一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型
Summary
我们介绍了一种在有机结构脑切片模型中测量乳腺癌脑转移细胞的实时药物和辐射反应的方案。这些方法提供了一种定量测定法，以研究各种治疗对脑微环境界面内乳腺癌脑转移瘤的治疗效果。
Abstract
脑转移瘤是乳腺癌对女性的严重后果，因为这些肿瘤难以治疗，并且与不良的临床结果有关。乳腺癌脑转移（BCBM）生长的临床前小鼠模型是有用的，但价格昂贵，并且很难跟踪活细胞和肿瘤细胞在脑实质内的侵袭。这里介绍的是来自异种移植小鼠的 离体 脑切片培养方案，其中包含颅内注射的乳腺癌寻脑克隆亚线。 将MDA-MB-231BR 荧光素酶标记的细胞颅内注射到Nu / Nu雌性小鼠的大脑中，肿瘤形成后，将大脑分离，切片并 离体培养。对肿瘤切片进行成像以鉴定表达荧光素酶的肿瘤细胞，并监测它们在脑实质中的增殖和侵袭长达10天。此外，该协议描述了使用延时显微镜对电离放疗或化疗后肿瘤细胞的生长和侵袭行为进行成像。肿瘤细胞对治疗的反应可以通过实时成像显微镜可视化，测量生物发光强度，并在含有BCBM细胞的脑切片上进行组织学。因此，这种 离体 切片模型可能是一个有用的平台，用于单独或与辐射联合使用新型治疗剂的快速测试，以鉴定个性化药物，以靶向个体患者的脑微环境中的乳腺癌脑转移生长。
Introduction
当细胞从原发性乳腺肿瘤扩散到大脑时，乳腺癌脑转移瘤（BCBM）就会发展。乳腺癌是仅次于肺癌的第二大脑转移原因，转移发生在10-16%的患者中1。不幸的是，脑转移瘤仍然无法治愈，因为>80%的患者在脑转移诊断后一年内死亡，并且由于神经功能障碍，他们的生活质量受损2。迫切需要确定更有效的治疗方案。单层二维或三维培养模型是在实验室中测试治疗剂最常用的方法。然而，它们不能模仿复杂的BCBM微环境，这是肿瘤表型和生长的主要驱动因素。虽然这些模型是有用的，但它们不能捕获复杂的肿瘤 – 基质相互作用，独特的代谢需求以及肿瘤的异质性3。为了更忠实地概括肿瘤 – 基质相互作用和微环境异质性，我们的小组和其他人已经开始用患者来源的肿瘤细胞（原发性或转移性）或癌细胞系4，5，6产生有机指型脑转移“切片”培养物。与传统的 体外 系统相比，这种短期 离体 模型可能为在大型动物队列的临床前评估之前筛选新疗法提供更多相关条件。
已经构建并成功用于鉴定各种癌症的成功治疗。它们需要几天的评估，此外还可以根据患者特定的药物筛查进行定制。例如，人膀胱癌和前列腺癌的离体组织已经显示出多西紫杉醇和吉西他滨7的剂量依赖性抗肿瘤反应。开发了类似的结直肠癌离体组织来筛选化疗药物奥沙利铂，西妥昔单抗和Pembrolizumab8。该应用已被广泛用于胰腺癌，考虑到基质环境与胰腺导管腺癌的基因型和表型特征之间的基本相互作用9，10。此外，已经开发了这种有机细胞模型，用于头，颈，胃和乳腺肿瘤的类似筛查11，12。
在这里，正在产生异种移植乳腺癌脑转移性肿瘤细胞在其微环境中的 离体 脑切片模型。将小鼠颅内注射乳腺癌脑转移性脑营养MDA-MB-231BR细胞13 在脑皮层顶叶 – TNBC转移的常见部位14，15 并允许发展肿瘤。从这些异种移植动物中产生脑切片，并作为有机细胞培养物进行 体外 保存，如16，17所述。这种新颖的 离体 模型可以分析BCBM细胞在脑实质内的生长，并可用于测试治疗剂和对脑微环境中肿瘤细胞的辐射作用。
Protocol
该协议已获得批准，并遵循德雷塞尔大学医学院机构动物护理和使用委员会（IACUC）的动物护理指南。本研究使用Nu / Nu无胸腺炎雌性小鼠（6-8周龄）。 1. 颅内注射肿瘤细胞 在高压灭菌器的干燥循环下，在灭菌袋中对所有设备（镊子，剪刀，缝合剪刀，手钻）进行灭菌长达45分钟的灭菌，包括灭菌指示剂。如果对多只动物进行手术，请使用加热的珠子灭菌器（最多3次）…
Representative Results
如上所述，将MDA-MB-231BR-GFP-荧光素酶细胞颅内注射到4-6周龄Nu / Nu小鼠的右半球（图1A）并允许生长12-14天，在此期间通过生物发光成像监测肿瘤生长（图1B）。正如其他小组19所报告的那样，我们在颅内注射了100，000个癌细胞，但有可能注射低至20，000个细胞20。如上所述，在脑切片生成（图1C）?…
Discussion
本研究建立了一种新的外植异种移植脑肿瘤的 离体 脑培养方法。我们表明，颅内注射在小鼠大脑中的BCBM细胞MDA-MB-231BR细胞可以在 离体 脑切片中存活和生长。该研究还测试了颅内注射的U87MG胶质母细胞瘤（GBM）细胞，还发现这些癌细胞在脑切片中存活和生长（数据未显示）。我们相信这个模型可以从BCBM和GBM扩展到其他容易转移到大脑的癌症，包括肺癌和黑色素瘤。选择颅内注射乳?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢Julia Farnan，Kayla Green和Tiziana DeAngelis的技术援助。这项工作得到了宾夕法尼亚州联邦通用研究增强资助计划（MJR，JGJ），UO1CA244303（MJR），R01CA227479（NLS），R00CA207855（EJH）和W.W.史密斯慈善信托基金（EjH）的部分支持。
Materials
| 1 mL syringe, slip tip | BD | 309659 | |
| 30 G1/2 Needles | BD | 305106 | |
| 6-well plates | Genessee | 25-105 | |
| Automated microscope and LUMAVIEW software | Etaluma | LS720 | |
| B27 (GEM21) | Gemini Bio-Products | 400-160 | |
| Beaker 50 mL | Fisher | 10-210-685 | |
| Blunt sable paintbrush, Size #5/0 | Electron Microscopy Sciences | 66100-50 | |
| Bone Wax | ModoMed | DYNJBW25 | |
| Brain injection Syringe | Hamilton Company | 80430 | |
| CaCl2 | Fisher Scientific | BP510-250 | |
