3维细胞培养系统研究膀胱癌侵袭和评价治疗方法
Summary
控制膀胱癌的过程代表了生物标志物和治疗发展的机会。在这里, 我们提出了一个膀胱癌入侵模型, 其中包括3维的肿瘤球体文化, 时间推移成像和共焦显微术。该技术可用于定义侵入过程的特征和筛选治疗药物。
Abstract
膀胱癌是一个重要的健康问题。据估计, 今年美国将有超过1.6万人死于膀胱癌。虽然75% 的膀胱癌是无创性的, 而且不太可能转移, 但有25% 的进展是侵入型生长模式。多达半数的侵袭性癌症患者将会产生致命的转移性复发。因此, 了解膀胱癌侵袭性进展的机制对于预测患者预后和预防致死性转移是至关重要的。在本文中, 我们提出了一个三维的癌症入侵模型, 允许合并肿瘤细胞和基质成分, 模仿在体内的条件发生在膀胱肿瘤微环境。该模型提供了利用时间推移成像实时观察侵入过程的机会, 并利用共焦荧光成像和筛查化合物, 以阻断入侵的可能性, 对所涉及的分子通路进行审问。虽然本议定书的重点是膀胱癌, 但也有可能采用类似的方法来检查其他肿瘤类型的侵袭和运动。
Introduction
侵袭是癌症进展的一个关键步骤, 这是转移的需要, 并与较低的生存和预后较差的患者。在人类膀胱癌中, 泌尿道最常见的恶性肿瘤每年导致大约16.5万人死亡, 癌症分期、治疗和预后直接关系到入侵1的存在或缺失。大约75% 的膀胱癌病例为非肌肉侵入, 并以局部切除术进行管理。相反, 肌肉侵袭性膀胱癌 (约25% 的所有病例) 是积极的肿瘤, 转移率高, 并以积极的综合治疗2,3治疗。因此, 了解触发入侵的分子通路, 对于更好地表征侵入性进展的危险和发展能够预防侵入性进展的治疗干预是至关重要的。
肿瘤侵袭性进展发生在复杂的三维 (3) 环境中, 与其他肿瘤细胞、基质、基底膜和其他类型的细胞 (包括免疫细胞、成纤维细胞、肌肉细胞和血管) 相互作用。内皮细胞。渗透性支持 (例如Transwell) 检测系统通常用于定量癌细胞入侵4, 但这些系统是有限的, 因为它们不允许对入侵过程进行实时的显微监测和对样品进行进一步染色和分子分析的检索具有挑战性。研制一种3维膀胱肿瘤球体系统来研究入侵是可取的, 因为它允许在体外系统的方便的情况下纳入已定义的微环境成分。
在本议定书中, 我们描述了一个系统, 以检查人体膀胱癌细胞的侵袭过程, 使用3维球形入侵检测, 包括胶原基凝胶基质和共焦显微镜, 以允许调查人员监测细胞活力和实时入侵 (图 1A)。该系统是多才多艺的, 可以修改, 以询问各种基质/肿瘤的设置。它可以纳入大多数膀胱癌细胞系或原发性膀胱肿瘤和其他基质细胞, 如癌症相关的成纤维细胞和免疫单元5,6,7。该协议描述了由 type-1 胶原组成的基质, 但可以被修改以加入其他分子, 如纤维粘连蛋白, 层粘连蛋白, 或其他胶原蛋白。侵入过程可以遵循72小时或更长的时间取决于显微镜和系统使用的能力。3维基质中嵌入的肿瘤的固定和免疫荧光染色允许对侵入细胞中的蛋白质上调进行审问, 从而提供通常不存在或难以收集的关键信息。使用其他3维文化模型。该系统还可用于屏蔽入侵的化合物, 并描绘受此类化合物影响的信号通路。
Protocol
Representative Results
Discussion
在这里, 我们描述了一个3维的肿瘤球体模型, 允许实时观察膀胱癌侵袭, 这是至关重要的癌症进展和转移。该系统可以将各种基质和细胞成分结合在一起, 使研究者能够更好地复述发生膀胱癌侵袭的组织微环境。膀胱癌球体可以从各种来源, 如细胞系 (包括基因修饰细胞线, 用于检查影响侵袭过程的信号通路) 和小鼠原发性肿瘤 (这里描述)。它也有可能适应人类原发性肿瘤的分析。根据所使用的成像?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢霍华德-克劳福德博士 (密歇根大学) 实验室的技术支持, 为这项研究提供材料和设备, 艾伦凯莱赫技术支持。
这项工作由密歇根大学 Rogel 癌症中心核心赠款 CA046592-26S3、nih K08 CA201335-01A1 (进工党)、BCAN YIA (进工党)、nih R01 CA17483601A1 (DMS) 资助。
Materials
| Human bladder cancer cell lines UM-UC9, UM-UC13, UM-UC14, UM-UC18, 253J | |||
| DMEM cell culture medium | Thermo Fisher Scientific | 11995065 | |
| Fetal bovine serum | Thermo Fisher Scientific | 26140079 | |
| Antibiotic-Antimycotic (100X) | Thermo Fisher Scientific | 15240062 | |
| Trypsin-EDTA (0.25%), phenol red | Thermo Fisher Scientific | 25200056 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A3803 | |
| Phosphate-buffered saline (PBS), pH 7.4 | Thermo Fisher Scientific | 10010023 | |
| Costar Ultral-low attachment 6-well cluster | Corning | 3471 | |
| Conventional inverted microscope | Carl Zeiss | 491206-0001-000 | General use for cell culture and checking spheroids |
| Collagen type 1 from rat tail, high concentration | Corning | 354249 | |
| Nunc Lab-Tek II Chambered Coverglass | Thermo Fisher Scientific | 155382 | |
| Confocal microscope | Carl Zeiss | LSM800 | A confocal miscoscope with climate chamber, multi-location imaging, and Z-stack scanning function |
| Cryostat micromtome | Leica Biosystems | CM3050 S | |
| Zen 2 Image processing software | Carl Zeiss | ||
| Paraformaldehyde solution | Electron Microscopy Sciences | 15710 | |
| ImmEdge Hydrophobic Barrier PAP Pen | Vector Laboratories | H4000 | |
| O.C.T compound | Thermo Fisher Scientific | 23730571 | |
| Hoechst 33342 solution | Thermo Fisher Scientific | 62249 | |
| Anti-ATDC (Trim29) antibody | Sigma-Aldrich | HPA020053 | |
| Anti-Cytokeratin 14 antibody | Abcam | ab7800 | |
| Anti-Vimentin antibody | Abcam | ab24525 | |
| ProLong Diamond | Mounting medium |
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