Name: stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts
Uploaded: 2018-09-01T13:00:00.000+00:00
Duration: 11 min 15 s
Description: Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

作者感谢南特大学医院动物设施 (犹特人) 的工作人员畜牧和护理, 南特大学的细胞和 tissular 成像核心设施 (MicroPICell) 的成像, 和从南特的细胞术设施 (Cytocell)他们的专家技术援助。这项工作由 INSERM、CNRS、大学 de 南特、国立杜肿瘤研究所 (INCa#PLBio2014-155)、国家民族反癌症 (AO 区域间 2017) 和欧洲联盟时代网 Transcan2 (Immunoglio) 资助。这支队伍由研究所 Medicale (DEQ20170839118) 资助。这项工作是在 LabEX 监察员办公室和 IHU Cesti 方案的范围内实现的, 该项目分别由艾文莉和 ANR-11-LABX-0016-01 的国家研究机构 Investissements d ' ANR-10-IBHU-005 支助。IHU-Cesti 项目也支持南特都市和支付 la 卢瓦尔地区。作者感谢 Chirine Rafia 为改正手稿提供帮助。

下列涉及动物的程序是根据机构指南 (协议 #00186) 执行的. 02; 意大利 (法国) 的区域道德委员会人类 PBMCs 从知情的健康捐献者的血液中分离出来 (景区法国法国南特)。所有步骤都是在无菌条件下进行的。
1. 细胞毒性效应 T 淋巴细胞的非特异扩张
<ol><li>准备并照射35的馈线细胞。为刺激 2 x 105 -4 x 105个效应细胞, 准备 10 x 106 PBMCs 和 1 x 106 BLCLs 从三个不同, 健康捐赠者。</li>
	<li>并用重悬15毫升 RPMI 中的饲养细胞和效应细胞, 辅以10% 热灭活 FCS、2毫米 l-谷氨酰胺、青霉素 (100 毫升/ml)、链霉素 (0.1 µg/毫升) 和300毫升/ml 重组 IL-2。</li>
	<li>在最终浓度为1µg/毫升, 仔细混合, 并分配150µL 的细胞悬浮每井在96井 U 底板。</li>
	<li>孵育在37°c 和与 5% CO2在一个湿润的大气。</li>
	<li>每日检查板材直到大细胞团簇形成在养殖井 (~ 天 7)。</li>
	<li>在新鲜培养基中将细胞转化为 1 x 106细胞/毫升的培养瓶。</li>
	<li>通过计数 (每周 2x) 确定细胞总数, 并在新鲜培养基中保持 1 x 106细胞/毫升。 注意: 效应免疫细胞应准备好治疗管理的休息状态 (通常3周后, 最初的放大刺激)。在体内注射前应检查这些效应细胞的纯度和反应性 (如体外测定)。</li>
</ol>2. 手术前效应细胞的制备
<ol><li>在检查效应细胞计数后, 通过离心 (300 x g 5 分钟) 收集50毫升管中的效应细胞。 注: 为弥补任何损失, 准备过量的细胞 (例如, 50 x 106)。</li>
	<li>小心取出上清液, 在15毫升的无菌 PBS 和离心机中并用重悬细胞, 在 300 x g 5 分钟进行洗涤。</li>
	<li>仔细和彻底清除上清, 然后并用重悬细胞颗粒在1毫升的无菌 PBS。</li>
	<li>将悬浮细胞转移到一个1.5 毫升的微细中, 以 300 x g的离心为5分钟。</li>
	<li>小心, 完全清除上清吹打缓慢。</li>
	<li>并用重悬细胞颗粒在8µL 不育 PBS 每只老鼠。 注意: 这是一个关键步骤。</li>
	<li>使用微测量细胞悬浮液的体积。如果需要, 添加无菌 pbs (20 x 106细胞在每剂量的 PBS 的15µL), 并仔细混合。</li>
	<li>使用微检查, 每个鼠标的最终音量介于15和 20 ul 之间 (势在必行 < 20 µL)。</li>
	<li>保持细胞在冰上, 直到立体定向注射。 注: 超过3小时 timepoints 未测试。</li>
</ol>3. 立体定向注射
<ol><li>
设备安装
	<ol>
<li>根据制造商的指示, 组装和校准小型动物立体定向框架, 以确保颅内注射的准确性 (如注射器大小、所需体积和注射率)。 注: 建议采用慢速输液 (即2-3 µL/分钟)。</li>
