在直肠癌原位小鼠模型中循环肿瘤细胞的分离
Summary
我们描述了通过将肿瘤细胞或有机体注入小鼠盲肠并随后从该模型中分离循环肿瘤细胞（CTC）来建立原位结肠直肠肿瘤。
Abstract
尽管皮下小鼠模型具有易于适用性和成本效益的优点，但其严重限制并不能准确模拟肿瘤生物学和肿瘤细胞传播。引入原位小鼠模型来克服这些局限性;然而，这种模型在技术上是苛刻的，特别是在诸如大肠的中空器官中。为了产生可靠地生长和转移的均匀的肿瘤，肿瘤细胞制备和注射的标准化技术至关重要。
我们已经开发了一种大肠癌（CRC）的原位小鼠模型，其发展高度均匀的肿瘤，并且可以用于肿瘤生物学研究以及治疗试验。将来自原发性肿瘤，二维（2D）细胞系或3维（3D）有机体的肿瘤细胞注射到盲肠中，并且根据注射的肿瘤细胞的转移潜能形成高度转移性肿瘤。此外，定期发现反恐委员会。我们这里描述来自2D细胞系和3D组织以及原发性肿瘤组织的肿瘤细胞制备技术，手术和注射技术以及来自荷瘤小鼠的CTC的分离以及目前的故障提示。
Introduction
结肠直肠癌（CRC）是西方国家癌症死亡的最常见原因之一。 1原发性肿瘤经常可以切除，远处转移的发生显着恶化预后，并常常导致死亡。 2，3转移的生物学相关成分被循环肿瘤细胞（CTC），其从肿瘤分离，生存中循环，附着在上皮在靶器官，侵入器官，并最终长大到新的病灶。 4虽然CTC的已知是预后相关，5，6，7，8的，9其生物学仅部分地理解为在CRC其极端罕见的结果。 10
鼠标模型是一个强大的tool研究癌症生物学的各个方面。通过将肿瘤细胞皮下注射到受体小鼠中产生经典的皮下肿瘤模型，其可以是免疫活性的（如果使用同基因鼠肿瘤细胞）或免疫缺陷。皮下肿瘤模型价格低廉，数据量快;它们的终点肿瘤生长可以容易地和非侵入性地测量。然而，在这些模型中已经证明抗肿瘤活性的新化合物中有88％在临床试验中失败。 11这部分是由于人和小鼠之间间差异;然而，这种失败的很大一部分是由于皮下小鼠模型的预测值低。
因此，肿瘤细胞注射入原发器官并因此在其原始微环境中生长的原位小鼠模型因此越来越多地用于癌症研究。 11，12， </s达> 13，14款原位不只是模拟局部肿瘤的生长条件;作为肿瘤生长的解剖学上正确的位点的结果，原位小鼠模型也允许转移的逼真的模拟，因此用于研究生物学CTC 8，15，16或它们的在CRC不同处理的响应。 13，17
原位小鼠模型的主要缺点是其技术复杂性。取决于注射细胞的器官，直到实验者能够诱导可再现肿瘤的学习曲线相当长。这尤其适用于结肠直肠癌模型，因为肿瘤细胞需要注射到肠壁中，这通常导致穿孔，肿瘤细胞渗漏或腔内肿瘤细胞损失。这是本文旨在描述从原代组织样品，2D细胞系和3D有机体培养物中注射细胞至小鼠盲肠的细胞制备方法。这里描述的技术导致高度均匀的肿瘤，并且取决于用于注射的细胞系的肿瘤生物学，在受体小鼠中可重复形成远处转移和CTC。 15
Protocol
这里提出的动物实验由机构和政府动物护理和使用委员会进行独立审查和许可，并根据实验动物科学协会联合会 （FELASA）指导进行。采取一切可能的措施，尽量减少包括麻醉和镇痛在内的痛苦，或必要时提早安乐死。 1.细胞和组织的制备注意：对于每次注射，使用20μL的体积，100,000个细胞。使用基底膜基质（BMM），以防止泄漏并确保标准化注射。?…
Representative Results
在该模型中成功和可重复地产生结肠直肠肿瘤严重依赖于细胞的精确注射而没有溢出或渗漏。如果这样做，这种模式是非常可靠的，很少造成人造腹膜传播。肿瘤的生长动力学及其传播模式取决于所用组织和细胞的生物学特性。 15在本模型中，HCT116细胞可靠地转移到肝脏，SW620细胞形成原位肿瘤，但不转移。 15 <p class="jove_content" fo:kee…
Discussion
尽管它们在皮下小鼠模型中具有临床前证实的活性，但大多数新型化合物在临床试验中失败，从未到达诊所。 11这明显皮下小鼠模型的功能不全，以精确地模拟肿瘤的生物学和生长模式导致基于喷射的肿瘤细胞直接导入原始器官原位小鼠模型的发展。
原位小鼠模型能够比皮下模型更准确地模拟实体瘤的生物学和传播。 15然而，主要的缺点?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了德国研究基金会（WE 3548 / 4-1）和Gandundheitswesen（1/14）的Roland-Ernst-Stiftung的支持。
Materials
| Cell culture Media and Components | |||
| Advanced DMEM F12 | Invitrogen | 12634010 | DMEM/ F12 +++ medium |
| HEPES (1 M) | Life Technologies GmbH | 15630056 | DMEM/ F12 +++ medium |
| Glutamax-I Supplement (200 mM) | Life Technologies GmbH | 35050038 | DMEM/ F12 +++ medium |
| Penicillin/Streptomycin (PenStrep) | Life Technologies GmbH | 15140122 | DMEM/ F12 +++ medium |
| DMEM | Life Technologies GmbH | 61965026 | basic medium of 2D cell lines (DMEM/10%FCS) |
| Fetal Calf Serum (FCS) | BIOCHROM AG | S 0115 | basic medium of 2D cell lines (DMEM/10%FCS) |
