这些模型在体外间皮间隙检测卵巢癌转移的早期步骤
Summary
这里描述的皮间隙检测，利用荧光标记的细胞和延时视频显微镜，可视化和定量测量卵巢癌多细胞球体和间皮细胞单层的相互作用。此法模拟卵巢癌转移的早期步骤。
Abstract
卵巢癌是第五次在美国癌症相关死亡的首要原因。尽管积极的初步反应治疗，70％至90％的卵巢癌的妇女开发新的转移，复发往往是致命的2。因此，有必要的，它是了解出现二次转移，以发展中间和晚期卵巢癌的更好的治疗。卵巢癌转移发生恶性细胞脱离原发肿瘤部位和传播整个腹腔。传播的细胞可以形成多细胞集群，或球体，将保持独立，或者植入到机关内的腹腔（图1，电影1）。
腹腔内的器官全部一字排开用一个单一的，连续的，一层4-6间皮细胞（图2）。然而，间皮细胞是从下面缺席腹膜肿块，切除人体肿瘤组织切片3,5-7（图2）的电子显微镜研究发现。这表明，由一个未知的过程中，间皮细胞排除从下面的肿块。
以前在体外实验表明，原发性卵巢癌的细胞更有效地附加比8间皮细胞外基质，最近的研究表明，原发性腹膜间皮细胞，实际上提供了一个屏障，卵巢癌的细胞粘附和侵袭（相比，黏附和侵袭不包括与间皮细胞）9,10基板上。这表明，间皮细胞对卵巢癌转移的障碍行动。卵巢癌细胞突破这一障碍，排除间皮有细胞和分子机制，直到最近，仍然不明。
在这里，我们描述日e为在体外试验方法，模型与卵巢癌的细胞球体和间皮细胞在体内的相互作用（图3，电影2）。我们的协议是改编自先前所描述的方法，分析卵巢肿瘤细胞的相互作用与8-16皮单层，并首次在报告中显示，卵巢肿瘤细胞利用整合依赖激活的肌球蛋白和牵引力，以促进排除描述从下间皮细胞肿瘤球体17。这种模式需要时间推移荧光显微镜的优势，在实时监控的两个细胞群，提供了空间和时间上的互动信息。卵巢癌的细胞表达红色荧光蛋白（RFP），而间皮细胞表达绿色荧光蛋白（GFP）。 RFP的表达卵巢癌的细胞球体连接到表达GFP的皮单层。球体的蔓延，侵入，迫使除了创造一个洞单层间皮细胞。这个洞是在GFP图像为负空间（黑）的可视化。洞的面积，然后可以测量，控制和卵巢癌和/或间皮细胞的实验种群之间的间隙活动的差异定量分析。此法只需要一个小数目，卵巢癌的细胞（100细胞每球体X 20-30％条件下球体），所以它是可行的，执行此法，采用珍贵的原发肿瘤细胞样本。此外，该法可以很容易地适应高通量筛选。
Protocol
1。卵巢癌的细胞球体的形成 RFP的表达卵巢癌的细胞培养在10％的基础培养基（自定义的细胞培养液中含有199和一个MCDB105 50:50的混合物，10％的灭活胎牛血清和1％笔链球菌）。来表达RFP的未标记卵巢癌的细胞，转染质粒含有利福平的细胞和细胞表达RFP的选择。另外，病毒载体可用于瞬时表达荧光蛋白或细胞可以预孵育与红色荧光的细胞追踪染料（Invitrogen公司）。 对卵巢癌的球体形?…
Discussion
“间皮间隙检测”这里使用的时间推移显微镜监测卵巢癌多细胞球体和间皮细胞单层的相互作用，在很大的空间和时间上的细节。此前，几组8-14端点检测表明，卵巢癌的细胞附着和侵入皮细胞单层。此法是独一无二的，它使用荧光标记的细胞来区分间皮细胞的肿瘤细胞，使这两个细胞群的动态，可以监测整个实验。插的过程中可以实时可视化，并随着时间的推移，皮清除率可以定量测定…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢哈佛医学院的尼康影像中心，尤其是珍妮弗水域，拉拉Petrak和温迪三文鱼，培训和使用他们的RSS 2.0订阅显微镜。我们也想感谢罗莎·吴和阿希姆·贝瑟有价值的讨论。这项工作是由NIH资助5695837（M. Iwanicki）GM064346来JSB的支持;由授予博士仪和Sheldon G.阿德尔森医学研究基金会（JSB的）。
Materials
| Reagent | Company | Catalog Number | Comments |
| OVCA433 Ovarian Cancer Cells | Gift from Dr. Dennis Slamon | ||
| ZT Mesothelial Cells | Gift from Dr. Tan Ince | ||
| Medium 199 | Gibco | 19950 | |
| MCDB105 | Cell Applications Inc. | 117-500 | |
| FBS-heat inactivated | Gibco | 10082 | |
| Pen-Strep | Gibco | 15070 | |
| 96 well plates | Corning Costar | 3799 | |
| Polyhydroxyethylmethacrylate (poly-HEMA) | Sigma Aldrich | 192066-25G | For poly-HEMA solution dissolve 6mg poly-HEMA powder in 1ml of 95% EtOH |
| EtOH | Pharmco-aaper | 111ACS200 | Dilute to 95% in dH20 |
