Name: a human peripheral blood mononuclear cell (pbmc) engrafted humanized xenograft model for translational immuno-oncology (i-o) research
Uploaded: 2019-08-15T14:00:00
Description: Watch this Scientific Journal Video about A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research at JoVE.com

我们感谢实验室成员进行有益的讨论。这项工作得到了北京市科委生物医学和生命科学创新与培养研究项目的一部分支持。Z151100003915070(项目"新免疫肿瘤抗肿瘤药物BGB-A317的临床前研究"),也部分得到公司内部临床前研究资金的支持。

在涉及人类参与者的研究中进行的所有程序都符合BeiGene和/或国家研究委员会的道德标准,以及1964年《赫尔辛基宣言》及其后来的修正或类似的道德标准。研究中的所有个人参与者都得到了知情同意。在涉及动物的研究中执行的所有程序都得到北金内部审查委员会的批准。该协议已针对人体化NOD/SCID小鼠的BGB-A317(Tislelizumab)的评估进行了专门调整。
1. 建立基于人PBMC的模型
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近年来,我们对癌症发展和进展的认识有了显著的进步,重点是全面了解肿瘤细胞及其相关频闪。利用宿主免疫机制可以引起对癌细胞的更大影响,这是一种有前途的治疗策略。具有完整小鼠免疫系统的鼠模型,如合成和GEM模型,已被广泛用于研究检查点介导的免疫。使用这些模型的功效评估主要取决于代理抗小鼠靶抗体13,14。然而,人类和鼠的免疫系统之间的...

免疫肿瘤学(I-O)是一个迅速扩大的癌症治疗领域。研究人员最近开始意识到调节免疫系统作用对肿瘤的攻击作用。免疫检查点封锁已经证明了各种癌症类型的令人鼓舞的活动,包括黑色素瘤,肾细胞癌,头和颈部,肺,膀胱和前列腺癌1,2。与直接杀死癌细胞的目标疗法相反,I-O疗法能增强人体免疫系统攻击肿瘤3。
迄今为止,已经建立了许多相关的I-O动物模型。这些包括:1)小鼠肿瘤细胞系或肿瘤同源性小鼠;2)来自基因工程小鼠(GEM)或致癌物的自发性肿瘤;3) 功能性鼠免疫系统中与人类药物靶点敲的嵌合的GEM;4)用人类癌细胞或患者衍生异种移植物(PDXs)移植的具有重组人体免疫力的小鼠。每种模型都有明显的优势和局限性,这些优点和局限性已在其他地方广泛描述和审查。
复生人类免疫免疫缺陷小鼠作为转化I-O研究的临床相关方法,已日益受到重视。这通常是通过成人免疫细胞(例如,外周血单核细胞 (PMBC))5、6 或 2) 从脐带血或胎儿的造血干细胞 (HSC) 的移植来实现的肝脏7,8。这些人性化的小鼠可以支持人类肿瘤的生长,从而允许在人类免疫和人类癌症的背景下评估I-O疗法。尽管这些优点,但人性化小鼠在I-O研究中的应用通常受到一些顾虑的阻碍,例如模型开发时间长,成本相当高。
在这里,我们描述了一个基于人类PBMC的模型,可以广泛应用于翻译I-O研究。该模型在疗效研究中具有较好的时间和成本效益,具有很高的可重复性。它已被内部用于评估目前正在临床前和临床开发中的几种I-O疗法。BGB-A317(Tislelizumab),一种研究性的人性化抗PD-1抗体9,作为例子,讨论模型开发、表征以及抗肿瘤疗效分析的可能应用。

