Name: image-guided resection of glioblastoma and intracranial implantation of therapeutic stem cell-seeded scaffolds
Uploaded: 2018-07-16T12:30:00
Description: Watch this Scientific Journal Video about Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds at JoVE.com

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...

<ol>
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L., Hingtgen, S., Shah, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+therapeutic+stem+cells+implanted+in+the+tumor+resection+cavity+induce+cell+death+in+gliomas.">Encapsulated therapeutic stem cells implanted in the tumor resection cavity induce cell death in gliomas.</a> Nature Neuroscience. 15 (2), 197-204 (2011).</li><li>Bag&#243;, J., Pegna, G., Okolie, O., Hingtgen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+matrices+enhance+the+transplant+and+efficacy+of+cytotoxic+stem+cell+therapy+for+post-surgical+cancer.">Fibrin matrices enhance the transplant and efficacy of cytotoxic stem cell therapy for post-surgical cancer.</a> Biomaterials. 84, 42-53 (2016).</li><li>Bag&#243;, J., Pegna, G., Okolie, O., Mohiti-Asli, M., Loboa, E., Hingtgen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+scaffolds+increase+the+efficacy+of+stem+cell-mediated+therapy+of+surgically+resected+glioblastoma.">Electrospun nanofibrous scaffolds increase the efficacy of stem cell-mediated therapy of surgically resected glioblastoma.</a> Biomaterials. 90, 116-125 (2016).</li><li>Loebinger, M., Eddaoudi, A., Davies, D., Janes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cell+Delivery+of+TRAIL+Can+Eliminate+Metastatic+Cancer.">Mesenchymal Stem Cell Delivery of TRAIL Can Eliminate Metastatic Cancer.</a> Cancer Research. 69 (10), 4134-4142 (2009).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J. Vis. Exp. (12), e680 (2008).</li><li>Ozawa, T., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Intracranial+Brain+Tumor+Xenografts+With+Subsequent+Analysis+of+Tumor+Growth+and+Response+to+Therapy+using+Bioluminescence+Imaging.">Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging.</a> J. Vis. Exp. (41), e1986 (2010).</li></ol>

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&#160;</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。

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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 of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

图像引导的对流增强交付到大脑的琼脂糖凝胶模型

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

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 ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

用机器学习预测图像引导疗法的治疗反应--肝细胞癌的跨动脉治疗实例

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...

Transaxillary First Rib Resection for Treatment of the Thoracic Outlet Syndrome

经腋窝第一肋骨切除术治疗胸廓出口综合征

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol ...