诱导和诊断肿瘤<em>果蝇</em> Imaginal Disc Epithelia
Summary
果蝇成像盘上皮的马赛克克隆分析是研究肿瘤发生的遗传和细胞机制的有力模型系统。在这里，我们描述了使用GAL4-UAS系统在果蝇翼成像盘中诱导肿瘤的方案，并介绍了分类肿瘤表型的诊断方法。
Abstract
在癌症的早期阶段，转化的突变细胞显示细胞学异常，开始不受控制的过度生长，并逐渐破坏组织组织。 黑腹果蝇已成为癌症生物学中流行的实验模型系统，用于研究肿瘤发生的遗传和细胞机制。特别地，用于果蝇成像盘（在幼虫中发育的上皮）的遗传工具使得能够在正常上皮组织内形成转化的前肿瘤细胞，这种情况类似于人类癌症的初始阶段。然而，最近关于果蝇翼成像圆盘中的肿瘤发生的研究表明，肿瘤起始取决于组织固有的细胞结构和局部微环境，这表明在评估肿瘤表型的成像中考虑到致瘤性刺激的区域特异性易感性是重要的光盘。促进肿瘤进展的表型分析在成像光盘中，我们描述了使用GAL4-UAS系统在机翼成像光盘中诱导肿瘤性肿瘤的遗传实验方案。我们进一步介绍一种诊断方法来分类在上皮细胞中诱导的克隆性病变的表型，因为以前没有描述过用于区分肿瘤进展的各个阶段（如增生，发育不良或瘤形成）的明确分类方法。这些方法可能广泛适用于果蝇各种器官的肿瘤表型的克隆分析。
Introduction
上皮组织是高度组织的系统，具有显着的稳态能力，通过发育和细胞周转维持其组织。然而，这种健壮的自组织系统在肿瘤发育过程中逐渐被破坏。在肿瘤发展开始时，由癌基因激活或肿瘤抑制基因失活引起的单个突变细胞出现在上皮层内。当这种转化的“前肿瘤细胞”逃避抑制环境时，会破坏上皮组织，并开始不受控制的增殖，发生肿瘤发生1 。在过去几十年中，遗传学和分子生物学方面的杰出技术进步在癌症研究方面取得了显着进展。特别是最近在黑腹果蝇中使用遗传马赛克分析工具的研究 ，如FLP-FRT（flippase重组酶/翻转酶重组酶靶）有丝分裂重组通货膨胀2和翻转出-GAL4-UAS（上游激活序列）系统3，已经极大地促进了更好地理解参与了肿瘤4，5，6的形成和转移的遗传机制。
一组保守的果蝇肿瘤抑制基因， 致死巨幼虫 （ lgl ）， 圆盘大 （ dlg ）和涂鸦 （ scrib ）的研究突出了上皮组织损失和肿瘤发展之间的关键关系，因为这些基因发挥关键作用在上皮组织中调节顶端细胞极性和细胞增殖7 。虽然果蝇成像圆盘通常是单层上皮，这三个基因中的任何一个的纯合突变导致细胞失去结构和极性，不能形成差异过度膨胀，最终形成与相邻组织融合的多层无定形块7 。同样，这些基因在哺乳动物的破坏是参与恶性肿瘤8,9的发展。由突变体组织表现出的表型的肿瘤已导致这三个基因的分类为保守的，肿瘤性的肿瘤抑制基因（nTSGs）7,8。然而，当纯合nTSG突变细胞开发使用FLP-FRT-介导的有丝分裂重组的野生型成虫盘被零散地生成，突变的细胞从组织至c-Jun N-末端激酶（JNK） -依赖性的细胞凋亡10,11消除，12，13，14，15挤压</suP>， 如图16所示 ，或通过邻居17吞没和吞噬作用。在该镶嵌基因的上皮细胞凋亡主要是在位于克隆边界nTSG突变体细胞中检测到，这表明相邻正常细胞触发nTSG突变的细胞10，11，12，18的细胞凋亡。在哺乳动物细胞中最近的研究已经证实，有利于肿瘤细胞的这种细胞依赖性竞争淘汰是对抗癌症的19，20，21，22，23的进化上保守的上皮细胞的自我防御机制。
然而，最近对果蝇成像圆盘的研究表明，镶嵌nTSG敲低克隆诱导特异性的肿瘤性肿瘤机翼成像光盘的ic区域16 。初始肿瘤形成始终存在于外周“铰链”区域，并且从未在翼盘上皮的中央“囊”区域观察到，这表明nTSG敲低细胞的致瘤潜力取决于局部环境。中央袋区域作为“肿瘤冷点”，其中肿瘤细胞不显示发育不良的过度生长，而外周铰链区则表现为“肿瘤热点” 16 。在“冷点”小袋区域，nTSG敲低细胞从基底侧分层并进行细胞凋亡。相比之下，由于“热点”铰链细胞在其基底具有强大的细胞骨架结构网络，所以nTSG敲低细胞从上皮的顶端分层并引发致瘤性过度生长16 。因此，成像光盘中肿瘤表型的分析需要仔细考虑区域特异性致瘤性刺激易感性的降容。
在这里，我们描述了使用GAL4-UAS- RNAi系统在果蝇翼成像盘中诱导肿瘤肿瘤形成的方案，通过其在正常翼盘上皮细胞中产生nTSG敲低细胞。虽然这些实验系统对于研究癌症的早期阶段是有用的，但是尚未清楚地描述用于评估成像盘上皮细胞肿瘤进展阶段的明确分类方法。因此，我们还提出了一种将翼片上皮诱导的前肿瘤克隆表型分为三类：增生（增殖增多的正常出现细胞数量过多），异常增生（异常出现的恶化前组织细胞）和瘤形成（良性或恶性肿瘤由具有异常外观和异常增殖模式的细胞组成）。
Protocol
Representative Results
Discussion
的GAL4-UAS系统是用于在果蝇 26靶基因表达的最有力的遗传工具之一，极大地方便了肿瘤细胞的诱导和分析在体内 4。该系统能够产生携带敲低肿瘤抑制基因的克隆或在野生型上皮组织内癌基因的过度表达，这种情况与人类癌症的初始阶段非常相似，其中转化的前肿瘤细胞被正常上皮细胞包围。由GAL4增强子捕获系或Janelia GAL4系27等增…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢J. Vaughen对手稿的批判性阅读。这项工作得到了JSPS KAKENHI Grant号26891025,15H01500和武田科学基金会YT研究奖学金的资助
