作者承认凯瑟琳· Pietrosimone 博士的社论贡献。PLA 脚手架是由 Loboa 博士在北卡罗来纳州立大学的实验室制造的。这项工作得到了 unc Lineberger 综合癌症中心的大学癌症研究基金和 unc 翻译和临床科学研究所 (KL2TR001109, UL1TR001111) 的支持。

这里描述的所有方法都得到了北卡罗来纳大学教堂山分校机构动物护理和使用委员会 (IACUC) 的批准。
注: 本程序是对小鼠进行开放式开颅手术, 作为修改版本的协议由 Mostany 和 Portera-Cailliau19 , 其中的骨组织, 手术切除被丢弃, 使大脑可供建立和肾小球肿瘤的最终切除术。开颅术后, 肿瘤是通过立体定向植入, 如小泽和詹姆斯20所述。我们植入 1 x 105 U87 妇幼保健-Fl 细胞在以下立体定向坐标 (毫米从 bregma): (2.5, 0,-0.5)。
1. 细胞培养和支架制备
注: 在小鼠植入前应准备48小时的支架。下面的卷是以每脚手架为基础提供的。根据需要增加额外的脚手架数量。
<ol><li>将 PLA 支架切割成切除腔大小 (约2毫米 x 2 毫米) 件。脚手架可以用剪刀手工切割, 或者用穿孔来进行重复。</li>
	<li>通过浸泡在70% 乙醇15分钟, 然后浸泡在 PBS 消毒。在 Dulbecco 的改良鹰的培养基 (DMEM) 中放置支架, 其中含有10% 胎牛血清 (血清) 和1% 青霉素-链霉素, 同时准备用于播种的细胞。</li>
	<li>用0.05% 胰蛋白酶 (3-5 毫升的 T75 瓶) 提升培养的 MSCs。孵育在37°c 5 分钟, 确保细胞已经解除, 然后添加7-10 毫升 DMEM 到瓶中, 以灭活胰蛋白酶。</li>
	<li>把烧瓶的内容转移到一个15毫升的离心管上。使用 hemocytometer 计数单元格。颗粒 5 x 105 MSCs 为每个脚手架通过离心在 100 x g 为5分钟。</li>
	<li>吸出上清和并用重悬 MSCs 在5µL DMEM 每 5 x 105细胞。</li>
	<li>用拍干的方法准备支架播种。<ol>
<li>取下一个脚手架, 从 DMEM 浸泡与镊子, 并临时把它放在盖子上的6井板。从盖子上抬起脚手架, 留下一滴多余的 DMEM。</li>
		<li>把脚手架再次放回盖子上, 在一个新的, 干燥的位置。重复3-5 次, 然后将部分干脚手架放在一个新的6井板中播种。对每个脚手架重复。 注: 部分干脚手架提供最佳的细胞播种结果。如果脚手架太湿, 细胞会从脚手架上滑下, 并坚持下面的井板。如果脚手架太干燥, 液滴不会扩散到整个脚手架上, 导致初始细胞分布不佳。</li>
	</ol>
</li>
	<li>使用吸管, 轻轻地混合的干细胞的小瓶, 以融汇暂停, 因为一些细胞可能已落户底部。</li>
	<li>慢慢地吸管2.5 µL 新鲜混合 MSC 悬浮直接地在脚手架上, 在脚手架的顶部创造一个小水滴。</li>
	<li>添加300µL DMEM 到每个井的边缘。这将防止细胞液滴迅速蒸发。孵育37°c 30 分钟, 使细胞附着在支架上。</li>
	<li>用镊子, 轻轻翻转在井板上的脚手架。种子2.5 µL 新鲜混合 MSC 悬浮 (这导致总共 5 x 105细胞种子每脚手架)。孵育37°c 30 分钟, 使细胞附着在支架上。</li>
	<li>在 DMEM 上盖上支架, 在6井板的每个井上加2毫升。轻轻举起支架, 让介质在它们下面流动。在植入手术前48小时在这个状态下孵育37摄氏度。</li>