| Cell culture hood | Nuaire | NU-425-300 | |
| Tissue culture incubator | Thermo Scientific | 3110 | |
| incubator for poly-HEMA plates | Labline Instruments | Imperial III 305 | |
| Tabletop centrifuge | Heraeus | 75003429/01 | |
| 6 well glass-bottom dish | MatTek corp. | P06G-1.5-20-F | |
| Fibronectin | Sigma | F1141-1MG | |
| PBS | Cellgro | 21-040-CV | |
| Timelapse Microscope: | |||
| Microscope | Nikon | Ti-E Inverted Motorized Fluorescence time-lapse microscope with integrated Perfect Focus System | |
| Lens | Nikon | 20X-0.75 numerical apeture | |
| Halogen transilluminator | Nikon | 0.52 NA long working distance condenser | |
| Excitation and emission filters | Chroma single pass filters in Nikon housing | GFP Ex 480/40, Em 525/50 RFP-mCherry Ex 575/50 Em 640/50 | |
| Transmitted and Epifluoresce light path | Sutter | Smart Shutters | |
| Linear-encoded motorized stage | Nikon | ||
| Cooled charged-coupled device camera | Hamamatsu | ORCA-AG | |
| Microscope incubation chamber with temperature and CO2 control | custom-built | ||
| Vibration isolation table | TMC | ||
| NIS-Elements software | Nikon | Version 3 |
Riferimenti
- Jemal, A. . CA Cancer J. Clin. 59, 225-249 (2009).
- Ries, L. G., Melbert, D., Krapcho, M., Stinchcomb, D. G., Howlader, N., Horner, M. J., Mariotto, A., Miller, B. A. . SEER Cancer Statistics Review, 1975-2005. , (2007).
- Burleson, K. M. Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers. Gynecol. Oncol. 93, 170-181 (2004).
- Birbeck, M. S., Wheatley, D. N. An Electron Microscopic Study of the Invasion of Ascites Tumor Cells into the Abdominal Wall. Cancer Res. 25, 490-497 (1965).
- Witz, C. A., Monotoya-Rodriguez, I. A., Schenken, R. S. Whole explants of peritoneum and endometrium: a novel model of the early endometriosis lesion. Fertil. Steril. 71, 56-60 (1999).
- Zhang, X. Y. Characteristics and growth patterns of human peritoneal mesothelial cells: comparison between advanced epithelial ovarian cancer and non-ovarian cancer sources. J. Soc. Gynecol. Investig. 6, 333-340 (1999).
- Kenny, H. A., Nieman, K. M., Mitra, A. K., Lengyel, E. The First Line of Intra-abdominal Metastatic Attack: Breaching the Mesothelial Cell Layer. Cancer Discovery. 1, 100-102 (2011).