Materials
| Reagents | |||
| Phosphate buffered saline (PBS) | Wako | 162-19321 | |
| TritonX-100 | Wako | 168-11805 | |
| Formaldehyde | Wako | 064-00406 | |
| bovine serum albumin | Sigma | A7906 | |
| normal goat serum | Sigma | G6767 | |
| mounting medium, Vectashield | Vector Laboratories | H-1000 | |
| DAPI | Sigma | D9542 | |
| mouse-anti-Dlg 4F3 | Developmental Studies Hybridoma Bank | 4F3 anti-discs large, RRID:AB_528203 | dilute in PBTG, 1:40 |
| mouse-anti-MMP1 | Developmental Studies Hybridoma Bank | 3A6B4, RRID:AB_579780 | 3 mixed 1:1:1 and dilute in PBTG, 1:40 |
| mouse-anti-MMP1 | Developmental Studies Hybridoma Bank | 3B8D12, RRID:AB_579781 | 3 mixed 1:1:1 and dilute in PBTG, 1:40 |
| mouse-anti-MMP1 | Developmental Studies Hybridoma Bank | 5H7B11, RRID:AB_579779 | 3 mixed 1:1:1 and dilute in PBTG, 1:40 |
| mouse-anti-atubulin | Developmental Studies Hybridoma Bank | AA4.3, RRID:AB_579793 | dilute in PBTG, 1:100 |
| Alexa Fluor 546 Phalloidin | Molecular probes | A22283 | dilute in PBS, 1:40 |
| goat anti-mouse IgG antibody, Alexa Fluor 546 | Molecular probes | A11030 | dilute in PBTG, 1:400 |
| Name | Company | Catalog Number | Comments |
| Fly strains | |||
| sd-Gal4 | Bloomington Drosophila Stock Center | #8609 | recombined with UAS-EGFP |
| upd-Gal4 | Bloomington Drosophila Stock Center | #26796 | recombined with UAS-EGFP |
| UAS-lgl-RNAi | Vienna Drosophila RNAi center | #51247 | |
| UAS-scrib-RNAi | Vienna Drosophila RNAi center | #105412 | |
| UAS-RasV12 | Bloomington Drosophila Stock Center | #64196 | |
| UAS-Yki3SA | Bloomington Drosophila Stock Center | #28817 | |
| hsFLP | Bloomington Drosophila Stock Center | #6 | |
| Act>CD2>GAL4 (flip-out GAL4) | Bloomington Drosophila Stock Center | #4780 | recombined with UAS-EGFP |
| UAS-EGFP | Bloomington Drosophila Stock Center | #5428 | X chromosome |
| UAS-EGFP | Bloomington Drosophila Stock Center | #6658 | third chromosome |
| UAS-Dicer2 | Bloomington Drosophila Stock Center | #24650 | second chromosome |
| UAS-Dicer2 | Bloomington Drosophila Stock Center | #24651 | third chromosome |
| vkg-GFP | Morin et al. 2001 | GFP protein trap |
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