	<li>检查支架后24小时, 并添加/改变介质, 如果过量蒸发或变色介质由于 pH 不平衡被观察。</li>
</ol>2. 荧光引导下切除和支架植入术
注: 在初始化手术前先消毒所有工具。管理您的机构 IACUC 协议中概述的预防性镇痛。
<ol><li>将立体定向的框架放置在荧光解剖显微镜的舞台上。</li>
	<li>麻醉在诱导腔内吸入异氟醚的小鼠。一旦麻醉后, 通过异氟醚鼻锥适配器连续吸入麻醉供应, 将鼠标固定在立体定向的框架内。用加热垫和/或探头保持体温。 注: 4-5% 异氟醚适用于诱导, 2-3% 适用于维护, 但必须对每只鼠标进行调整和监测。</li>
	<li>将眼膏涂抹于眼部, 防止角膜干燥。用一系列三酒精和三 betadine 湿巾消毒头皮切口部位。</li>
	<li>对每一个肢体进行脚趾捏反射试验, 确认阴性反应, 以确保正确的麻醉。确保呼吸速率保持在大约55-65 呼吸/分钟左右。</li>
	<li>使用镊子, 捏和轻轻地抬起头皮。用手术剪刀做中线线延髓-尾鳍切口。用 PBS 冲洗切口部位, 用棉尖涂抹器去除真皮下脂肪。安排皮肤, 使以前建立的颅骨窗口完全可见。</li>
	<li>用18克针轻轻地将硬脑膜穿透到颅骨窗户的边框内。重复, 直到切口完全跟踪窗口的内部。</li>
	<li>用细钳剥去硬脑膜, 露出底层的软组织和肿瘤。</li>
	<li>通过关闭房间灯和转动显微镜荧光模式对 U87 妇幼肿瘤进行定位。通过将1-200 µL 吸管尖端装入真空泵的油管末端准备切除。</li>
	<li>打开真空泵。轻吸荧光组织切除肿瘤, 直到没有信号残留。关闭真空泵并丢弃吸管尖端。把荧光关掉, 房间灯亮着。 注: 控制出血, 用冷 PBS 冲洗, 用棉尖涂抹固定压力。在更严重的情况下, 切除腔也可以临时包装与止血剂, 只要它被删除后, 出血停止后, 另一个 PBS 灌溉。</li>
	<li>在植入之前, 在 PBS 中慢慢地蘸一个 MSC 播种的 PLA 脚手架, 以去除不需要的介质和相关组件。将支架植入切除腔内。如果需要, 添加1µL 纤维蛋白原 (67-106 毫克/毫升), 其次是1µL 凝血酶 (400-625 U/毫升), 以确保支架到位。</li>
	<li>随着硬脑膜的去除和颅骨瓣从头骨窗口已经丢弃, 只需关闭皮肤和使用手术胶水密封伤口。将动物从吸入麻醉剂中取出, 并允许在受热的表面恢复。</li>
	<li>一旦走动, 将鼠标返回笼子。对 IACUC 批准的附表施行镇痛。对每个鼠标重复过程。</li>
</ol>3. 术后影像学
<ol><li>使用28克胰岛素注射器, 注射 d-荧光素 (150 毫克/千克腹腔) 小鼠。</li>
	<li>等待10分钟。这将使荧光素在整个身体中循环, 并与荧光素酶-表达大脑中癌细胞的反应, 以获得峰值表达。</li>
	<li>在异氟醚麻醉下 (如前所述), 图像小鼠在生物发光成像 (BLI) 系统中可视化肿瘤。根据肿瘤大小的不同需要 (几秒到几分钟) 改变曝光时间。</li>
	<li>根据需要重复成像过程来确定肿瘤的生长动力学。继续监测动物健康状况。 注: 治疗骨髓间充质干细胞将组成性表达, 杀死癌细胞, 抑制肿瘤的整体生长。成像每2-5 天提供足够的频率为此目的。</li>
	<li>当 IACUC 协议中概述的预定端点被满足时, 通过 transcardial 灌注和收集组织来牺牲小鼠进行分析。</li>
</ol>

