肿瘤进展和血管生成的双生物发光成像
Summary
该协议描述了通过双生物发光成像实时监测肿瘤进展和血管生成情况的肿瘤携带小鼠模型。
Abstract
血管生成作为肿瘤进展的关键环节,已成为抗肿瘤治疗的研究热点和目标。然而,没有可靠的模型来同时以视觉和敏感的方式追踪肿瘤进展和血管生成。生物发光成像具有灵敏度高、特异性强、测量准确等优点,在活成像中具有独特的优势。这里介绍的一个协议,通过注射一个雷尼拉荧光酶标记的鼠乳腺癌细胞系4T1到转基因小鼠与血管生成诱导萤火酶酶表达建立肿瘤小鼠模型。该小鼠模型提供了一个有价值的工具,通过单只小鼠的双生物发光成像,实时监测肿瘤进展和血管生成。该模型可广泛应用于抗肿瘤药物筛选和肿瘤研究。
Introduction
血管生成是癌症从小,局部肿瘤到更大的,潜在的转移肿瘤1,2进展的一个基本过程。肿瘤生长与血管生成的相关性成为肿瘤学研究领域的重点之一。然而,传统的测量形态变化的方法未能同时监测活体动物的肿瘤进展和血管生成。”
肿瘤细胞的生物发光成像(BLI)是监测肿瘤生长的一种特别合适的实验方法,因为它的非侵入性、敏感性和特异性3、4、5、6.BLI技术基于荧光素酶在排放生物发光时可以催化特定基质氧化的原理。植入的肿瘤细胞中表达的荧光素酶与注射的基质发生反应,这种基质可以通过活的成像系统检测,信号间接反映细胞数或细胞定位在体内6、7的变化。
除了肿瘤生长,肿瘤血管生成(癌症进展的关键步骤)也可以通过BLI技术使用Vegfr2-Fluc-KI转基因小鼠8,9,10可视化。血管内皮生长因子(Vegf)受体2(Vegfr2),一种类型的Vegf受体,主要表达在成年小鼠的血管内皮细胞11。在Vegfr2-Fluc-KI转基因小鼠中,萤火虫荧光素酶(Fluc)的DNA序列被敲入内源性Vegfr2序列的第一个外子。因此,Fluc以与小鼠血管生成水平相同的方式表示(显示为 BLI 信号)。为了生长超过几毫米的大小,肿瘤从现有的血管中招募新的血管,这高度表达由肿瘤细胞1的生长因子触发的Vegfr2。这为使用Vegfr2-Fluc-KI转基因小鼠通过BLI非侵入性监测肿瘤血管生成开辟了可能性。
在此协议中,建立了一个带肿瘤的小鼠模型,分别通过萤火虫荧光素酶(Fluc)和雷尼拉荧光酶(Rluc)成像来监测单个小鼠的肿瘤进展和血管生成(图1)。创建 4T1 细胞系 (4T1-RR),通过 Rluc 成像可稳定增长 Rluc 和红色荧光蛋白 (RFP) 来跟踪细胞生长。为了进一步调查血管生成在肿瘤的进展和回归的动态变化,另一个4T1细胞系(4T1-RRT)被创建,表达自杀基因单纯疱疹病毒截断胸腺激酶(HSV-ttk),Rluc和RFP。通过对甘西洛韦(GCV)的分管,HSV-ttk表达细胞被选择性地消融。基于这些细胞系,建立了Vegfr2-Fluc-KI小鼠的肿瘤承载模型,作为连接肿瘤进展和体内肿瘤血管生成的实验模型。
Protocol
Representative Results
Discussion
在本协议中,描述了一种非侵入性双BLI方法,用于监测肿瘤发育和血管生成。BLI报告器系统是首次开发,包含HSV-ttk/GCV自杀基因,用于跟踪肿瘤进展和通过Rluc成像在体内的回归。同时,通过氟成像利用Vegfr2-Fluc-KI小鼠评估肿瘤血管生成。这种带肿瘤小鼠模型能够提供一个实用的平台,通过双BLI在单一小鼠具有高相关性、可重复性和可翻译性,为连续和非侵入性跟踪肿瘤发育和肿瘤血管生成提供实用平台。
…Divulgaciones
The authors have nothing to disclose.
Acknowledgements
本研究得到国家重点研发项目(2017YFA0103200)、国家自然科学基金(81671734)和天津市科技支撑项目重点项目(18YFZCSY00010)的支持。中央大学 (63191155)。我们感谢格洛丽亚·南斯的修订,这些修订对于提高我们手稿的质量很有价值。
Materials
| 0.25% Trypsin-0.53 mM EDTA | Gibco | 25200072 | |
| 1.5 mL Tubes | Axygen Scientific | MCT-105-C-S | |
| 15 mL Tubes | Corning Glass Works | 601052-50 | |
| 293T | ATCC | CRL-3216 | |
| 4T1 | ATCC | CRL-2539 | |
| 60 mm Dish | Corning Glass Works | 430166 | |
| 6-well Plate | Corning Glass Works | 3516 | |
| Biosafety Cabinet | Shanghai Lishen Scientific | Hfsafe-900LC | |
| Blasticidine S Hydrochloride (BSD) | Sigma-Aldrich | 15205 | |
| Cell Counting Kit-8 | MedChem Express | HY-K0301 | |
| CO2 Tegulated Incubator | Thermo Fisher Scientific | 4111 | |
| Coelenterazine (CTZ) | NanoLight Technology | 479474 | |
| D-luciferin Potassium Salt | Caliper Life Sciences | 119222 | |
| DMEM Medium | Gibco | C11995500BT | |
| Fetal Bovine Serum (FBS) | BIOIND | 04-001-1A | |
| Fluorescence Microscope | Nikon | Ti-E/U/S | |
| Ganciclovir (GCV) | Sigma-Aldrich | Y0001129 | |
| Graphics Software | GraphPad Software | Graphpad Prism 6 | |
| Insulin Syringe Needles | Becton Dickinson | 328421 | |
| Isoflurane | Baxter | 691477H | |
| Lentiviral Packaging System | Biosettia | cDNA-pLV03 | |
| Liposome | Invitrogen | 11668019 | |
| Living Imaging Software | Caliper Life Sciences | Living Imaging Software 4.2 | |
| Living Imaging System | Caliper Life Sciences | IVIS Lumina II | |
| MEM Medium | Invitrogen | 31985-070 | |
| Penicillin-Streptomycin | Invitrogen | 15140122 | |
| Phosphate Buffered Saline (PBS) | Corning Glass Works | R21031399 | |
| Polybrene | Sigma-Aldrich | H9268-1G | |
| RPMI1640 Medium | Gibco | C11875500BT | |
| SORVALL ST 16R Centrifuge | Thermo Fisher Scientific | Thermo Sorvall ST 16 ST16R | |
| Ultra-low Temperature Refrigerator | Haier | DW-86L338 | |
| XGI-8 Gas Anesthesia System | XENOGEN Corporation | 7293 |
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