| TrypLE Express enzymatic dissociation buffer | Life Technologies GmbH | 12604021 | |
| Matrigel basement membrane matrix (BMM, phenol red free) | CORNING B.V. Life Sciences | 356231 | |
| Dulbecco's Phosphate Buffered Saline | Life Technologies GmbH | 14190169 | |
| Trypsin-EDTA (0,25%, Phenol-Red) | Life Technologies GmbH | 25200072 | |
| 6-/48-well plates with lid | CORNING | 3516/3548 | |
| cell culture flask 75cm², 250 mL | VWR International GmbH | 734-2066 | |
| cell culture flask 150cm², 600 mL | Corning B.V. Life Sciences | 355001 | |
| Eppendorf tubes 1,5 mL / 2 mL | Sarstedt AG & Co. | 72.706.400/ 72.695.400 | |
| 15 ml, 50 ml centrifuge tubes | Greiner-Bio-One GmbH | 188271/227270 | |
| TC10 Counting Slides (for TC20 Counting Machine) | Bio-Rad Laboratories GmbH | 1450016 | |
| Pasteur pipettes (glass, 150 mm) | Fisher Scientific GmbH | 11546963/ FB50251 | thinly pulled by using a bunsen burner |
| gentleMACS Dissociator | Miltenyi Biotec | 130-093-235 | for primary tumor tissue preparation |
| MACSmix Tube Rotator | Miltenyi Biotec | 130-090-753 | for primary tumor tissue preparation |
| gentleMACS C Tubes | Miltenyi Biotec | 130-093-237 | for primary tumor tissue preparation |
| Human Tumor Dissociation Kit | Miltenyi Biotec | 130-095-929 | for primary tumor tissue preparation |
| Falcon 70µm Cell Strainer | Corning B.V. Life Sciences | 352350 | for primary tumor tissue preparation |
| Name | Company | Catalog Number | Comments |
| Surgical Equipment | |||
| Sevoflurane | AbbVie Germany GmbH & Co. KG | – | |
| Medical oxygen | Air Liquide Medical GmbH | – | |
| Buprenorphine | Temgesic | – | |
| Bepanthen – opthalmic ointment | Bayer Vital GmbH | 10047757 | |
| Normal saline 0.9% (E154) | Serumwerk Bernburg AG | 10013 | |
| Aqua ad injectabilia | Braun | 235144 | |
| 1 mL Syringe (without dead volume) – Injekt-F SOLO | Braun/neoLab | 194291661 | |
| 30G injection needle | BECTON DICKINSON | 304000 | |
| cellulose swabs | Lohmann & Rauscher Deutschland | 13356 | |
| Micro-Adson Forceps | FST – Fine Science Tools | 11018-12 | |
| Iris Scissor – ToughCut | FST – Fine Science Tools | 14058-11 | |
| Olsen-Hegar Needle Holder | FST – Fine Science Tools | 12002-12 | |
| AutoClip Kit | FST – Fine Science Tools | 12020-00 | |
| PDS Z1012H 6/0 C1 (surgical suture) | Johnson & Johnson Medical GmbH | Z1012H | |
| Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer | Parkland Scientific | V3000PS/PK | |
