesicles by flow cytometry of splenic t cells
Uploaded: 2021-09-23T14:30:00
Description: Watch this Scientific Journal Video about In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells at JoVE.com

这项研究得到了Deutsche Forschungsgemeinschaft（DFG，德国研究基金会）的支持 - Projektnummer 360372040 - SFB 1335和Projektnummer 395357507 - SFB 1371（给惠普），一个来自DKMS给予生命基金会的Mechtild Harf研究资助（给惠普），一个黑色素瘤研究联盟的年轻研究员奖（到S.H.），一个由Else-Kröner-Fresenius-Stiftung（给F.S.）的奖学金， 慕尼黑工业大学的种子基金（向S.H.）和Wilhelm Sander基金会的研究资助（2021.041.1，给S.H.）。H. P.得到了EMBO青年调查员计划的支持。
作者贡献：
F.S.，H.P.和S.H.设计了研究，分析和解释结果。F.S.和S.H.撰写了手稿。H.P.和S.H.指导了这项研究。

在实验开始时，小鼠至少6周龄，并在特定的无病原体条件下维持。本议定书符合机构道德标准和现行地方法规。动物研究得到了当地监管机构（德国慕尼黑上巴伐利亚大学）的批准。这些研究中没有调查可能的性别相关偏倚。
1. 化疗后肿瘤细胞衍生EV的生成和分离
<ol><li>在DMEM（含有4mM L-谷氨酰胺和4.5g / L D-葡萄糖）中培养表达卵清蛋白（B16-OVA）的小鼠B16黑色素瘤细胞，并?...

<ol>
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该协议提供了在化疗诱导的压力下从黑色素瘤细胞衍生的EV的免疫 学体内 评估，同时适应各种治疗下各种癌症释放的EV。例如，用来自奥沙利铂处理的B16-OVA细胞的EV免疫小鼠，扩增脾脏中产生IFN-γ的CD8 + T细胞，这些细胞通过与卵清蛋白 的离体 孵育进一步刺激，表明肿瘤特异性免疫反应。因此，通过该协议筛查免疫原性EV有助于更全面地了解常规癌症疗法，并能够对EV在癌症?...

