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利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

Published: June 26, 2019
doi:
10.3791/59633
, , , , , , ,
1Department of Anatomy and Structural Biology,Albert Einstein College of Medicine, 2Gruss-Lipper Biophotonics Center,Albert Einstein College of Medicine, 3Integrated Imaging Program,Albert Einstein College of Medicine, 4Department of Surgery,Montefiore Medical Center, 5Department of Pathology,Montefiore Medical Center

Summary

我们描述了两种方法,用于评估与转移(TMEM)的肿瘤微环境(TMEM)和癌细胞内溢相关的瞬态血管渗透性,使用静脉注射高分子量(155 kDa)dextran小鼠。这些方法包括活体动物的活体成像和使用免疫荧光的固定组织分析。

Abstract

癌症相关死亡的最常见原因是转移,这个过程需要将癌细胞从原发性肿瘤传播到继发位点。最近,我们确定,原发性乳腺癌和肺部转移部位的癌细胞传播只发生在称为肿瘤微环境(TMEM)的门口。TMEM 门数是乳腺癌患者转移性疾病远程复发的预后。TMEM 门由癌细胞组成,该细胞在与血管周围、原兆噬细胞直接接触时过度表达行为蛋白调节蛋白 Mena,该细胞表示高水平的 TIE2 和 VEGF,其中这两个细胞都与血液紧密结合血管内皮细胞。癌细胞可以通过TMEM门内血管,由于由TMEM相关巨噬细胞和TMEM相关的梅纳表达癌细胞的联合活动编排的瞬态血管渗透。在本手稿中,我们描述了两种评估 TMEM 介导的瞬态血管渗透性的方法:生命内成像和固定组织免疫荧光。虽然这两种方法各有优缺点,但两种方法的结合可以最完整地分析 TMEM 介导的血管渗透性以及 TMEM 功能的微观环境先决条件。由于乳腺癌的转移过程,可能还有其他类型的癌症,涉及通过TMEM门传播癌细胞,因此必须采用成熟的方法来分析TMEM门活动。此处描述的两种方法为分析 TMEM 门性提供了一种全面的方法 , 无论是幼稚的还是药物治疗的动物 , 这对预防癌细胞的制剂的临床前试验至关重要通过TMEM传播。

Introduction

最近对癌症转移的理解的进步发现,上皮到间质过渡(EMT)和诱导迁移/侵入性癌细胞亚群本身不足以进行造血传播1.确实,以前人们认为,通过整个与癌症相关的内皮物转移癌细胞,因为肿瘤新血管学通常具有低围肠覆盖率的特点,因此具有很强的渗透性和渗透性。不稳定2,3,4。虽然肿瘤内功能缺陷的暗示性很强,但致癌过程中的血管修饰本身并不提供肿瘤细胞能够以不受控制的方式轻易穿透血管的证据。从生命内成像(IVI)研究的见解,其中肿瘤细胞荧光标记和血管标记通过静脉注射荧光探针(如dextran或量子点),表明,虽然肿瘤血管是均匀的可渗透至低分子量的extrans(例如70 kD)、高分子量的extrans(155 kD)和肿瘤细胞只能在位于血管分支点5的专门内血管内分部穿过内皮。6,7.使用动物模型和人类患者衍生材料进行的免疫组织化学(IHC)分析表明,这些部位是专门调节局部和瞬态血管渗透性的”门道”,提供了一个简短的窗口。迁徙/侵入性癌细胞进入循环的机会。这些门道被称为”转移的肿瘤微环境”或”TMEM”,而且,相当预料的是,它们的密度与乳腺癌患者患转移性疾病的风险增加有关8,9, 10.

每个TMEM门由三种不同类型的细胞组成:血管内大噬细胞,肿瘤细胞过度表达行为蛋白哺乳动物启用(Mena),和内皮细胞,所有直接物理接触对方1, 5,9,10,11,12,13。TMEM作为血管内门的功能的关键事件是血管内皮生长因子(VEGF)通过血管内大噬细胞14向基础血管的局部释放。VEGF 可以破坏内皮细胞 15、16、17、18、19 之间的同型结点,这种现象会导致瞬态血管泄漏,也称为”爆裂”渗透性,如IVI研究5所述。 TMEM 巨噬细胞已被证明表达酪氨酸激酶受体TIE2,这是VEGF介导的TMEM功能和这些巨噬细胞的定位到血管周围利基5,20,21,22.除了调节癌细胞传播和转移外,TIE2+巨噬细胞已被证明是肿瘤血管生成21、22、23的中央调节器, 24,25,26,27,28,29,30,31。因此,TIE2+巨噬细胞是肿瘤微环境的重要组成部分,是转移级联的主要调节器。

为了更好地概念化TMEM介导的血管渗透性(即”爆裂”),将它与与内皮细胞-细胞结的溶解无关的其他血管渗透性模式区分开来非常重要。在完整的内皮内皮(其紧和粘结未中断),有三种主要类型的血管渗透性:(a) 细胞化,可能,也可能不,与摄入的物质的转细胞;(a) 细胞虫症,可以,也可能不,与摄入的物质的转细胞;((d) 细胞内渗透。(d) 血管渗透性。(d) 血管渗透性。(a) 血管渗透性。(a) 血管渗透性。(a) 血管渗透性。(d) 血管渗透性。(d) 血管渗透性。(a) 血管渗透性。(a) 血管渗透性,可能或可能不会与摄入材料的转细胞结合;(b) 通过内皮芬斯特雷运输材料;和(c) 通过副细胞通路运输材料,由内皮紧接15、16、17、18、19、32调节,33,34.虽然在许多肿瘤中解除管制,但上述血管渗透模式主要在正常组织生理学和平衡的背景下加以描述,其极端是渗透性有限的组织(例如,血脑屏障,血检屏障),或丰富的渗透性(例如,肾肾肾小球仪的芬芳毛细血管)34,35,36,37 。