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Drs、Bagó和 Hingtgen 在猎鹰疗法方面拥有公平性, 他们从 UNC 教堂山获得了干细胞和脚手架技术方面的许可。

手术通常可以在每只老鼠30分钟内完成, 考虑到以下几点, 以最大限度地提高精确度和避免耗时的陷阱。首先, 确保鼠标在开始程序之前正确定位在立体定向仪器中。不需要的头部运动将限制开颅手术的准确性, 肿瘤植入的位置和肿瘤切除的程度。切除前, 充分去除覆盖肿瘤的硬脑膜部分。坚韧, 纤维硬脑膜硬化, 因为它脱水在渴望。如果不完全删除, 硬化硬脑膜可能限制吸头移动和防止完整的肿瘤切?...

胶质瘤 (肾小球) 是成人最常见的原发性脑癌, 平均存活12-15 月1,2,3,4,5。自2005年以来, 当目前最大手术切除的临床标准, 其次为放疗和伴随和佐剂 temozolomide 化疗采用6,7, 生存没有显著改善。虽然这种治疗为患者提供暂时的症状缓解, 治疗标准总是导致复发, 因为侵袭性癌症病灶逃避切除, 并保护不受系统治疗的血脑屏障 (BBB)。针对侵袭性肿瘤灶而规避脑屏障的策略, 迫切需要对这种好斗和衰弱的疾病进行牵引。
人骨髓间充质干细胞 (MSCs) 由于其原发的肿瘤取向8、9, 显示出有望成为肾小球的药物传递载体。MSCs 拥有受体并迁移到肿瘤分泌的可溶性因子, 包括基质细胞衍生因子 1α (SDF-1α)、基质 metalloproteinase-1 (MMP-1) 和单核细胞趋化蛋白 protein-1 (MCP-1) 等10,11,12,13. 工程间充质干细胞用于表达和分泌细胞毒药物, 使其能够被用作肿瘤自导的药物运载工具。设计的 MSCs 向侵袭性肿瘤灶移动并提供治疗蛋白。这种方法已经证明了在各种临床前肾小球模型9,14的可行性。然而, 绝大多数这些模型不包括手术切除, 尽管临床相关性的这一组成部分。使用新的切除模型进行的研究显示, 手术切除肿瘤可以减少直接注射到手术腔15的干细胞的持续性。生存能力的丧失导致疗效降低, 可能是由于药物的剂量和持续时间减少到侵袭性肿瘤灶。
为了增加干细胞的生存能力和药物的传递, MSCs 在植入前可以播种到支架上。在本协议中, 生物相容性和可吸收静电纺丝纤维结构聚乳酸 (PLA) 被用作 MSCs 的支架. PLA 在植入物上弯曲并符合切除腔的形状, 最大限度地提高治疗覆盖率, 最大限度地减少距离 MSCs 必须去到达肿瘤细胞。骨髓间充质干细胞在植入过程中保持在支架上, 然后在植入16、17后移离支架向肿瘤细胞转移。MSCs 和他们携带的细胞毒药物在肿瘤灶处积聚。肿瘤细胞毒性药物的传递需要 MSC 的生存能力和持久性, 这两个都是在支架上植入的辅助。
在这一过程中, 慢病毒载体载体被用来诱导稳定的荧光 (体外跟踪) 和生物荧光 (在体内跟踪) 标记的肿瘤和干细胞系。人肾小球系 U87 感染了 mCherry 和萤火虫荧光素酶 (U87 mCh), 和非治疗性 MSCs 与 gfp 和 renilla 荧光素酶 (gfp-Rluc)。干细胞表达 TNFα相关凋亡诱导配体 (msc) 的治疗变异。踪迹是一种组成性分泌的蛋白质, 它与癌细胞附近的死亡受体结合, 并启动 caspase 介导的凋亡18。
在这里, 我们提供了一个协议, 为临床前图像引导肾小球手术切除和植入 MSC 种子支架。简而言之, 裸鼠在三天后通过立体定向原位注射 U87 妇幼保健, 建立原发性肿瘤。嫁接肿瘤的生长周期约为一周。在切除手术前, PLA 支架植入48小时的 MSCs。然后在荧光引导下切除肿瘤, 将 MSC 加载的支架植入切除腔内。肿瘤的负担和老鼠的生存, 然后跟踪手术后, 生物发光成像 (BLI)。下面提供了这些过程的时间线 (图 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57452/57452fig1.jpg"> 图 1: 过程的时间线.老鼠最初收到一个头骨窗口 (0 天)。在三天的恢复期后, 肿瘤被植入 (3 天) 并生长大约一周。支架在肿瘤切除和植入术前两天 (8 天) 播种骨髓间充质干细胞 (10 天)。肿瘤进展和治疗效果通过术后影像学评估 (10 天 +)。<a href="//cloudflare.jove.com/files/ftp_upload/57452/57452fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>Just for mouse stereotaxic instrument</td><td>Stoelting</td><td>51730</td><td>Maintains steady head positioning during surgery</td></tr><tr><td>Fluorescence dissecting stereomicroscope</td><td>Leica</td><td>M165 FC&amp;nbsp;</td><td>Allows real-time imaging of tumor during resection</td></tr><tr><td>Motorized integrated stereotaxic injector (ISI) system</td><td>Stoelting</td><td>53315</td><td>Allows precise tumor cell injection volume and rate</td></tr><tr><td>Vetbond tissue adhesive</td><td>3M</td><td>1469</td><td>Sugical glue to close skin wound</td></tr><tr><td>Artificial tears</td><td>Akorn</td><td>664268</td><td>Prevents eyes from drying during surgery</td></tr><tr><td>Webcol alcohol preps</td><td>Covidien</td><td>6818</td><td>Sterilize incision site</td></tr><tr><td>Betadine surgical scrub</td><td>Purdue Fredick Company</td><td>6761815117</td><td>Sterilize incision site</td></tr><tr><td>Cotton-tipped applicators</td><td>Fisherbrand</td><td>23-400-115</td><td>Surgery tool</td></tr><tr><td>E-vac aspirating system</td><td>Argos</td><td>EV310</td><td>Vacuum pump used to resect tumor</td></tr><tr><td>Fibrinogen and thrombin extracted from as-received TISSEEL</td><td>Baxter</td><td></td><td>To temporarily secure the scaffold in the resection cavity</td></tr><tr><td>IVIS Kinetic in vivo optical imaging system</td><td>Caliper Life Science</td><td></td><td>Bioluminescent Imager</td></tr><tr><td>D-Luciferin potassium salt</td><td>PerkinElmer</td><td>122799</td><td>In vivo imaging agent</td></tr></tbody></table>