		<li>在微生物安全柜 (MSC) 下安装材料, 以维持器械的不育, 保护小鼠不受感染。 注: 在37摄氏度的水浴中放置 "等温集团"。这个系统限制了小鼠在手术中的体温过低。加热垫, 这是必要的后程序护理, 必须使用在连续的温度监测。</li>
	</ol>
</li>
	<li>
手术前动物制剂
	<ol>
<li>麻醉小鼠腹腔注射氯胺酮 (10 毫克/毫升) 和甲苯噻嗪 (0.1 毫克/毫升) 在10µL/克体重的鼠标。</li>
		<li>执行脚趾捏试验, 以确保完全麻醉和镇痛的动物。 注意: 任何运动都是不深镇痛的征兆, 如果出现这种情况, 在重复手术前需要几分钟的时间。</li>
		<li>一旦鼠标被正确麻醉, 使用剪刀从手术部位 (两个耳朵之间, 由鼻子) 去除皮毛。</li>
	</ol>
</li>
	<li>
手术前细胞准备
	<ol>
<li>在每次注射前用吸管 (几次) 仔细并用重悬细胞, 以防止细胞结块。</li>
		<li>仔细绘制所需的细胞悬浮量 (15-20 µL) 到22克微量泵, 以避免气泡的渴望。 注意: 这个单元格加载步骤进入微量泵是很重要的, 以尽量减少差异的注入量。在程序之间重新加载每个单独注入的单元格, 以防止细胞聚集, 并确保队列中的效应细胞管理甚至数量。</li>
		<li>然后, 把注射器放进适应的注射器泵中。</li>
	</ol>
</li>
	<li>
程序护理
	<ol>
<li>使用聚维酮碘5% 溶液浸泡的拭子对手术部位进行消毒, 至少3x。</li>
		<li>在鼠标的眼睛里放一个润滑的眼膏, 防止角膜的任何干燥。</li>
		<li>将麻醉的鼠标放在立体定向的框架上, 在一个温暖的等温块上, 覆盖着无菌塑料包装, 以维持在手术中的老鼠的体温, 并限制死亡率。 注: 鼠标的鼻子和牙齿应适当地放置在牙杆上方, 以确保在手术过程中有足够的呼吸流量。</li>
		<li>一旦鼠标定位在牙杆上方, 将耳棒牢牢地拧紧在鼠标的耳朵下, 以固定头部。 注意: 小心不要破坏耳膜或损害呼吸。</li>
		<li>用无菌剪刀在颅骨的上半部, 从前到后, 使头骨显露出 1-2 厘米中线矢状皮肤切口。</li>
		<li>确定矢状和冠状缝线 (Bregma) 的交叉点, 作为注射前立体定向定位的地标 (如图 1所示)。</li>
		<li>把微量泵放在这一点上。</li>
		<li>移动微量泵2毫米右侧面和0.5 毫米前的 Bregma。</li>
		<li>使用 microdrill, 在预定坐标处用无菌钻头在头骨上做一个小孔。小心保持肤浅, 以避免任何外伤的大脑。 注: 在本议定书中, 免疫细胞注射在一个已建立的肿瘤 (肿瘤细胞注射后一周)。皮肤应重新打开 (疤痕) 和注射是执行在同一坐标用于肿瘤细胞植入 (孔通常仍然存在2周后注射)。在脑实质13、14中为注射肿瘤和效应细胞选择了坐标。</li>
	</ol>
</li>
	<li>
免疫效应细胞的注射
	<ol>
<li>小心地将微量泵插入钻孔中, 缓慢移动, 将针3毫米向下的硬脑膜, 然后向后0.5 毫米到最后深度2.5 毫米之前注入效应细胞。</li>
		<li>在 2-3 µL/分钟内运行效应细胞注射, 并在注射时间内监测小鼠。</li>
		<li>一旦注射完成, 取出针仅1毫米, 并保持微量泵在一个额外的分钟, 然后慢慢地完全撤回微量泵, 以防止任何渗漏从输液现场。 注: 在将动物从立体定向装置移走后, 立即清洁注射设备进行实验。</li>
	</ol>
</li>
	<li>
小鼠术后护理及随访
	<ol>
<li>将动物从立体定向的框架中取出, 立即在切口部位应用聚维酮碘5% 溶液, 用适当的手术缝合闭合皮肤。</li>
		<li>在伤口上直接应用2% 利多卡因凝胶, 用皮下注射治疗丁丙诺啡0.15 µg/克, 术后镇痛。</li>
		<li>将被麻醉的老鼠放回它的笼子上面的加热垫设置为37°c, 以保持适当的老鼠体温和避免任何体温过低。 注: 不需要单独的住房。</li>
		<li>监视鼠标直到它完全从麻醉中恢复, 并将其转移到一个住房室。 注意: 到目前为止, 该协议得到很好的支持, 因为很少发生意外并发症 (< 5% 的注射小鼠)。</li>
		<li>每日监测小鼠和弄死, 当任何下降的健康迹象被观察 (例如, 驼背的姿势, 减少流动性, 虚脱, 或重大体重下降 [≥ 15%])。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