- Niedbala, M. J., Crickard, K., Bernacki, R. J. Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix. An in vitro model system for studying tumor cell adhesion and invasion. Exp. Cell. Res. 160, 499-513 (1985).
- Kenny, H. A., Krausz, T., Yamada, S. D., Lengyel, E. Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells to the omentum. Int. J. Cancer. 121, 1463-1472 (2007).
- Ksiazek, K. Senescent peritoneal mesothelial cells promote ovarian cancer cell adhesion: the role of oxidative stress-induced fibronectin. Am. J. Pathol. 174, 1230-1240 (2009).
- Burleson, K. M., Boente, M. P., Pambuccian, S. E., Skubitz, A. P. Disaggregation and invasion of ovarian carcinoma ascites spheroids. J. Transl. Med. 4, 6-6 (2006).
- Heyman, L. Vitronectin and its receptors partly mediate adhesion of ovarian cancer cells to peritoneal mesothelium in vitro. Tumour. Biol. 29, 231-244 (2008).
- Heyman, L. Mesothelial vitronectin stimulates migration of ovarian cancer cells. Cell. Biol. Int. 34, 493-502 .
- Lessan, K., Aguiar, D. J., Oegema, T., Siebenson, L., Skubitz, A. P. CD44 and beta1 integrin mediate ovarian carcinoma cell adhesion to peritoneal mesothelial cells. Am. J. Pathol. 154, 1525-1537 (1999).
- Leroy-Dudal, J., Heyman, L., Gauduchon, P., Carreiras, F. Adhesion of human ovarian adenocarcinoma IGROV1 cells to endothelial cells is partly mediated by the alphav integrins-vitronectin adhesive system and induces an alteration of endothelial integrity. Cell. Biol. Int. 29, 482-488 (2005).
- Leroy-Dudal, J. Transmigration of human ovarian adenocarcinoma cells through endothelial extracellular matrix involves alphav integrins and the participation of MMP2. Int. J. Cancer. 114, 531-543 (2005).
- Iwanicki, M. Ovarian cancer spheroids use myosin-generated force to clear the mesothelium. Cancer Discovery. 1, 144-157 (2011).
- Folkman, J., Moscona, A. Role of cell shape in growth control. Nature. 273, 345-349 (1978).
- Gregoire, L., Munkarah, A., Rabah, R., Morris, R. T., Lancaster, W. D. Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis. In Vitro Cell Dev. Biol. Anim. 34, 636-639 (1998).
- Roberts, P. C. Sequential molecular and cellular events during neoplastic progression: a mouse syngeneic ovarian cancer model. Neoplasia. 7, 944-956 (2005).
- Okada, T., Okuno, H., Mitsui, Y. A novel in vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and alpha 3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells. Clin. Exp. Metastasis. 12, 305-314 (1994).
- Zervantonakis, I. K., Kothapalli, C. R., Chung, S., Sudo, R., Kamm, R. D. Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments. Biomicrofluidics. 5, 13406-1310 (2011).
- Brandt, B. 3D-extravasation model — selection of highly motile and metastatic cancer cells. Semin. Cancer Biol. 15, 387-395 (2005).
- Condeelis, J., Segall, J. E. Intravital imaging of cell movement in tumours. Nat. Rev. Cancer. 3, 921-930 (2003).
- Dai, J., Ting-Beall, H. P., Hochmuth, R. M., Sheetz, M. P., Titus, M. A. Myosin I contributes to the generation of resting cortical tension. Biophys. J. 77, 1168-1176 (1999).
- Laferriere, J., Houle, F., Taher, M. M., Valerie, K., Huot, J. Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activated protein kinase-2 (SAPK2/p38) in the tumor cells. J. Biol. Chem. 276, 33762-33772 (2001).
- Dong, C., Slattery, M. J., Rank, B. M., You, J. In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation. Ann. Biomed. Eng. 30, 344-355 (2002).
Citazione di questo articolo
Davidowitz, R. A., Iwanicki, M. P., Brugge, J. S. In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis. J. Vis. Exp. (60), e3888, doi:10.3791/3888 (2012).