利用活体中鸡胆膜研究妇科和泌尿癌
Summary
我们提出了鸡胆膜模型作为替代的，可移植的，体内模型，用于妇科和泌尿细胞系和患者衍生肿瘤的移植。
Abstract
小鼠模型是体内癌症研究的基准测试。然而，成本、时间和道德方面的考虑导致了对替代体内癌症模型的呼吁。鸡胆膜 （CAM） 模型提供了一种廉价、快速的替代方案，允许直接可视化肿瘤发育，并适用于体内成像。因此，我们寻求开发一个优化的协议，将妇科和泌尿肿瘤移植到这个模型中，我们在这里介绍。受精后约7天，空气细胞移动到卵子的血管侧，在壳中形成开口。然后，来自鼠和人体细胞系和原发组织的肿瘤可以移植。这些通常播种在细胞外基质和介质的混合物，以避免细胞分散，并提供营养支持，直到细胞招募血管供应。然后，肿瘤在卵子孵化前可能再长14天。通过植入用萤火虫荧光素酶稳稳地转导的细胞，生物发光成像可用于敏感检测膜上的肿瘤生长和整个胚胎的癌细胞。该模型可能用于研究肿瘤原性、入侵、转移和治疗效果。与传统的鼠种模型相比，鸡CAM模型需要的时间和财政资源要少得多。由于卵子具有免疫功能低下和免疫耐受性，任何生物体的组织都有可能被植入，而无需植入人体组织所需的昂贵的转基因动物（如小鼠）。然而，该模型的许多优点也可能有局限性，包括肿瘤生成时间短和免疫功能低下/免疫耐受状态。此外，虽然这里呈现的所有肿瘤类型在鸡胆膜模型中，它们这样做与肿瘤生长的程度不同。
Introduction
老鼠是研究人类疾病（包括恶性肿瘤）的经典模型有机体。作为哺乳动物，它们与人类有许多相似之处。它们的遗传相似性很高，使得转基因操作小鼠基因组提供了对人类疾病基因控制的巨大洞察力。在小鼠的处理和实验方面拥有丰富的经验，因此它们成为生物医学研究的首选模式。然而，除了伦理和科学的关注，关于鼠模型，他们也可以是相当昂贵和耗时2，3。肿瘤的发展可能需要数周甚至数月的时间。仅在一个典型的机构的住房可以运行在数百到数千美元，而肿瘤正在发展。卵巢癌是这个缺点的一个例子，因为它在鼠模型中的生长很容易需要几个月。研究进展的延迟可能会影响卵巢癌患者持续低的5年生存率只有47%（即，在30年内只增加10%的存活率）4。同样，泌尿癌（肾癌、前列腺癌和膀胱癌）占美国所有癌症病例的19%，占癌症相关死亡的11%4。因此，研究妇科和泌尿癌的新型体内方法可以节省实验室大量的时间、人力和资金，即使这种模式只应用于最初的筛选实验。此外，由此带来的研究结果的加速可能对每年被诊断患有这些癌症的177，000人产生重大影响。
鸡CAM模型提供了许多优势，解决上述问题。研究血管生成5，6，肿瘤细胞入侵7，8，和转移7，9，小鸡胚胎CAM模型已经用于研究多种形式的癌症，包括胶质瘤10，11，12，头颈部鳞状细胞癌13，14，白血病15，16，胰腺癌17，结肠直肠癌18。此外，CAM模型已经生成神经母细胞瘤19，伯基特淋巴瘤20，黑色素瘤21，和猫纤维肉瘤22。先前的研究也提出了膀胱癌23和前列腺癌细胞系24的移植，但协议细节有限。鸡蛋不仅比老鼠便宜得多，而且能产生高度可重复的结果25，26。它们显示血管发育迅速，肿瘤移植可在几天内发生，并通过打开的窗口纵向可视化。卵子受精和孵化之间的21天时间，实验可以在几周内完成。此外，成本低、住房需求有限、面积小，很容易进行大型实验，使小鼠研究望而却步。
因此，我们寻求优化CAM模型，用于妇科和泌尿癌的移植。由于早期鸡胚胎27的免疫功能低下，小鼠和人体细胞都可以轻松植入。因此，我们已经成功地移植了卵巢癌、肾癌、前列腺癌和膀胱癌。对于每种肿瘤类型，CAM 欣然接受已建立的小鼠和/或人类肿瘤细胞系。重要的是，新收获的原发性人类肿瘤组织也可以从消化细胞或固体组织碎片中移植，成功率高。每种癌症类型和细胞源都需要优化，我们在这里分享。
Protocol
此处介绍的所有实验都经过加州大学洛杉矶分校 （UCLA） 相关道德委员会的审查和批准。UCLA 机构审查委员会已批准使用非识别的初级人类肿瘤（协议编号 17-000037、17-001169 和 11-001363）。在加州大学洛杉矶分校，动物研究委员会审查不需要使用鸡胚胎的实验;仅当卵子孵化时，才需要协议批准。然而，最佳实践，如AVMA动物安乐死指南，被用来处理鸡胚胎的道德和尽可能避免疼痛。在使用 CAM 模型进?…
Representative Results
到目前为止，我们发现这种植入方法对于卵巢癌、肾癌、前列腺癌和膀胱癌是成功的。每种产品都经过优化，以确定植入的特定条件，尽管可能具有灵活性。在已测试的肿瘤类型中，卵巢癌的生长远没有那么明显，如果没有生物发光成像的帮助，通常不可见（图1）。然而，在植入区域，用钳子可以感觉到CAM的僵硬。这可能有助于确定在缺乏生物发光…
Discussion
使用CAM模型的肿瘤扩张和移植允许比体内动物模型更快速和直接地观察到肿瘤生长。此外，一旦完成设备的初始购买，成本就会大大降低，特别是与免疫功能低下小鼠的成本相比。鸡胚胎的初始免疫功能低下状态很容易允许人体和鼠群组织移植。即使有这些优势，CAM 模型也有局限性。如果有必要进行长期治疗研究，短期可能是有益的，也可能有害。CAM模型的免疫功能低下/免疫耐受状态可能使肿?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢Tamanoi博士和Binh Vu博士对这种方法的初步培训。与Eva Koziolek博士的讨论有助于优化这种方法，并对此非常赞赏。如果没有以下来源的资助，这项工作就不可能进行：烟草相关疾病研究计划博士后研究金（27FT-0023， 到ACS，国防部（国防部）卵巢癌研究计划（W81XWH-17-1-0160），NCI/NIH（1R21CA216770），烟草相关疾病研究计划高影响试点奖（27IR-0016）和加州大学洛杉矶分校机构支持， 包括JCCC种子赠款（NCI/NIH P30CA016042）和从研究部副部长办公室到LW的3R补助金。
Materials
| -010 Teflon (PTFE) White 55 Duro Shore D O-Rings | The O-Ring Store | TEF010 | Nonstick ring for cell seeding. 1/4"ID X 3/8"OD X 1/16"CS Polytetrafluoroethylene (PTFE). |
| C4-2 | ATCC | CRL-3314 | Human prostate cancer cell line. |
| CWR22Rv1 | CWR cells were the kind gift of Dr. David Agus (Keck Medicine of University of Southern California) | ||
| Cytokeratin 8/18 Antibody (C-51) | Novus Biologicals | NBP2-44929-0.02mg | Used at a dilution of 1:100 for immunohistochemical analysis of human ovarian CAM tumors. |
| D-Luciferin Firefly, potassium salt | Goldbio | LUCK-1G | |
| Delicate Operating Scissors; Curved; Sharp-Sharp; 30mm Blade Length; 4-3/4 in. Overall Length | Roboz Surgical | RS6703 | This is provided as an example. Any similar curved scissors would work as well. |