经选择的本机或工程免疫效应细胞的移植, 是有效治疗肿瘤的一种有希望的方法, 如浸润性脑癌, 注意限制活性对非转化细胞15, 16,17,18。然而, 包括大脑在内的中枢神经系统具有特殊的免疫状态, 特别是由于血脑屏障的存在以及缺乏典型的淋巴引流系统19、20。...

肾小球 (IV. 型星形细胞瘤) 是成人最常见和最有攻击性的原发性脑癌。尽管有积极的治疗, 结合手术和射频化疗, 肾小球仍然与一个非常糟糕的预后 (中位存活率14.6 月和2年死亡率 > 73%)1。这一证据表明, 在过去十年2中, 没有有效的治疗进展得到证实。在设计更有效的治疗策略3,4,5, 免疫疗法6的候选者中, 目前正在探索追踪和消除高度侵入性和无线电/抗化疗肿瘤细胞, 怀疑他们的关键贡献迅速和致命的肿瘤复发7。对免疫疗法的各种潜在免疫指标进行了鉴定和建议, 涉及天然或改良的αβ或υδ T 淋巴细胞, 如肾小球特异肿瘤抗原或应力诱导分子8,9, 10。管理选定的肾小球活性免疫细胞效应器的可能性代表了一个独特的机会, 提供高数量的效应淋巴细胞直接进入残余恶性肿瘤的部位。为支持这一策略, 我们最近发现, 基于 immunodeficient 小鼠的同种异体肾小球移植的模型忠实地重述了肾小球肿瘤的发展9,11。此外, 这些模型被用来证明过继转输转移异体人 vυ9vδ2t 淋巴细胞的强抗癌功效。
本协议描述了在同种异体 T 淋巴细胞的移植基础上实现脑肿瘤立体定向免疫疗法的关键实验步骤。本文说明: (i) 治疗同种异体免疫效应 T 淋巴细胞 (如人 vυ9vδ2t 淋巴细胞) 的扩增;(ii) 制备注射用的这些效应 T 淋巴细胞;(iii) 大脑内立体定向管理的程序, 靠近肿瘤。本文还介绍了立体定向注射后这些细胞效应的行为。
这里提出的治疗方法是基于每剂量 20 x 106效应细胞为每个脑肿瘤-轴承 immunodeficient 鼠注射。一个初始的体外扩张步骤是需要产生大量的免疫细胞。因此, 非特异细胞扩张是使用 phytohemagglutinin (PHA L) 和辐照异基因饲养细胞: 外周血单个核细胞 (PBMCs) 的健康捐献者和 eb 病毒 (eb) 转换的 B-永生细胞线 (BLCLs), 从 PBMCs 的体外感染, 从狨 B95-8 细胞系的 eb 病毒的培养上清液中提取, 在1µg/毫升环孢素 a 的存在。
从体外测定9中比较和选择了肾小球活性效应免疫细胞。这些效应细胞的激活和放大使用标准协议, 根据其性质 (例如, 人类 Vγ9Vδ2 T 淋巴细胞9或人抗疱疹病毒αβ T 淋巴细胞12) 与最低纯度 > 80%, 例行由细胞分析检查。下面详述的细胞扩张程序适用于各种人类淋巴细胞子集。