image-guided resection of glioblastoma and intracranial implantation of therapeutic stem cell-seeded scaffolds

胶质瘤 (肾小球) 是最常见和最具攻击性的原发性脑癌, 其预期寿命为12-15 月。短期寿命的部分原因是目前的治疗能力不足, 包括手术切除术后放疗和化疗, 以消除侵袭性肿瘤病灶。杀伤人骨髓间充质干细胞 (MSCs) 可以改善这些病灶的治疗。MSCs 表现出强有力的肿瘤取向, 可以被设计用来表达杀死肿瘤细胞的治疗蛋白。临床前模型的进展表明, 手术切除可诱发早期 MSC 丢失, 降低疗效。在生物降解聚乳酸 (PLA) 支架上播种 MSCs 可以改善 msc 的治疗效果。在解放军脚手架上的手术切除腔的交付恢复细胞的保留, 持久性和肿瘤的杀戮。为了研究 PLA 种子植入对肾小球的影响, 需要一个准确的临床前模型。在这里, 我们提供了一个临床前手术协议的图像引导肿瘤切除免疫缺陷小鼠的肾小球, 其次是 MSC 种子支架植入。MSCs 的设计与慢病毒载体构造组成性表达和分泌治疗 TNFα相关的凋亡诱导配体 (踪迹) 以及绿色荧光蛋白 (GFP), 以允许荧光跟踪。同样, U87 肿瘤细胞的设计, 以表达 mCherry 和萤火虫荧光素酶, 提供双荧光/发光跟踪。虽然目前用于调查干细胞介导的治疗方法, 这项议定书可以修改, 以调查的影响, 手术切除的其他肾小球的干预。

U87 肿瘤细胞被设计来表达 mCherry 和萤火虫荧光素酶 (U87 mCh) 在注射之前, 并允许图像引导切除和生物发光跟踪 (图 2A-C)。干细胞同样是用诊断 gfp-Rluc (gfp) 或治疗 gfp 试验 (msc), 并播种到 PLA 支架上以确认附着和增殖 (图 2D-F)。
<img alt="Figure 2" class="xfigimg" src="/fil...

Watch this Scientific Journal Video about Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds at JoVE.com

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

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

肿瘤寻求治疗间充质干细胞 (MSCs) 显示有望作为一种治疗侵袭性胶质瘤。最佳移植包括将 MSCs 送到支架上的肿瘤切除腔内。在这里, 研究理学硕士治疗的临床前技术, 包括: 图像引导肿瘤切除;植入 MSC 种子支架;和术后治疗跟踪。

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 ...

image-guided-resection-glioblastoma-intracranial-implantation

Research

JoVE Journal

Medicine

8.2K 次观看.  University of North Carolina at Chapel Hill. 肿瘤寻求治疗间充质干细胞 (MSCs) 显示有望作为一种治疗侵袭性胶质瘤。最佳移植包括将 MSCs 送到支架上的肿瘤切除腔内。在这里, 研究理学硕士治疗的临床前技术, 包括: 图像引导肿瘤切除;植入 MSC 种子支架;和术后治疗跟踪。

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

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

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). 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 ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

成像神经胶质瘤启动<em在体内</em&gt;通过抛光和增强的薄膜头骨的颅窗

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

用于胶质母细胞瘤脉冲电场治疗的柔性有机电子器件

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

癌症研究

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

在小鼠中使用 2P-IVM 实时跟踪脑肿瘤中的纳米颗粒

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

作者聚焦：优化胶质母细胞瘤治疗中药物递送和早期检测的多模式成像策略

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

小鼠模型中胶质母细胞瘤的双光子活体显微镜

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

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

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

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

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

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

摘要

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此视频中的章节

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

随后的三维颅内植入在体内

立体定向颅内植入和

成像神经胶质瘤启动

用于胶质母细胞瘤脉冲电场治疗的柔性有机电子器件

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

小鼠模型中胶质母细胞瘤的双光子活体显微镜

小鼠模型中胶质母细胞瘤的双光子活体显微镜

小鼠模型中胶质母细胞瘤的双光子活体显微镜

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