研究癌症转移至肺部的永久窗口
1Department of Anatomy and Structural Biology,Einstein College of Medicine / Montefiore Medical Center, 2Gruss-Lipper Biophotonics Center,Einstein College of Medicine / Montefiore Medical Center, 3Department of Surgery,Einstein College of Medicine / Montefiore Medical Center, 4Integrated Imaging Program,Einstein College of Medicine / Montefiore Medical Cente, 5Department of Pathology,Einstein College of Medicine / Montefiore Medical Center
Summary
在这里，我们提出了一种用于为小鼠胸部手术植入永久留置光学窗口的方案，该方案可实现高分辨率的肺活体成像。窗口的持久性使其非常适合研究肺部的动态细胞过程，特别是那些缓慢进化的过程，例如播散性肿瘤细胞的转移性进展。
Abstract
转移占癌症相关死亡率的约90%，涉及癌细胞从原发性肿瘤到继发部位（如骨骼，大脑和肺）的全身扩散。尽管经过了广泛的研究，但这一过程的机制细节仍然知之甚少。虽然常见的成像方式，包括计算机断层扫描（CT），正电子发射断层扫描（PET）和磁共振成像（MRI），提供不同程度的粗略可视化，但每种方式都缺乏检测单个肿瘤细胞动力学所需的时间和空间分辨率。为了解决这个问题，已经描述了许多技术用于常见转移部位的活体成像。在这些部位中，肺已被证明特别难以进入活体成像，因为它的细腻性和维持生命的关键作用。虽然之前已经描述了几种用于完整肺的单细胞活体内成像的方法，但所有方法都涉及高度侵入性和终末期手术，将最大可能的成像持续时间限制在6-12小时。这里描述的是一种改进的技术，用于永久植入用于肺部高分辨率成像（WHRIL）的微创胸腔光学窗口。结合经过调整的显微血管造影方法，创新的光学窗口有助于在多个成像会话和数周内以单细胞分辨率对完整肺进行连续活体成像。鉴于可以收集成像数据的前所未有的时间，WHRIL可以促进加速发现转移进展的动态机制和肺部许多其他生物过程。
Introduction
造成~90%的死亡，转移是癌症相关死亡的主要原因1。在临床观察到的转移的主要部位（骨，肝，肺，脑）2中，肺已被证明对通过活体显微镜进行体内成像特别具有挑战性。这是因为肺是永动机中一个微妙的器官。肺部的连续运动，进一步由胸内心脏运动加剧，是准确成像的重要障碍。因此，由于其相对难以获得高分辨率活体内光学成像的模式，肺内的癌症生长通常被认为是一个隐匿的过程3。
在临床环境中，计算机断层扫描（CT），正电子发射断层扫描（PET）和磁共振成像（MRI）等成像技术可以在完整的重要器官（如肺）深处进行可视化4。然而，虽然这些模式提供了对大器官的良好观察（通常甚至在临床症状发作之前揭示病理学），但它们的分辨率不足以检测单个播散的肿瘤细胞，因为它们在转移的早期阶段进展。因此，当上述方式提供任何肺转移迹象时，转移灶已经建立并增殖。由于肿瘤微环境在癌症进展和转移形成5、6中起着举足轻重的作用，因此对研究体内转移性播种的最早步骤有很大的兴趣。这种兴趣进一步被进一步激发了人们的意识，即癌细胞甚至在检测到原发性肿瘤之前传播7，8，并且越来越多的证据表明它们作为单个细胞存活并且处于休眠状态数年到数十年，然后才生长成宏观转移9。
以前，单细胞分辨率下的肺成像必然涉及离体或外植体制剂10，11，12，13，将分析限制在单个时间点。虽然这些制剂确实提供了有用的信息，但它们并没有提供任何关于连接到完整循环系统的器官内肿瘤细胞动力学的见解。
最近在成像方面的技术进步使得在长达12小时14，15，16的时间内以单细胞分辨率对完整肺进行活体内可视化。这是在小鼠模型中完成的，使用涉及机械通气，胸腔切除术和真空辅助肺固定的方案。然而，尽管提供了生理完整肺的第一个单细胞分辨率图像，但该技术具有高度侵入性和终末性，从而排除了索引程序之外的进一步成像过程。因此，这种限制阻止了其应用于研究需要超过12小时的转移步骤，例如休眠和生长的重新启动14，15，16。此外，使用这种成像方法观察到的细胞行为模式必须谨慎解释，因为真空诱导的压差可能导致血流转移。
为了克服这些限制，最近开发了一种用于肺部高分辨率成像的微创窗口（WHRIL），便于在数天至数周的延长时间内进行连续成像，而无需机械通气17。该技术需要创建一个具有密封胸腔的”透明胸腔”，以保持正常的肺功能。该过程具有良好的耐受性，允许小鼠恢复而不会对基线活动和功能进行有意义的改变。为了在每次相应的成像过程中可靠地定位完全相同的肺区域，将一种称为显微刻图的技术应用于该窗口18。通过这个窗口，可以捕获细胞到达肺血管床，穿过内皮，经历细胞分裂并生长成微转移的图像。
在这里，该研究详细介绍了用于植入WHRIL的改进手术方案，该方案简化了手术，同时提高了其可重复性和质量。虽然该协议旨在能够研究转移背后的动态过程，但该技术可以替代地应用于肺生物学和病理学的众多过程的研究。
Protocol
本议定书中描述的所有程序均按照脊椎动物使用的指南和法规进行，包括阿尔伯特爱因斯坦医学院机构动物护理和使用委员会的事先批准。 1. 窗户钝化 用1%（w / v）酶活性洗涤剂溶液冲洗光学窗框（补充图2）。 在玻璃罐内，将光学窗框浸没在5%（w / v）氢氧化钠溶液中，在70°C下30分钟。 取下并用去离子水清洗窗框。 在新的玻璃罐内…
Representative Results
图1总结了本方案中描述的外科手术步骤并进行了说明。简而言之，在手术前，麻醉小鼠并去除左胸部的毛发。对小鼠进行插管和机械通气，以便在胸腔破裂后存活。切除覆盖肋骨的软组织，并产生一个小的圆形缺陷，横跨第6和第7肋骨。将光学窗框插入缺陷中，其底部（透明孔外）粘附在肺组织上。然后用缝合线和粘合剂的组合固定窗框，重新密封胸腔，并…
Discussion
在肺等远处转移部位，高分辨率光学成像可以深入了解肿瘤细胞转移的复杂动力学。通过实现单个癌细胞及其与宿主组织的相互作用的 体内 可视化，高分辨率的活体成像已被证明有助于理解转移的潜在机制。
这里描述的是一种改进的手术方案，用于永久胸腔植入光学窗口，旨在通过高分辨率多光子显微镜对小鼠肺进行连续成像。使用该协议创建的窗口具有良好的耐受…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了以下资助的支持：CA216248，CA013330，Montefiore的Ruth L. Kirschstein T32培训补助金CA200561，METAvivor早期职业奖，Gruss-Lipper生物光子学中心及其综合成像计划，以及Jane A.和Myles P. Dempsey。我们要感谢爱因斯坦医学院的分析成像设施（AIF）的成像支持。