使用多光子生命内成像和多路免疫荧光显微镜,我们能够区分TMEM介导的血管渗透性(“爆裂”)和乳腺肿瘤中其他血管渗透性模式。为此,我们在小鼠中单次静脉注射高分子量荧光标记的探针。然后,使用活小鼠的活体成像捕获自发性爆裂事件;或者,通过血脉(如CD31+或内杜木明+)和使用免疫荧光显微镜的TMEM门分,可以量化探针的外化。此处介绍的协议描述了这两种技术,这些技术可以独立使用,也可以相互结合使用。

Protocol

所有使用活体动物的实验必须按照动物使用和护理准则和条例进行。本研究中描述的程序是按照国家卫生研究院关于照料和使用实验动物的规定,并经阿尔伯特·爱因斯坦医学医学学院动物护理和使用学院批准进行的。委员会。 1. 利用活体动物成像评估”爆裂渗透性” 将合成乳腺肿瘤移植到具有荧光巨噬细胞的小鼠宿主中 生成适合移植的肿瘤组织片段…

Representative Results

本文中描述的实验过程在图1A-C中进行了简要的总结和说明。 为了测量TMEM介导的血管渗透性(“爆裂活性”)和减少其他血管渗透模式(如导言所述的跨细胞和准细胞)的实验噪音,我们进行了静脉注射(即v.)注入高分子量探针,如155 kDa Dextran,与四甲基罗丹胺结合。低分子量的Dextrans没有区分三种血管渗透模式。我们以往的经验显示,155 kDa TMR-Dextran 的全?…

Discussion

在这里,我们概述了两个协议,可用于可视化和量化一种特定类型的血管渗透性,这是存在于TMEM门口,并与血管紧和粘附结的中断相关。这种类型的血管渗透性是瞬态的,由三方TMEM细胞复合体控制,如上文5所述。识别和量化 TMEM 相关血管渗透性的能力对于评估亲转移性癌细胞微环境以及检查传统细胞毒性疗法对肿瘤的影响的临床前研究至关重要微环境以及靶向和非靶向疗法的抗转移潜?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们要感谢阿尔伯特·爱因斯坦医学院的分析成像设施(AIF)提供成像支持。这项工作得到了NCI(P30CA0133330、CA150344、CA 100324和CA216248)、SIG 1S10OD019961-01、格鲁斯-利珀生物光子学中心及其综合成像项目以及蒙特菲奥雷的露丝·基尔施施泰因T32外科医生培训资助项目的支持。肿瘤微环境研究(CA200561)。

葛兰素史克共同撰写稿件,对图1C和3B进行成像,开发固定组织分析协议,分析并解释所有数据;JMP共同撰写了手稿,并为图1B、2C和3A进行了手术和生命内成像;LB & AC为图 2B 进行了手术和生命内成像;RJ为图2A进行了手术和生命内成像;JSC共同撰写稿件,并分析和解释所有数据;MHO共同撰写手稿,并分析和解释所有数据;和DE为图2D进行了手术和生命内成像,共同撰写了手稿,开发了固定组织分析和生命内成像方案,并分析和解释了所有数据。

Materials

Anti-rabbit IgG (Alexa 488) Life Technologies Corporation A-11034
Anti-rat IgG (Alexa 647) Life Technologies Corporation A-21247
Bovine Serum Albumin Fisher Scientific BP1600-100
Citrate Eng Scientific Inc 9770
Cover Glass Slips Electron Microscopy Sciences 72296-08
Cyanoacrylate Adhesive Henkel Adhesive 1647358
DAPI Perkin Elmer FP1490
Dextran-Tetramethyl-Rhodamine Sigma Aldrich T1287
DMEM/F12 Gibco 11320-033
Endomucin (primary antibody) Santa Cruz Biotechnology sc-65495
Enrofloxacin Bayer 84753076 v-06/2015
Fetal Bovine Serum Sigma Aldrich F2442
Fish Skin Gelatin Fisher Scientific G7765
Insulin Syringe Becton Dickinson 309659
Isofluorane Henry Schein NDC 11695-6776-2
Matrigel Corning CB40234 Artificial extracellular matrix
Needle (30 G) Becton Dickinson 305128
Phosphate Buffered Saline Life Technologies Corporation PBS
Polyethylene Tubing Scientific Commodities Inc BB31695-PE/1
Pulse Oximeter Kent Scientific MouseOx
Puralube Vet Ointment Dechra NDC 17033-211-38
Quantum Dots Life Technologies Corporation Q21561MP
Rubber McMaster Carr 1310N14
TMR (primary antibody) Invitrogen A6397
Tween-20 MP Biologicals TWEEN201
Xylene Fisher Scientific 184835
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Tags

Tumor MicroenvironmentTMEMVascular PermeabilityCancer Cell DisseminationIntravital ImagingDextranTumor TransplantationMacrophagesMammary Fat PadExtracellular MatrixTail Vein Catheter
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Karagiannis, G. S., Pastoriza, J. M., Borriello, L., Jafari, R., Coste, A., Condeelis, J. S., Oktay, M. H., Entenberg, D. Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis. J. Vis. Exp. (148), e59633, doi:10.3791/59633 (2019).
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