<table><tbody><tr><td>PBMCs</td><td></td><td></td><td>from 3 different healthy donors</td></tr><tr><td>BLCLs</td><td></td><td></td><td>from 3 different donors</td></tr><tr><td>Roswell Park Memorial Institute medium (RPMI)</td><td>Gibco</td><td>31870-025</td><td></td></tr><tr><td>FCS</td><td>Dutscher</td><td>S1810-500</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>IL-2</td><td>Novartis</td><td>proleukin</td><td></td></tr><tr><td>PHA-L</td><td>Sigma</td><td>L4144</td><td></td></tr><tr><td>Stereotaxic frame</td><td>Stoelting Co.</td><td>51600</td><td></td></tr><tr><td>Mouse adaptator for stereotaxic frame&amp;nbsp;&amp;nbsp;</td><td>Stoelting Co.</td><td>51624</td><td></td></tr><tr><td>microsyringe pump injector&amp;nbsp;</td><td>WPI</td><td>UMP3-4</td><td></td></tr><tr><td>NanoFil Syringe</td><td>WPI</td><td>NF34BV-2</td><td></td></tr><tr><td>NSG mice</td><td>Charles River</td><td>NSGSSFE07S</td><td></td></tr><tr><td>Ketamine</td><td>Merial</td><td>Imalg&amp;egrave;ne 1000</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>Rompur 2%</td><td></td></tr><tr><td>Scissors</td><td>WPI</td><td>201758</td><td></td></tr><tr><td>Forceps</td><td>WPI</td><td>501215</td><td></td></tr><tr><td>OmniDrill 115/230V</td><td>WPI</td><td>503598</td><td></td></tr><tr><td>Vicryl 4-0</td><td>Ethicon</td><td>VCP397H</td><td></td></tr><tr><td>Xylocaine</td><td>Astrazeneca</td><td>3634461</td><td></td></tr></tbody></table>

stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts

胶质瘤多形性 (肾小球) 是成人中最常见和最有攻击性的原发性脑癌, 通常与预后较差有关, 在过去十年中提出了缺乏有效的治疗方法。在设计新的治疗策略的有希望的候选者中, 细胞免疫疗法已被用来消除高度侵袭性和化疗放射抗拒肿瘤细胞, 可能涉及迅速和致命复发的癌症。因此, 在肿瘤附近的同种异体肾小球-反应性免疫细胞效应剂 (如人 vυ9vδ2 T 淋巴细胞) 的管理, 将是一个独特的机会, 以提供高效和高度集中的治疗药物直接进入脑恶性肿瘤现场。在这里, 我们提出了一项协议, 以制备和立体定向管理的异基因人淋巴细胞在 immunodeficient 小鼠携带原位人原发性脑肿瘤。本研究为这些细胞免疫疗法的可行性和抗肿瘤效果提供了一个临床前的概念证明, 它们依赖于 intrabrain 肿瘤床内的异基因人淋巴细胞的立体定向注射。

这项研究描述了在肿瘤携带的小鼠脑内进行细胞免疫效应细胞转移的策略, 其基础是直接在瘤床内进行的立体定向注射。
为了尽量减少与大注射量相关的脑损伤的风险, 需要将效应细胞悬浮液集中 (20 x 106细胞在 PBS 的 15-20 µL 中)。为了检查这个细胞浓度步骤是否可能影响效应细胞的生存能力, 这些细胞是根据所描述的协...

Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

在这里, 我们提出了一项协议, 以制备和立体定向管理的异基因人淋巴细胞在 immunodeficient 小鼠携带原位人原发性脑肿瘤。本研究为 intrabrain 细胞免疫疗法的可行性和抗肿瘤疗效提供了概念验证。

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

stereotactic-adoptive-transfer-cytotoxic-immune-cells-murine-models

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  CRCINA. 在这里, 我们提出了一项协议, 以制备和立体定向管理的异基因人淋巴细胞在 immunodeficient 小鼠携带原位人原发性脑肿瘤。本研究为 intrabrain 细胞免疫疗法的可行性和抗肿瘤疗效提供了概念验证。

视频: Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

科研

医学

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

原正畸胶质血异草回顾渗透生长和同源脱氢酶 I 突变

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

免疫与感染

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

多形性胶质母细胞瘤的转化原位模型

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

癌症研究

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

用于胶质母细胞瘤激光间质热疗的免疫活性小鼠模型

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

使用立体定位装置建立脑肿瘤的原位患者来源的异种移植模型

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

影像引导切除治疗干细胞种子支架的胶质母细胞瘤和颅内植入术

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...