| Cleaved caspase 3 Antibody | Cell Signaling | 14220S | |
| DAPI | Invitrogen | P36935 | |
| D-Luciferin Potassium Salt | Perkin Elmer | 122799 | |
| Double edge razor blade | VWR | 55411-060(95-0043) | |
| Filter Paper (#1), quantitative circles, 4.25 cm | Fisher | 09-805a (1001-042) | |
| Fine sable paintbrush #2/0 | Electron Microscopy Sciences | 66100-20 | |
| Forceps | Fine Science Tools | 11251-20 | |
| Gamma-H2AX antibody | Millipore | 05-636 | |
| GFAP antibody | Thermo Fisher | 13-0300 | |
| GFP antibody | Santa Cruz | SC-9996 | |
| Glucose | Sigma Aldrich | G8270 | |
| Glutamine (200 mM) | Corning cellgrow | 25-005-Cl | |
| H&E and KI-67 | Jefferson Core Facility Pathology staining | ||
| Hand Drill Set with Micro Mini Twist Drill Bits | Amazon | YCQ2851920086082DJ | |
| HEPES, free acid | Fisher Scientific | BP299-1 | |
| Just for mice Stereotaxic Frame | Harvard Apparatus (Holliston, MA, USA). | 72-6049, 72-6044 | |
| KCl | Fisher Scientific | S271-10 | |
| Large surgical scissors | Fine Science Tools | 14001-18 | |
| MDA-MB-231BR cells | Kindly provided by Dr. Patricia Steeg | Ref 14 | |
| MgCl2·6H2O | Fisher Scientific | M33-500 | |
| Mice imaging device | Perkin Elmer | IVIS 200 system | |
| Mice imaging software | Caliper Life Sciences (Waltham, MA, USA). | Living Image Software | |
| Microplate Reader | Tecan Spark | ||
| Mounting solution | Invitrogen | P36935 | |
| MTS reagent | Promega CellTiter 96 Aqueous One Solution | (Cat:G3582) | |
| N2 supplement | Life Technologies | 17502-048 | |
| Neurobasal medium | Life Technologies | 21103049 | |
| Nu/Nu athymic mice | Charles Rivers Labs (Wilmington, MA, USA) | ||
| Paraformaldehyde | Affymetrix | 19943 | |
| Pen/Strep | Life Technologies | 145140-122 | |
| Polypropylene Suture | Medex supply | ETH-8556H | |
| Povidone Iodine Swab sticks | DME Supply USA | Cat: 689286X | |
| Scalpel blade #11 (pk of 100) | Fine Science Tools | 10011-00 | |
| Scalpel handle #3 | Fine Science Tools | 10003-12 | |
| Sodium Pyruvate | Sigma Aldrich | S8636 | |
| Spatula/probe | Fine Science Tools | 10090-13 | |
| SS Double edge uncoated razor blades (American safety razor co (95-0043)) | VWR | 55411-060 | |
| Sucrose | Amresco | 57-50-1 | |
| Surgical Scalpel | Exelint International | D29702 | |
| Tissue Chopper | Brinkman | (McIlwain type) | |
| Tissue culture inserts | Millipore | PICMORG50 or PICM03050 | |
| X-ray machine | Precision 250 kVp |
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Cite This Article
Ciraku, L., Moeller, R. A., Esquea, E. M., Gocal, W. A., Hartsough, E. J., Simone, N. L., Jackson, J. G., Reginato, M. J. An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth. J. Vis. Exp. (175), e62617, doi:10.3791/62617 (2021).