| Dremel 8050-N/18 Micro 8V Max Tool Kit | Dremel | 8050-N/18 | This kit contains all necessary tools. |
| Fertilized chicken eggs (Rhode Island Red – Brown, Lab Grade) | AA Lab Eggs Inc. | N/A | A local egg supplier would need to be identified, as this supplier only delivers regionally. |
| HT-1376 | ATCC | CRL-1472 | Human bladder cancer cell line. |
| Hovabator Genesis 1588 Deluxe Egg Incubator Combo Kit | Incubator Warehouse | HB1588D-NONE-1102-1588-1357 | Other egg incubators may be used, but their reliability would need to be verified. After implantation, a cell incubator with the CO2 disabled may also be used. |
| ID8 | Not commercially available, please see PMID: 10753190. | ||
| Incu-Bright Cool Light Egg Candler | Incubator Warehouse | 1102 | Other candlers may be used; however, this is preferred among those that we have tested. This candler is included in the aforementioned incubator kit. |
| Iris Forceps, 10cm, Curved, Serrated, 0.8mm tips | World Precision Instrument | 15915 | This is provided as an example. Any similar curved forceps would work as well. Multiple brands have been used for this method. |
| Isoflurane | Clipper Distributing | 0010250 | |
| IVIS Lumina II In Vivo Imaging System | Perkin Elmer | ||
| Matrigel Membrane Matrix HC; LDEV-Free | Corning | 354248 | Extracellular matrix solution |
| MyC-CaP | ATCC | CRL-3255 | Murine prostate cancer cell line. |
| Portable Pipet-Aid XP Pipette Controller | Drummond Scientific | 4-000-101 | Any similar pipet controller would be appropriate. |
| PrecisionGlide Hypodermic Needles | BD | 305196 | This is provided as an example. Any 18G needle would work similarly. |
| RENCA | ATCC | CRL-2947 | |
| Semken Forceps | Fine Science Tools | 11008-13 | This is provided as an example. Any similar forceps or another style that suits researcher preference would be appropriate. |
| SKOV3 | ATCC | HTB-77 | Human ovarian cancer cell line. |
| Specimen forceps | Electron Microscopy Sciences | 72914 | This is provided as an example. The forceps used for pulling away the shell for bioluminescence imaging are approximately 12.8 cm long with 3 mm-wide tips. |
| Sterile Cotton Balls | Fisherbrand | 22-456-885 | This is provided as an example. Any sterile cotton balls would suffice. |
| Stirring Rods with Rubber Policeman; 5mm diameter, 6 in. length | United Scientific Supplies | GRPL06 | This is provided as an example. Any similar glass stir rods would work as well. |
| T24 | ATCC | HTB-4 | Human bladder cancer cell line. |
| Tegaderm Transparent Dressing Original Frame Style 2 3/8" x 2 3/4" | Moore Medical | 21272 | |
| Tissue Culture Dishes, 10 cm diameter | Corning | 353803 | This is provided as an example. Any similar, sterile 10-cm dish may be used. Tissue culture treatment is not necessary. |
| Tygon Clear Laboratory Tubing – 1/4 x 3/8 x 1/16 wall (50 feet) | Tygon | AACUN017 | This is provided as an example. Any similarly sized tubing would work as well. |
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Cite This Article
Sharrow, A. C., Ishihara, M., Hu, J., Kim, I. H., Wu, L. Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers. J. Vis. Exp. (155), e60651, doi:10.3791/60651 (2020).