免疫系统在抗击癌症方面起着举足轻重的作用，无论是在免疫检查点抑制的刺激下，还是在常规癌症治疗的功效下。肿瘤细胞屈服于基因毒性疗法，如化疗药物奥沙利铂和阿霉素，或电离放射治疗，可以释放抗原和危险相关分子模式（DAMPs），从而可能引发适应性抗肿瘤免疫反应1。在免疫原性细胞死亡的背景下，最突出的DAMP包括fin-me信号，如趋化ATP，eat-me信号，如钙网蛋白的暴露，促进抗原呈递细胞对肿瘤细胞的摄取，以及HMGB1的释放，激活模式识别受体，从而增强肿瘤抗原的交叉呈递2。此外， 通过 肿瘤来源的免疫原性核酸或其他刺激诱导的I型干扰素（IFN-I）被树突状细胞感知，使它们能够有效地启动肿瘤特异性细胞毒性T细胞3，4。临床上，活化和增殖的CD8 + T细胞浸润肿瘤为许多癌症患者的生存期提供了独立的预后因素。IFN-γ从这些活化的T细胞中释放出来，介导对癌细胞的直接抗增殖作用，并驱动Th1极化和细胞毒性T细胞分化，从而有助于对癌症的有效免疫监测5，6。奥沙利铂是一种真正的免疫原性细胞死亡诱导剂，介导这种针对癌症的适应性免疫反应7。然而，肿瘤细胞在治疗诱导的压力下释放的大量初始免疫原性信号仍有待完全揭开。尽管癌症免疫疗法取得了重大进展，但将其益处扩大到更大比例的患者仍然是一个挑战。更详细地了解启动T细胞活化的免疫原性信号可以指导新疗法的开发。
一组异质的膜封闭结构，称为细胞外囊泡（EV），似乎充当细胞间通信设备。几乎所有细胞类型都发射的EV将功能蛋白，RNA，DNA和其他分子携带到受体细胞中，或者可能通过与细胞表面的受体结合来改变细胞的功能状态。它们的生物活性货物因生成细胞的类型和功能状态而显着变化8。在癌症免疫学中，从肿瘤细胞释放的EV主要被视为对抗免疫疗法，因为它们最终会促进侵入性生长，形成转移性生态位9并抑制免疫反应10。相比之下，一些研究表明，EV可以将肿瘤抗原转移到树突状细胞上，以实现有效的交叉呈现11，12。如果EV在治疗诱导的压力下出现，则可提供免疫刺激性核酸，从而促进抗肿瘤免疫反应13，14。最近，在肿瘤微环境中感知这种RNA和DNA先天免疫配体已被证明可以显着调节对检查点阻断的反应性15，16，17。因此，肿瘤细胞在不同治疗诱导应激下释放的EV的免疫原性作用需要进一步阐明。由于电动汽车是一个年轻但不断增长的研究领域，因此方法的标准化仍在进行中。因此，共享知识对于提高电动汽车与癌症免疫学之间相互作用的研究可重复性至关重要。考虑到这一点，本文描述了一种简单的方案来评估肿瘤衍生的EV 在体内的免疫原性作用。
该评估是通过生成肿瘤衍生的EV，用这些EV免疫受体小鼠， 并通过 流式细胞术分析脾T细胞来进行的。理想情况下，EV的产生是通过在无EV的细胞培养基中接种小鼠肿瘤细胞以获得高纯度来进行的。用特定的细胞应激刺激（例如化疗）处理细胞，以比较治疗诱导的EV与相应肿瘤衍生EV的基线免疫原性的影响。电动汽车的分离可以通过各种技术进行，这些技术应根据 体内 适用性和当地可用性进行选择。以下方案描述了使用用于EV纯化的商用试剂盒的基于沉淀的测定。用这些EV对小鼠进行两次免疫。第一次注射后14天，从脾脏中提取T细胞， 并通过 流式细胞术分析IFN-γ的产生，以评估全身免疫反应。有了这个，在不同治疗方案下出现的肿瘤衍生的EV诱导抗肿瘤T细胞反应的潜力被相对容易，快速和高有效性地评估13。因此，该方法适用于在各种条件下对衍生自癌细胞的EV进行免疫学筛选。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-CD3 FITC</td>
			<td>Biolegend</td>
			<td>100204</td>
			<td>Clone 17A2</td>
		</tr>
		<tr>
			<td>Anti-CD4 PacBlue</td>
			<td>Biolegend</td>
			<td>100428</td>
			<td>Clone GK1.5</td>
		</tr>
		<tr>
			<td>Anti-CD8 APC</td>
			<td>Biolegend</td>
			<td>100712</td>
			<td>Clone 53-6.7</td>
		</tr>
		<tr>
			<td>Anti-IFN&#947; PE</td>
			<td>eBioscience</td>
			<td>RM90022</td>
			<td>Clone XMG1.2</td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Biolegend</td>
			<td>420601</td>
			<td>Brefeldin A Solution (1,000x)</td>
		</tr>
		<tr>
			<td>Cell Strainer, 100 &#181;m</td>
			<td>Greiner</td>
			<td>542000</td>
			<td>EASYstrainer 100 &#181;m</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium with D-glucose (4.5 g/L) and L-glutamine (4 mM)</td>
		</tr>
		<tr>
			<td>FBS Good Forte</td>
			<td>PAN BIOTECH</td>
			<td>P40-47500</td>
			<td>Fetal Calf Serum (FCS)</td>
		</tr>
		<tr>
			<td>Fixable Viability Dye eFluor 506</td>
			<td>eBioscience, division of Thermo Fischer Scientific</td>
			<td>65-0866-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Concentrate</td>
			<td>eBioscience</td>
			<td>00-5123-43</td>
			<td>Fixation/Permeabilization Concentrate (10x)</td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Diluent</td>
			<td>eBioscience</td>
			<td>00-5223-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>407952</td>
			<td>From Streptomyces conglobatus - CAS 56092-82-1, &#8805; 97% (HPLC)</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-032</td>
			<td>L-Glutamine (200 mM)</td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>InvivoGen</td>
			<td>vac-pova</td>
			<td>Ovalbumine with &#60; 1 EU/mg endotoxin - CAS 9006-59-1</td>
		</tr>
		<tr>
			<td>Oxaliplatin</td>
			<td>Pharmacy of MRI hospital</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Phosphate Buffered Saline without calcium chloride and magnesium chloride</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>1514-122</td>
			<td>Mixture of penicillin (10,000 U/mL) and streptomycin (10,000 ug/mL)</td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma-Aldrich</td>
			<td>P1585</td>
			<td>Phorbol 12-myristate 13-acetate, &#8805; 99% (HPLC)</td>
		</tr>
		<tr>
			<td>PVDF filter, 0,22 &#181;m, for syringes</td>
			<td>Merck Millipore</td>
			<td>SLGV033RS</td>
			<td>Millex-GV Filter Unit 0.22 &#181;m Durapore PVDF Membrane</td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Invitrogen</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fischer Scientific</td>
			<td>11875</td>
			<td>Roswell Park Memorial Institute 1640 Medium with D-glucose (2.00 g/L) and L-glutamine (300 mg/L), without HEPES</td>
		</tr>
		<tr>
			<td>Syringe, 26 G</td>
			<td>BD Biosciences</td>
			<td>305501</td>
			<td>1 mL Sub-Q Syringes with needle (0.45 mm x 12.7 mm)</td>
		</tr>
		<tr>
			<td>Total Exosome Isolation Reagent</td>
			<td>Invitrogen</td>
			<td>4478359</td>
			<td>For isolation from cell culture media</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Thermo Fischer Scientific</td>
			<td>31350</td>
			<td>&#946;-Mercaptoethanol (50 mM)</td>
		</tr>
	</tbody>
</table>