Materials
| 1% (w/v) solution of enzyme-active detergent | Alconox Inc | N/A | concentrated, anionic detergent with protease enzyme for manual and ultrasonic cleaning |
| 2 µm fluorescent microspheres | Invitrogen | F8827 | |
| 5 mm coverslip | Electron Microscopy Sciences | 72296-05 | |
| 5% (w/v) solution of sodium hydroxide | Sigma-Aldrich | S8045 | |
| 5% Isoflurane | Henry Schein, Inc | 29405 | |
| 5-0 braided silk with RB-1 cutting needle | Ethicon, Inc. | 774B | |
| 7% (w/v) solution of citric acid | Sigma-Aldrich | 251275 | |
| 8 mm stainless steel window frame | N/A | N/A | Custom made, Supplementary Figure 2 |
| 9 cm 2-0 silk tie | Ethicon, Inc. | LA55G | |
| 5 mm disposable biopsy punch | Integra | 33-35-SH | |
| Blunt micro-dissecting scissors | Roboz | RS-5980 | |
| Buprenorphine | Hospira | 0409-2012-32 | |
| Cautery pen | Braintree Scientific | GEM 5917 | |
| Chlorhexidine gluconate | Becton, Dickinson and Company | 260100 | ChloraPrep Single swabstick 1.75 mL |
| Compressed air canister | Falcon | DPSJB-12 | |
| Cyanoacrylate adhesive | Henkel Adhesives | LOC1363589 | |
| Fiber-optic illuminator | O.C. White Company | FL3000 | |
| Bead sterilizer | CellPoint Scientific | GER 5287-120V | Germinator 500 |
| Graefe forceps | Roboz | RS-5135 | |
| Infrared heat lamp | Braintree Scientific | HL-1 | |
| Insulin syringes | Becton Dickinson | 329424 | |
| Isoflurane vaporizer | SurgiVet | VCT302 | |
| Jacobson needle holder with lock | Kalson Surgical | T1-140 | |
| Long cotton tip applicators | Medline Industries | MDS202055 | |
| Nair | Church & Dwight Co., Inc. | 40002957 | |
| Neomycin/polymyxin B/bacitracin | Johnson & Johnson | 501373005 | Antibiotic ointmen |
| Ophthalmic ointment | Dechra Veterinary Products | 17033-211-38 | |
| Paper tape | Fisher Scientific | S68702 | |
| Murine ventilator | Kent Scientific | PS-02 | PhysioSuite |
| Rectangular Cover Glass | Corning | 2980-225 | |
| Rodent intubation stand | Braintree Scientific | RIS 100 | |
| Small animal lung inflation bulb | Harvard Apparatus | 72-9083 | |
| Stainless steel cutting tool | N/A | N/A | Custom made, Supplementary Figure 1 |
| Sulfamethoxazole and Trimethoprim oral antibiotic | Hi-Tech Pharmacal Co. | 50383-823-16 | |
| SurgiSuite Multi-Functional Surgical Platform for Mice, with Warming | Kent Scientific | SURGI-M02 | Heated surgical platform |
| Tracheal catheter | Exelint International | 26746 | 22 G catheter |
| Vacuum pickup system metal probe | Ted Pella, Inc. | 528-112 |
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Citar este artigo
Borriello, L., Traub, B., Coste, A., Oktay, M. H., Entenberg, D. A Permanent Window for Investigating Cancer Metastasis to the Lung. J. Vis. Exp. (173), e62761, doi:10.3791/62761 (2021).