我们描述了源自患者的异种移植物(PDXs)用表达绿色荧光蛋白和荧光素酶记者慢病毒颗粒的稳定标记的方法。该方法允许用于跟踪PDXs的生长在主站点,以及检测使用体内成像系统自发和实验转移。
The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients’ tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.
源自患者的肿瘤异种移植物(PDXs),其中,手术切除肿瘤样品被移入直接进入免疫妥协小鼠的发展,提供了超过标准细胞系异种移植模型几个优点,并代表在癌症研究1,2的一大进步。 PDXs可以保持,并通过与在第一通道生长的肿瘤的遗传和生物学特性最小改变连续传代扩大;和更准确地反映肿瘤的异质性比来自人肿瘤细胞系3-8衍生异种移植物。这些模型现在广泛用作个性化癌症治疗9,10一个平台,如在药物开发6,11的临床前平台和作为研究癌症生物学4,12的实验工具。
最PDXs植入皮下传播,其中使用卡尺可行允许肿瘤生长的测量随着时间的推移。然而,转移性疾病已经越来越难以用PDXs建模。专门为乳腺癌,转移性能力不同器官异种移植物已被描述3,5,13,但自发传播到转移部位的频率极低。凡报道,转移性负担的定性和定量依赖于靶器官验尸费力组织学检查。表达生物发光(荧光素酶,卢克)或荧光癌细胞系(绿色荧光蛋白,GFP)基因记者常用于乳腺癌转移到脑,肺,骨和肝腔内后,尾静脉,intrafemoral和脾注射的实验模型中使用14-16。虽然这些模型绕过从原发性肿瘤传播,它们是研究器官趋向性和转移性定植的机制有价值。然而,从初级病人肿瘤和PDXs来源的细胞可以具有低转染或转导率全光照g标准程序。一个替代方案是建立在体外 17,其可使用常规的组织培养协议来然后标记的PDX-来源的细胞系。然而,这种方法不适合用于标记最PDXs,为此细胞系派生是困难的,可以改变细胞的表型。在这里,我们提出了一个协议对PDX-解离的肿瘤细胞与合适的用于体内成像的慢病毒载体的转导。此外,我们将介绍使用分离的吕克 – 绿色荧光蛋白标记的PDX细胞心内注射免疫功能低下小鼠的实验性转移。
用于与基因-报道表达慢病毒PDX-解离组织体转导的基本协议先前已经描述18。在当前的协议中,我们描述了其他的方法来富集人类肿瘤细胞,将获得接近100%的转导效率,以及用于检测试验乳腺癌使用标记PDXs的转移。这个协议可以适于标记与各种发光和荧光标记PDXs的多种癌症类型,以及基因的表达( 即 ,shRNA的目的基因的敲低)的调制。
该协议中的关键步骤:
使用高滴度的慢病毒颗粒(> 10 8 TU /毫升)是这个协议的成功的一个关键步骤,如允许体外转期间媒体组成的严格控制。虽然生产高滴度的病毒颗粒的多种方法得到了很好的描述18,19;该协议使用于中详细描述产生的慢病毒颗粒www.kottonlab.com 。对于肿瘤的消化和分解癌细胞的温和方法是?…
The authors have nothing to disclose.
作者感谢达雷尔Kotton博士在波士顿大学提供噬菌体EF1aL – 红色荧光蛋白-UBC-GFP-W在这些研究中使用高滴度的慢病毒载体的生产和协议。这项工作是由国防部BCRP W81XWH-11-1-0101(DMC),ACS IRG#57-001-53(DMC),NCI K22CA181250(DMC)和R01 CA140985(CAS)资助.NCI P30CA046934中心授予支持活体成像和组织培养内核,AMC科罗拉多大学。
DMEM/F12 (1:1) | Hyclone | SH30023.01 | |
bFGF | BD Biosciences | 354060 | |
EGF | BD Biosciences | 354001 | |
Heparin | Sigma | H4784 | |
B27 | Gibco/Thermo Fisher | 17504-44 | |
Anti-fungi-antibiotics | Hyclone | SV30010 | |
Accumax | Innovative Cell Technologies | AM-105-500 | Digestion Buffer |
FBS | Atlanta Biologicals | S11550 | |
HBSS Red Ca++/Mg++ free | Hyclone | SH30031.02 | |
Hepes | |||
10X PBS | Hyclone | SH30258.01 | |
Cultrex | Cultrex | 3433-005-01 | Basement Matrix Extract (BME) |
30C shaker | NewBrunswick Scientific CO. INC | Series 25 Incubator Shaker | |
70um filters | Falcon | 7352350 | |
scalpels | Fisher | 22079690 | |
Clorhexidine disinfectant | Durvet | NDC# 30798-624-35 | |
Red blood cell lysis reagent | Sigma | R7757 | |
Neuraminidase | Sigma | N7885-1UN | |
EpCAM (CD326+) microbeads* | Miltenyil Biotec | 130-061-101 | |
Lineage cell depletion Kit, mouse* | Miltenyil Biotec | 130-090-858 | |
MiniMACS Separator | Miltenyil Biotec | 130-042-102 | |
Mini MACS Magnetic Stand | Miltenyil Biotec | 130-042-303 | |
MS Columns | Miltenyil Biotec | 130-042-201 | MS or LS columns can be used, adjust to number of cells. |
Illumatool Tunable light system | Lightools research | Various | For in vivo fluorescence imaging |
Xenogen IVIS200 imaging device | Xenogen | Various | For in vivo luminiscence imaging |
Human Cytokeratin Clone MNF116 Monoclonal antibody | DAKO | M0821 | Pan-cytokeratin |
Epidermal Growth factor receptor antibody | Cell signaling | 4267S | EGFR |