in vivo immunogenicity screening of tumor-derived extracellular vesicles by flow cytometry of splenic t cells

由化疗或照射引起的肿瘤免疫原性细胞死亡可以通过释放危险相关的分子模式并诱导I型干扰素的产生来触发肿瘤特异性T细胞反应。免疫疗法，包括检查点抑制，主要依靠预先存在的肿瘤特异性T细胞来展开治疗效果。因此，利用免疫原性细胞死亡作为内在抗癌疫苗的协同治疗方法可以提高其反应性。然而，细胞在治疗诱导的压力下释放的免疫原性因子谱仍然不完全表征，特别是关于细胞外囊泡（EV）。EV，即几乎所有细胞发射的纳米级膜颗粒，被认为可以促进细胞间通讯，并且在癌症中已被证明可以介导对肿瘤抗原的交叉启动。为了评估衍生自肿瘤的EV在各种条件下的免疫原性作用，寻求适应性强，可扩展和有效的方法。因此，本文提出了一种相对简单和稳健的方法来评估EV的 体内 免疫原性。该协议基于对EV小鼠 进行体内 免疫后脾T细胞的流式细胞术分析，通过基于沉淀的测定在治疗或稳态条件下从肿瘤细胞培养物中分离。例如，这项工作表明，B16-OVA小鼠黑色素瘤细胞的奥沙利铂暴露导致免疫原性EV的释放，这些EV可以介导肿瘤反应性细胞毒性T细胞的激活。因此， 通过体内 免疫和流式细胞术筛查EV可确定免疫原性EV可能出现的条件。确定免疫原性EV释放的条件为测试EV对癌症的治疗功效和探索潜在的分子机制提供了必要的先决条件，以最终揭示EV在癌症免疫学中的作用的新见解。

该协议旨在促进对肿瘤衍生EV的免疫原性进行直接且易于重复的评估。因此，将小鼠接种来自表达模型抗原鸡卵清蛋白（OVA）的肿瘤细胞 体外 培养物的EV。随后的免疫反应 通过 流式细胞术在脾T细胞中分析。
图 1 概述了整个协议的实际步骤。由于该工作侧重于免疫原性细胞死亡，交叉展示和EV诱导的抗肿瘤免疫，因此该方案仅限于CD8 +细胞?...

Watch this Scientific Journal Video about In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells at JoVE.com

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

本手稿描述了如何使用流式细胞术评估肿瘤细胞来源的细胞外囊泡（EV）的 体内 免疫原性。来自接受治疗诱导的免疫原性细胞死亡的肿瘤的EV似乎与肿瘤免疫监测特别相关。该协议是奥沙利铂诱导的免疫刺激性肿瘤EV评估的例证，但可以适应各种环境。