<table border="0" cellpadding="0" cellspacing="0" style="width:955px;" width="955">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">PBMC separation /cell culture</td>
			<td style="width:211px;"></td>
			<td style="width:193px;"></td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Histopaque-1077</td>
			<td style="width:211px;">Sigma</td>
			<td style="width:193px;">10771</td>
			<td style="width:143px;">Cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">DMEM</td>
			<td style="width:211px;">Corning</td>
			<td style="width:193px;">10-013-CVR</td>
			<td style="width:143px;">Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">DPBS</td>
			<td style="width:211px;">Corning</td>
			<td style="width:193px;">21-031-CVR</td>
			<td style="width:143px;">Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">FBS</td>
			<td style="width:211px;">Corning</td>
			<td style="width:193px;">35-076-CV</td>
			<td style="width:143px;">Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Penicillin-Streptomycin, Liquid</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:193px;">15140-163</td>
			<td style="width:143px;">Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:193px;">25200-114</td>
			<td style="width:143px;">Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Matrigel</td>
			<td style="width:211px;">Corning</td>
			<td style="width:193px;">356237</td>
			<td style="width:143px;">CDX inoculation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">FACS analysis</td>
			<td style="width:211px;"></td>
			<td style="width:193px;"></td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Deoxyribonuclease I from bovine pancreas</td>
			<td style="width:211px;">Sigma</td>
			<td style="width:193px;">DN25</td>
			<td style="width:143px;">Sample preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Collagenase Type I</td>
			<td style="width:211px;">Sigma</td>
			<td style="width:193px;">C0130</td>
			<td style="width:143px;">Sample preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-mouse/human CD11b (M1/70) antibody</td>
			<td style="width:211px;">BioLegend</td>
			<td style="width:193px;">101206</td>
			<td style="width:143px;">FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-mouse Ly-6C (HK1.4) antibody</td>
			<td style="width:211px;">BioLegend</td>
			<td style="width:193px;">128008</td>
			<td style="width:143px;">FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-mouse Ly-6G (1A8) antibody</td>
			<td style="width:211px;">BioLegend</td>
			<td style="width:193px;">127614</td>
			<td style="width:143px;">FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-human CD8 (OKT8) antibody</td>
			<td style="width:211px;">Sungene Biotech</td>
			<td style="width:193px;">H10082-11H</td>
			<td style="width:143px;">FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-human CD279 (MIH4) antibody</td>
			<td style="width:211px;">eBioscience</td>
			<td style="width:193px;">12-9969-42</td>
			<td style="width:143px;">FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-human CD3 (HIT3a) antibody</td>
			<td style="width:211px;">4A Biotech</td>
			<td style="width:193px;">--</td>
			<td style="width:143px;">FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Guava easyCyte 8HT Benchtop Flow Cytometer</td>
			<td style="width:211px;">Millipore</td>
			<td style="width:193px;">0500-4008</td>
			<td style="width:143px;">FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Tumor/PDX implantation /dosing / measurement</td>
			<td style="width:211px;"></td>
			<td style="width:193px;"></td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Cyclophosphamide</td>
			<td style="width:211px;">J&#38;K</td>
			<td style="width:193px;">Cat#419656, CAS#6055-19-2</td>
			<td style="width:143px;">In vivo efficacy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Disulfiram</td>
			<td style="width:211px;">J&#38;K</td>
			<td style="width:193px;">Cat#591123, CAS#97-77-8</td>
			<td style="width:143px;">In vivo efficacy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Syringe</td>
			<td style="width:211px;">BD</td>
			<td style="width:193px;">300841</td>
			<td style="width:143px;">CDX inoculation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Hypodermic needles (14G)</td>
			<td style="width:211px;">Shanghai SA Mediciall &#38; Plastic Instruments Co., Ltd.</td>
			<td style="width:193px;">0.7*32 TW SB</td>
			<td style="width:143px;">PDX inoculation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Vernier Caliper (MarCal)</td>
			<td style="width:211px;">Mahr</td>
			<td style="width:193px;">16ER</td>
			<td style="width:143px;">Tumor measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">IVC individual ventilated cages</td>
			<td style="width:211px;">Lingyunboji Ltd.</td>
			<td style="width:193px;">IVC-128</td>
			<td style="width:143px;">Animal facility</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">IHC</td>
			<td style="width:211px;"></td>
			<td style="width:193px;"></td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Leica ASP200 Vacuum tissue processor</td>
			<td style="width:211px;">Leica</td>
			<td style="width:193px;">ASP200</td>
			<td style="width:143px;">IHC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Leica RM2235 Manual Rotary Microtome for Routine Sectioning</td>
			<td style="width:211px;">Leica</td>
			<td style="width:193px;">RM2235</td>
			<td style="width:143px;">IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Leica EG1150 H Heated Paraffin Embedding Module</td>
			<td style="width:211px;">Leica</td>
			<td style="width:193px;">EG1150 H</td>
			<td style="width:143px;">IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Ariol-Clinical IHC and FISH Scanner</td>
			<td style="width:211px;">Leica</td>
			<td style="width:193px;">Ariol</td>
			<td style="width:143px;">IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-human CD8 (EP334) antibody</td>
			<td style="width:211px;">ZSGB-Bio</td>
			<td style="width:193px;">ZA-0508</td>
			<td style="width:143px;">IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-human PD1 [NAT105] antibody</td>
			<td style="width:211px;">Abcam</td>
			<td style="width:193px;">ab52587</td>
			<td style="width:143px;">IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Anti-human PD-L1 (E1L3N) antibody</td>
			<td style="width:211px;">Cell Signaling Technology</td>
			<td style="width:193px;">13684S</td>
			<td style="width:143px;">IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Polink-2 plus Polymer HRP Detection System</td>
			<td style="width:211px;">ZSGB-Bio</td>
			<td style="width:193px;">PV-9001/9002</td>
			<td style="width:143px;">IHC</td>
		</tr>
	</tbody>
</table>

