临床前小鼠模型的外科手术和方法<em> De Novo</em>乳腺癌转移
Summary
评估靶向乳腺癌转移的辅助治疗的临床前模型缺乏。为了解决这个问题，我们开发了从头肺乳腺癌转移，其特征在于，在辅助设定（后手术切除原发肿瘤的）给药的治疗可以为疗效影响先前接种的肺转移进行评估的鼠模型。
Abstract
A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.
Introduction
北美地区的女性患乳腺癌的终生风险约为12％ 2 ;大多数这些个体将通过手术去除原发性肿瘤，并且依赖于癌症亚型，然后将在佐剂设置中接受靶向的内分泌，化疗和/或放射治疗3 。诊断为接受抗雌激素治疗的激素受体阳性癌症妇女和接受HER2靶向治疗的HER2阳性肿瘤的妇女与放射治疗/化疗相关的实例包括：三重阴性肿瘤尚无靶向治疗3 。尽管在放疗，化疗，个性化和基于激素疗法的进步是补充手术切除，复发的疾病诊断为II期或III期疾病有4名妇女的30％-70％，由于治疗是在远处器官转移根除疾病很大程度上是无效的，包括肺，骨，b雨和/或肝脏5 。这是特别重要的，因为当没有原发性肿瘤再生长时发生转移性疾病时，这意味着在确定性手术时传播的恶性细胞可能已经存在于二级器官中。因此迫切需要能够消除或减缓转移性肿瘤生长的治疗方法。
虽然乳腺癌发生的从头鼠模型在揭示调节肿瘤进展1的机制方面已经非常显着，但现有模型也有一些限制。其中之一是事实， 从头转基因模型通常在多个乳腺中发展原发性肿瘤，其中原发性肿瘤负担限制了研究的持续时间。虽然原发性肿瘤细胞逃逸和转移性种子可能在这些模型的肿瘤进展早期发生，但是转移性肿瘤的坦率发展发生在晚期，而且依赖于n小鼠模型和应变背景，经常部分渗透1 。这进一步限制了从头模型发现调节继发器官转移的分子的效用，以及用于评估治疗剂在辅助治疗中的临床前功效。
为了规避这些局限性，我们开发了一种乳腺癌转移到肺的从头自体模型。携带晚期从头乳腺肿瘤的父母转基因雌性（ 即 ，本文所述用于研究的FVB / n株系背景上的MMTV-PyMT）老化至〜100天6 ，此时，其原发性肿瘤经手术切除并酶解解为单细胞悬液悬浮液（1×10 6个细胞）又原位移植到6-7周龄的受体同基因雌性小鼠中，其中单一原发性乳腺肿瘤发生在38至60天的时间段<strong class ="“xfig”">图1A）。在确定的肿瘤大小（172至450mm 3 ）处，将受体小鼠麻醉并手术切除原发性肿瘤，使得在手术部位的肿瘤再生长被最小化，与女性的手术一致（ 补充图1 ）。在FVB / n株系背景下，小鼠在手术后〜115天内以45％的外显率在肺中发展组织学检测的转移灶（ 图1B ）。随着转移性肿瘤生长的这种延长的延迟，该模型在辅助治疗递送中是独特的，并且用于阐明和评估影响原发性肿瘤手术切除后的转移进展的潜在生物学。
Protocol
Representative Results
Discussion
修改和故障排除：
当钝器从腹壁解剖肿瘤时，肿瘤可能仍然粘附在腹壁上。在<5％注射肿瘤的小鼠中观察到这种情况（数据未显示）。对于具有粘附于腹壁的肿瘤的小鼠，应当对小鼠进行安乐死，因为切除难度而没有原发性肿瘤再生长。
模型/技术的限制：
除了肺外，其他研究者报告除了肺外，分布在肝?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢Jo Hill的组织病理学帮助，John Gleysteen博士在外科技术方面的教学，Tessa Diebel的摄影辅助，Wong和Coussens实验室的所有成员进行重要的洞察和讨论，以及OHSU Knight Cancer Institute的财务支持。作者承认T32GM071388-10和T32CA106195-11支持CEG，NCI / NIH，国防部乳腺癌研究计划，苏珊·戈曼斯基金会，乳腺癌研究基金会，以及一个支持癌症 – Lustgarten基金会胰腺癌融合梦想团队转化研究基金（SU2C-AACR-DT14-14）向LMC提供MHW颁发基金奖的妇女健康圈，以及Brenden-Colson对MHW和LMC的胰腺健康中心。
Materials
| Isofluorane | Piramal Healthcare | N/A | Prescription order |
| Collagenase A | Roche | 11088793001 | |
| DNase I | Roche | 10104159001 | |
| DMEM | ThermoFisher | 12634010 | |
| 25 mL Pyrex bottle | Sigma-Aldrich | CLS139525 | |
| Fetal Bovine Serum | Atlanta Bio | S11150 | |
| 0.7 µm nylon strainer | Corning | 352350 | |
| 50 mL conical tube | VWR | 89039-658 | |
| Dimethyl sulfoxide | Sigma-Aldrich | D2650 | |
| Growth factor-reduced Matrigel | BD | 354230 | Growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma |
| Poly(vinylpyrrolidone)–Iodine complex | Sigma-Aldrich | PVP1 | |
| 29 gauge 0.3 mL insulin syringe | BD | 324702 | |
| Small Vessel Cauterizer Kit | FST | 18000-00 | |
| Wound clips | Texas Scientific | 205016 | |
| AutoClip wound clip applier | BD | 427630 | |
| AutoClip wound clip remover | BD | 427637 | |
| Bromodeoxyuridine | Roche | 10280879 | |
| Heparinized capillary tubes | Fisher | 22362566 | |
| Microtainer tubes with dipotassium EDTA | BD | 365974 | |
| 20 mL syringe | BD | 309661 | |
| DPBS | Thermo-Fisher | 14190-250 | |
| OCT-freezing medium | VWR | 25608930 | |
| Formalin, Buffered, 10% (Phosphate Buffer) | Fisher | SF100-4 | |
| 23g needle | Fisher | 14-826-6B | |
| FVB/n mouse | Jackson Laboratories | 001800 |
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