| UltraMicro Pump with Micro4 Controller | World Precision Instruments | UMP3-4 | equipment for highly controlled orthotopic injection |
| Footswitch for SYS-Micro4 Controller | World Precision Instruments | 15867 | equipment for highly controlled orthotopic injection |
| Three-axis Manual Micromanipulator | World Precision Instruments | M325 | equipment for highly controlled orthotopic injection |
| Magnetic Stand for Micromanipulator | World Precision Instruments | M10 | equipment for highly controlled orthotopic injection |
| Steel Base Plate for M10 Magnetic Stand | World Precision Instruments | 5479 | equipment for highly controlled orthotopic injection |
| Hot Plate 062 | Labotect | 13854 | |
| Isis – Hair shaver | AESCULAP – Braun | – | |
| Binocular Surgical Microscope | Parkland Scientific | VS-2Z | |
| Name | Company | Catalog Number | Comments |
| CTC isolation | |||
| EDTA | Roth | 8040.1 | |
| Density gradient medium – Ficoll | StemCell – Lymphoprep | 7801 | |
| Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4 | BioLegend | 324210 | |
| Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8 | BioLegend | 118210 | |
| Petri Dish, ø 60 x 15 mm, 21 cm², Vent | Greiner bio-one | 628102 | |
| Fluorescence Cell Culture Microscope | Leica | ||
| Transferman 4r Micromanipulator | Eppendorf | ||
| CellTram Air | Eppendorf | aspiration pump connected to the micromanipulator | |
| Dmz Universal Microelectrode Puller | Dagan Corporation | required for the manufacturing of micro capillaries for single cell aspiration | |
| Prism Glass Capillaries | Dagan Corporation | ||
| PAP pen | Abcam | ab2601 | |
| Dulbecco's Phosphate Buffered Saline | Life Technologies GmbH | 14190169 | picking buffer |
| Fetal Calf Serum (FCS) | BIOCHROM AG | S 0115 | picking buffer |
| Penicillin/Streptomycin (PenStrep) | Life Technologies GmbH | 15140122 | picking buffer |
| EDTA | Roth | 8040.1 | picking buffer |
| Name | Company | Catalog Number | Comments |
| Immunohistochemistry | |||
| Purified anti-human CD326 (EpCAM) antibody clone 9C4 | BioLegend | 324201 | EpCAM immunohistochemistry (cf, fig 2C) |
| HRP rabbit anti-mouse IgG | Abcam | ab97046 | EpCAM immunohistochemistry (cf, fig 2C) |
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Citazione di questo articolo
Kochall, S., Thepkaysone, M., García, S. A., Betzler, A. M., Weitz, J., Reissfelder, C., Schölch, S. Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer. J. Vis. Exp. (125), e55357, doi:10.3791/55357 (2017).