a human peripheral blood mononuclear cell (pbmc) engrafted humanized xenograft model for translational immuno-oncology (i-o) research

近年来免疫肿瘤学(I-O)疗法的发现和发展是癌症治疗的一个里程碑。然而,治疗挑战依然存在。健壮且与疾病相关的动物模型是继续临床前研究和开发的重要资源,以解决一系列额外的免疫检查点。在这里,我们描述了人类外周血单核细胞(PBMC)-基于人性化异种移植模型。BGB-A317(Tislelizumab)是一种在晚期临床开发中研究性的人性化抗PD-1抗体,作为讨论平台设置、模型特征和药物疗效评估的例子。这些人性化的小鼠支持大多数经过测试的人类肿瘤的生长,从而允许在人类免疫和人类癌症的背景下评估I-O疗法。一旦建立,我们的模型是比较时间和成本效益,通常产生高度可重复的结果。我们建议本文中概述的协议可以作为建立小鼠模型的一般指南,该模型与人类PBMC和肿瘤重组为I-O研究。

按照此处介绍的程序,成功建立了基于 PBMC 的人性化异种移植模型。总之,在CP和DS治疗后,通过对嗜中性粒细胞和单细胞种群的流细胞测定分析,确定了NOD/SCID小鼠的CP骨髓消融效应(图1)。100毫克/千克CP加125毫克/千克DS被确定为最佳剂量,并在后来的研究中使用,因为该方案可导致嗜中性粒细胞和单核细胞的最大耗竭,而不会对小鼠造成严重毒性。其次,进行了人体PBMC和肿瘤移植。IHC?...

Watch this Scientific Journal Video about A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research at JoVE.com

