在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪
Summary
We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.
Abstract
高档浆液性卵巢癌（HGSC），广泛腹膜转移的原因，仍然有一个非常预后较差;妇女只有不到30％是活确诊5年后。大网膜是HGSC转移形成一个首选站点。尽管这样的微环境的临床重要性，网膜脂肪组织卵巢癌进展的贡献仍充分研究。网膜脂肪是不寻常的，因为它含有一种被称为乳斑，这是由B，T，以及NK细胞，巨噬细胞和祖细胞周围的脉管的稠密巢结构。乳斑起到网膜的生理功能，这需要进行腹膜稳态中起关键作用。我们已经表明，乳斑也促进腹膜脂肪的卵巢癌的转移性定植，在腹膜转移的发展中的关键步骤。在这里，我们描述的方法我们开发评估和量化每个乳斑itoneal脂肪和学习他们的体内和体外大网膜组织的卵巢癌细胞转移的殖民功能的贡献。这些方法推广到更多的小鼠模型和细胞系，从而使卵巢癌转移形成的研究，从细胞的初始定位，以乳白色斑结构的广泛腹膜转移的发展。
Introduction
不像大多数实体瘤，从高档浆液性卵巢癌（HGSC）转移限于腹膜腔1。因此，有效的腹腔疗法可能控制或消除HGSC。目前，标准的治疗方法是手术细胞减灭术联合化疗1-3。不幸的是，绝大多数患者体验并死于疾病复发的并发症。这些令人沮丧的统计数据显示，需要转移的殖民认识的提高，这个过程由癌细胞本地化，利用和扩散宿主组织内，形成转移灶2。
网膜是HGSC转移4-7的一个优选的早期站点。不像其他的腹膜脂肪，网膜脂肪组织含有称为乳斑，其含有B，T和NK细胞和巨噬细胞，这在腹膜homeos发挥关键作用不寻常的免疫结构TASIS 8,9。除了 他们的生理功能，我们发现，乳斑发挥卵巢癌转移定植4了积极的作用。在实验性转移测定法，SKOV3ip.1，CAOV3，和HeyA8（人）和ID8（鼠; C57BL / 6）的卵巢癌细胞迅速家乳斑，这表明细胞正在朝着分泌趋化因子。有趣的是，癌细胞不定居腹膜脂肪缺乏乳斑（ 即性腺和子宫脂肪）4。
为了鉴定调节乳斑定植机制，我们优化异种移植模型中，使过转移性定植4的时间过程的细胞和分子事件的询问。这里所描述的方法的具体优点是它强调组织结构和功能，使用户能够验证假设的完全融合在体内和 m的体外模型etastatic殖民4,10。通过在腹膜脂肪库其中或者含有或缺乏乳斑比较癌细胞本地化和生长，研究者可以测试脂肪细胞和细胞的乳斑的倒伏和卵巢癌的细胞中生理相关组织进行性生长中的相对贡献（多个）。
Protocol
Representative Results
Discussion
发展疗法靶向播散细胞需要一个机械理解转移性定植，在腹膜疾病发展的关键的第一步。为了解决这些问题，我们报告，可以用来辨别网膜独特的组织构成和架构如何促进卵巢癌转移定植的方法。我们的方法的显着特征是：1）专注于在殖民化过程中的早期事件;乳白色斑和含缺陷的脂肪库2）比较; 3）可重复技术的可用性，以定量的组织乳白色斑量; 体内互补和体外4）可?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Supported by grants from the Department of Defense (W81XWH-09-1-0127), the NIH (2-R01-CA089569), the Elsa U. Pardee Foundation, a Marsha Rivkin Center for Ovarian Cancer Research Pilot Study Award, and generous philanthropic support from Section of Urology and Section of Research in the Department of Surgery, University of Chicago.
Materials
| Dissection Tools | Fine Science Tools | NA | |
| Geimsa | Fluka/Sigma Aldrich | 48900 | |
| 5% Formalin | Sigma | HT501320 | |
| DMEM | Corning | 10-013-CV | |
| Trypsin | Gibco | 25200-056 | |
| PBS | Corning | 21-040-CV | Without calcium and Magnesium |
| 26 gauge needle | BD | 329652 | |
| BSA | Sigma | A7906 | |
| Collagenase | Worthington | LS004196 | |
| Stomacher | Seward Labsystems | Stomacher 80 Biomaster | |
| Microstomacher bag | Stomacher Lab Systems | BA6040/Micro | |
| ACK Lysis Buffer | Gibco | A10492-01 | |
| Millicell culture plate insert | Millipore | PICM01250 | |
| Cell-Tak | BD | 354240 |
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