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

该视频展示了如何使用流式细胞术评估肿瘤来源的细胞外囊泡或EV的体内免疫原性。本文所述的筛选方法相对容易执行，并且可以适应各种设置。在癌症中，鉴定具有特殊翻译相关性的免疫原性细胞外囊泡释放的条件。因此，自体电动汽车可以用作个性化的抗癌治疗。演示程序的第一部分的将是我实验室的技术助理Tatiana Nedelko。为了开始制备细胞外囊泡或EV所需的细胞培养基，通过在4摄氏度下以100，000次g超速离心24小时来耗尽FCS培养基中的牛EV。离心后丢弃沉淀。从培养物中收获表达卵清蛋白或B16-OVA细胞的小鼠B16黑色素瘤细胞，并用PBS洗涤细胞两次。然后，在EV耗尽的培养基中以每毫升400， 000个细胞的浓度接种细胞。接下来，用每毫升30微克奥沙利铂处理B16-OVA细胞，并在37摄氏度下与未处理的对照样品孵育24小时。24小时后，收集上清液，在4摄氏度下以400次g离心5分钟。在以2， 000次g再次离心之前丢弃沉淀，在4摄氏度下离心30分钟。丢弃沉淀后，通过220纳米PVDF膜过滤器过滤上清液到新鲜管中。然后，将1毫升上清液与0.5毫升特定的市售外泌体分离试剂混合。涡旋并将混合物转移到1.5毫升管中，然后在4摄氏度下孵育过夜。第二天，将混合物以10， 000次g在4摄氏度下离心60分钟。小心地丢弃上清液后，通过将1.5毫升管倒置在纸巾上，并通过细尖移液管抽吸而不接触底部的EV沉淀来除去剩余的滴剂。通过上下移液而不用吸头刮擦沉淀，将沉淀重悬于冷PBS中。然后，从所有管中汇集EV。如果不立即使用，请将EV悬浮液以80摄氏度存放在硅化容器中长达28天，直到应用。为了用从4.0 x 10到第五个B16-OVA细胞分离的EV对治疗组中的每只小鼠进行免疫，将5微升EV悬浮液与55微升冷PBS混合。接下来，用26至30号针头填充注射器，并加入60微升稀释的EV或PBS，然后立即将注射器放在冰上。将EV或PBS皮下接种到小鼠大腿的内侧。七天后重复免疫接种。第一次治疗后14天，牺牲小鼠切除脾脏。然后，用湿润的100微米细胞过滤器和注射器的塑料柱塞捣碎切除的脾脏。将脾细胞冲洗到50毫升管中，其中有5至10毫升的冷却完全RPMI。接下来，在4摄氏度下以400次g离心细胞五分钟，然后丢弃上清液。为了从细胞悬浮液中除去红细胞，在室温下用2毫升红细胞裂解缓冲液重悬并孵育细胞沉淀5分钟。然后，通过加入5至10毫升完全RPMI来停止反应，然后如前所述离心。在完全RPMI中重悬细胞沉淀后，计数来自每只小鼠的细胞，并将200， 000个脾细胞的一式三份和200微升cRPMI放入具有U形底部的96孔板的每个孔中。然后，将板在37摄氏度下孵育48小时。48小时后，用每毫升5纳克Brefeldin A，每毫升PMA20纳克和每毫升1微克离子红霉素在37摄氏度下处理细胞培养物4小时。孵育后，将脾细胞转移到具有V形底部的96孔板中，并用PBS洗涤细胞两次。将沉淀的脾细胞重悬于新制备的染色溶液中，并在4摄氏度下孵育细胞悬浮液30分钟，使其避光。对于固定和透化，在FACS缓冲液中洗涤脾细胞两次，然后将脾细胞重悬在每孔100微升的固定和透化溶液中，然后在黑暗中孵育，如前所述。为了染色细胞内干扰素γ，在固定或透化缓冲液中洗涤脾细胞，然后在缓冲液中用1：200稀释的针对干扰素γ的荧光抗体重新发送细胞。在黑暗中以4摄氏度孵育脾细胞1至12小时。在FACS缓冲液中彻底洗涤和重悬后，根据手稿中解释的门控策略分析细胞毒性T细胞的活化。在代表性图像中，显示了分析细胞毒性T细胞活化的门控策略。这些细胞被门控为单细胞，淋巴细胞亚群，活的CD3阳性T细胞和CD4阴性CD8阳性细胞毒性T细胞。评估干扰素γ的细胞内积累作为激活的替代标志物。用来自肿瘤细胞的EV进行免疫后，研究了受体小鼠抗原特异性T细胞反应的诱导。与未治疗的EV相比，在遗传毒性应激条件下培养的肿瘤衍生的EV（例如奥沙利铂治疗）诱导脾细胞毒性T细胞的有效活化。经过车辆处理的动物组表现出最低的反应。脾T细胞的活化是在离体再刺激后确定的，无论是否存在卵清蛋白。当肿瘤EV治疗动物的脾细胞在分析前用模型肿瘤抗原卵清蛋白重新刺激时，干扰素γ的产生增加。请注意，可重复的结果取决于电动汽车的持续隔离效率。因此，确保EV颗粒完全及时地重悬以避免干燥。通过该协议，我们能够分析肿瘤细胞内的特定途径，以确定肿瘤细胞衍生EV的免疫原性。这可能为在癌症治疗中利用这种免疫原性EV铺平道路。

Research

JoVE Journal

Cancer Research

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

科研

癌症研究

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

A Spheroid Killing Assay by CAR T Cells

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Extraction of Extracellular Vesicles from Whole Tissue

全组织细胞外囊的提取

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Cell Cycle-specific Measurement of ÃƒÅ½Ã‚Â³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

分离外体富集的细胞外囊泡携带颗粒细胞-巨噬细胞-胚胎干细胞的细胞外刺激因子

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

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 ...

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

体内通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性