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

用于转化免疫肿瘤学(I-O)研究的人类外周血单核细胞(PBMC)的人工化异种移植模型

我们描述了人类外周血单核细胞(PBMC)–基于人体的异种移植小鼠模型,用于转化免疫肿瘤学研究。该协议可作为建立和描述类似I-O治疗评估模型的一般准则。

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

我们的协议可以作为建立小鼠模型的一般指南，重建与人类PBMC和肿瘤免疫肿瘤学研究。这些程序提供了一种经济高效且高度可重复的方法，通过皮下和将人类PBMC与癌症异种移植物混合，部分重组人类肿瘤小鼠的人体免疫力。与我一起演示手术的将是崔欣欣、张燕菊、白惠晨和杨小龙，他们从药理科送来。对于骨髓细胞耗竭内腹膜内交付100毫克每公斤环磷酰胺和口头交付125毫克每公斤二硫化素到6至8周大的NOD/SCID雌性小鼠每天一次，为期两天。在第二剂环磷酰胺和硫化剂24小时后，重新注射6个新鲜分离的人类PBMC中的5倍10和6个人类肿瘤细胞系细胞中的2.5倍和200微升PBS加50%的PBS，每个动物将整个体积皮下注射到每个实验动物的右侧。每周测量和记录原发性肿瘤体积两次，为期四到六周。当肿瘤达到平均体积200至500毫米的立方体，使用光剪刀收获整个肿瘤从每个动物。PBMC供方肿瘤细胞系和患者衍生的异种移植，导致中度肿瘤生长与相对较高的PD-1，PD-L1和CD8表达应用于后续分析。对于收获的肿瘤组织的免疫组织化学分析，在脱水和将组织嵌入石蜡之前24至72小时固定样品和形式。在聚硅素涂层幻灯片上获取三微米内嵌组织，用三次七分钟二甲苯浸入将样品脱酸。第三次治疗后，用三分钟分级酒精浸入水合。然后用去压水冲洗幻灯片三次，然后从滑梯中去除多余的液体。要执行抗原检索，请将幻灯片放在容器中，用 10 毫摩尔高二酸化缓冲液盖住幻灯片。将滑梯、容器在微波炉中加热三分钟，然后将滑梯放在 95 摄氏度的水浴中加热 30 分钟。冷却至室温后，用去压水冲洗幻灯片三次，从滑梯中吸入多余的液体。用0.3%过氧化氢孵育10分钟，以阻止内源性过氧化物酶活性，然后用3%BSA和PBS进行一小时的阻断。在过氧化氢孵育结束时，在四摄氏度的温度下标记幻灯片，并贴上适当的原抗体。第二天早上，用马萝卜过氧化物酶结合二次抗体在室温下治疗一小时，然后将基板滴到幻灯片上，直到通过光显微镜观察到适当水平的棕色染色。接下来，用赤氧林染色样品。一分钟后，停止与蒸馏水的反应，然后在0.5%盐酸醇中下潜5秒，在0.5%氨水中吸收5秒。然后用三升三分钟乙醇和三、五分钟二甲苯浸入使样品脱水。要评估感兴趣的候选药物的抗肿瘤活性，在细胞接种当天根据计划的实验方案开始治疗，并每周两次监测原发性肿瘤体积，为期四至六周。对于Proncode动态分析肿瘤渗透免疫细胞，将肿瘤组织切碎成小块，用1型胶原蛋白和DNase I和RPMI-1640介质加5%FBS消化片段，在37摄氏度下30分钟。在消化结束时，通过40微米细胞过滤器过滤消化的组织，以获得单个细胞悬浮液，并通过离心收集细胞。每毫升冰冷传真缓冲液浓度的1倍10的7细胞将颗粒重新填充，并转移等量的细胞到96孔圆形底板中适当数量的孔。通过离心收集细胞，并在人类 IGG 每毫升 20 微克中重新给颗粒 30 分钟，以阻止任何非特异性结合。然后，在4摄氏度下用适当的原抗体染色细胞30分钟，然后根据标准方案通过流式细胞分析进行分析。如证明，每公斤环磷酰胺100毫克，每千克125毫克解硫磷酰胺导致中性粒细胞和单核细胞在治疗后四天大量枯竭。人体PBMC和肿瘤移植后，通过免疫组织化学在肿瘤微环境内验证人体免疫细胞渗透的存在。可以对PBMC捐赠者小组进行体内筛查，以确认在可接受的肿瘤生长速率下，有相对较高的免疫细胞渗入肿瘤微环境。也可以评估不同癌症类型患者衍生异种移植体内的人类癌细胞系，以评估肿瘤生长速率和免疫细胞渗透。在这个具有代表性的实验中，用人化抗PD1抗体对PBMC和移植人化小鼠进行人工化小鼠的治疗产生了显著的抗肿瘤活动。二硫化素可降低环磷酰胺的尿毒毒性，这种组合可能在小鼠中产生更持久的中性粒细胞减少效应。环磷酰胺和二硫化硫化物的确切剂量方案可能需要预先确定。较新的免疫缺陷小鼠菌株，以更容易的人体免疫重组和减少异源GvHD效应已经建立，并正在等待进一步的研究，我们的文件协议。我们的模型可用于评估T细胞参与癌症免疫疗法，特别是当工作时间短或选择代理之前移动到更复杂的多线免疫模型。

Research

JoVE Journal

Cancer Research

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

A Mouse Model of Fatigue Induced by Peripheral Irradiation

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

科研

癌症研究

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

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

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 ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

患者衍生的胰腺癌异质异种移植模型,使用斑马鱼幼虫作为比较药物评估的宿主

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from SÃƒÆ’Ã‚Â©zary Syndrome Patients for Transcriptomic Profiling

从Sézary综合征患者中分离人外周血单核细胞和CD4 + T细胞以进行转录组学分析

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

循环肿瘤细胞系：基础和转化研究的创新工具

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...