胶质瘤移植中体内成像的荧光分子层析成像研究
Summary
原位颅内注射肿瘤细胞已用于癌症研究, 研究脑肿瘤生物学, 进展, 进化和治疗反应。本文提出了肿瘤移植的荧光分子层析成像技术, 为临床前的胶质瘤模型提供了实时活体成像和肿瘤质量定量。
Abstract
致瘤性是癌细胞形成肿瘤肿块的能力。一种广泛使用的方法来确定细胞是否是癌变是通过注射 immunodeficient 小鼠皮下肿瘤细胞和测量肿瘤肿块后, 它变得可见和明显。原位注射癌细胞的目的是在微环境中引入异种异体, 最接近于所研究的肿瘤起源组织。脑癌研究需要颅内注射癌细胞, 以使肿瘤形成和分析在独特的微环境中的大脑。颅内移植的体内成像能瞬间监测原位嫁接小鼠的肿瘤质量。在这里, 我们报告的使用荧光分子层析成像 (裂变材料) 的脑肿瘤移植。癌细胞首先转基因近红外荧光蛋白, 然后注入免疫缺陷小鼠的大脑。然后对这些动物进行扫描, 以便在较长的时间内获得肿瘤肿块的定量信息。细胞预标记允许对每只老鼠的肿瘤负担进行有效、可重复和可靠的定量。我们消除了注射成像基质的需要, 从而减少了对动物的压力。这种方法的局限性表现为无法检测到非常小的质量;但是, 它比其他技术更能解决较大的质量问题。可用于评价胶质瘤细胞系和患者源性样品的药物治疗或基因改变的疗效。
Introduction
癌症是工业化世界人类疾病相关死亡的主要原因之一。由于死亡人数极高, 迫切需要新的治疗方法。胶质瘤多形性 (肾小球) 是一种极其致命的脑癌类型, 由脑肿瘤、基质和免疫细胞的异质群组成。根据美国中央脑肿瘤登记处的数据, 原发性恶性和非恶性脑肿瘤的发病率约为 10万, 约为22例。大约1.1万新的案件预计将被诊断在美国在 2017年1。
前临床研究调查的可能性, 药物, 程序, 或治疗是有效的之前, 在人类的测试。临床前研究的最早的实验室步骤之一是通过植入宿主生物体中的癌细胞来确定药物治疗的潜在分子靶点, 定义为人移植模型。在此背景下, 利用患者源性异体移植 (PDXs) 的颅内脑肿瘤移植模型已被广泛应用于研究脑肿瘤生物学、进展、进化和治疗反应, 最近对生物标志物的开发、药物筛选和个性化医学2,3,4。
最负担得起和无侵入性的体内成像方法之一监测颅内移植是生物发光成像 (BLI)5,6,7,8。然而, 一些 BLI 限制包括基质的管理和可用性, 酶的稳定性, 和光淬火和散射在成像采集9。在这里, 我们报告红外裂变材料作为一种替代成像方法, 以监测前胶质瘤模型。在这种方法中, intracranially 植入 PDXs 的信号采集和量化, 表达近红外荧光蛋白 iRFP72010,11 (从今以后称为 FP720) 或 turboFP635 (从今以后称为 FP635),是用裂变材料成像系统执行的。使用裂变材料技术, 可以在治疗前、期间或之后以无创性、无基质和定量的方式监测原位肿瘤的体内。
Protocol
Representative Results
Discussion
肿瘤移植已被广泛应用于癌症研究中, 已经建立了一些成熟的成像技术: BLI;磁共振成像 (MRI);正电子发射层析成像 (PET), 计算机断层扫描 (CT);Fmt。每种方法都有利弊, 但最终会与提供的信息类型相辅相成。最常用的体内成像技术之一是 BLI5,6,7,8。BLI 和裂变材料条约都要求细胞工程 (与荧光素酶或红?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢弗雷德里克. 朗博士, PDX neurospheres 肿瘤中心。这项工作得到了失败的研究合作的支持, 美国脑肿瘤协会 (弗兰克 Furnari) 的子公司, R01-NS080939 (弗兰克 Furnari), 詹姆斯的麦基金会 (弗兰克 Furnari);贝尼特斯获得美国脑肿瘤协会 (ABTA) 奖的支持;Zanca 被美国-意大利癌症基金会博士后研究奖学金部分支持。弗兰克 Furnari 接受了来自路德维希癌症研究研究所的工资和额外支持。
Materials
| DMEM/High Glucose | HyClone/GE | SH30022.1 | |
| DMEM/F12 1:1 | Gibco | 11320-082 | |
| FBS | HyClone/GE | SH30071.03 | |
| Accutase | Innovative cell technologies | AT-104 | |
| Trypsin | HyClone/GE | SH30236.01 | |
| B27 supplement | Gibco | 17504044 | |
| human recombinant EGF | Stemcell Technologies | 2633 | |
| human recombinant FGF | Stemcell Technologies | 2634 | |
| DPBS | Corning | 21-031-00 | |
| FACS tubes | Falcon | 352235 | |
| DAPI | ThermoFisher Scientific | 62248 | |
| Blasticidin | ThermoFisher Scientific | A1113903 | |
| p24 ELISA | Clontech | 632200 | |
| Xylazine | Akorn | NDC 59399-110-20 | |
| Ketamine | Zoetis | NADA 043-403 | Controlled substance |
| Ointment | Dechron | NDC 17033-211-38 | |
| Absorbable suture | CpMedical | VQ392 | |
| 5 ul syringe | Hamilton | 26200-U | Catalog number as sold by Sigma-Aldrich |
| Cell Sorter | Sony | SH8007 | |
| Mouse stereotaxic frame | Stoelting | 51730 | |
| Motorized stereotaxic injector | Stoelting | 53311 | |
| Micromotor hand-held drill | Foredom | K1070 | |
| Mouse warming pad | Ken Scientific Corporation | TP-22G | |
| Fluorescence Tomography System | PerkinElmer | FMT 2500 XL | |
| TrueQuant Imaging Software | Perkin Elmer | 7005319 | |
| Ultra-centrifuge Optima L-80 XP | Beckman Coulter | 392049 | |
| Tissue Culture 100mm Dishes | Olympus Plastics | 25-202 | |
| Tissue Culture 150mm Dishes | Olympus Plastics | 25-203 | |
| Tissue Culture Flasks T75 | Corning | 430720U | |
| 50 mL conical tubes | Corning | 430290 | |
| 15 mL conical tubes | Olympus Plastics | 28-101 | |
| Centrifuge Avanti J-20 | Beckman Coulter | J320XP-IM-5 | |
| Tube, Polypropylene, Thinwall, 5.0 mL | Beckman Coulter | 326819 | |
| Tube, Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm | Beckman Coulter | 326823 | |
| Athymic nude mice | Charles River Laboratories | Strain Code 490 (Homozygous) | Prior approval by the Institutional Animal Care Program and by the Institutional Biosafety Committee required. |
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