Name: in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis
Uploaded: 2012-02-17T13:00:00
Description: Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

نود أن نشكر التصوير نيكون المركز في كلية الطب بجامعة هارفارد، ووترز على وجه التحديد جينيفر، لارا Petrak والسلمون ويندي، لتدريب واستخدام المجاهر timelapse بهم. ونود أيضا أن نشكر روزا نج وبيسير أكيم لإجراء مناقشات ذات قيمة. وأيد هذا العمل من قبل المعاهد الوطنية للصحة جرانت 5695837 (إلى م. Iwanicki) وGM064346 إلى JSB، عن طريق منحة من ميريام والدكتور شيلدون اديلسون زاي للبحوث الطبية مؤسسة (لJSB).

1. المبيض خلية سرطان تشكيل الجسم الشبه الكروي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تستزرع طلب تقديم العروض في التعبير عن خلايا سرطان المبيض في قاعدة متوسطة 10٪ (ثقافة الخلية المخصصة المتوسطة التي تحتوي على خليط 50:50 من 199 وMCDB105، المعطل الجنين بنسبة 10٪ مصل بق?...

<ol>
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Cancer. 114, 531-543 (2005).</li><li>Iwanicki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+spheroids+use+myosin-generated+force+to+clear+the+mesothelium.">Ovarian cancer spheroids use myosin-generated force to clear the mesothelium.</a> Cancer Discovery. 1, 144-157 (2011).</li><li>Folkman, J., Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Gregoire, L., Munkarah, A., Rabah, R., Morris, R. T., Lancaster, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+human+ovarian+surface+epithelial+cells:+a+potential+model+for+ovarian+carcinogenesis.">Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis.</a> In Vitro Cell Dev. Biol. Anim. 34, 636-639 (1998).</li><li>Roberts, P. 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في &quot;الفحص التخليص الظهارية&quot; المعروضة هنا يستخدم الوقت الفاصل بين المجهري لرصد تفاعلات الأجسام الشبه الكروية سرطان المبيض المتعددة الخلايا والخلايا الظهارية monolayers، في رائعة من التفصيل المكاني والزماني. سابقا، كانت عدة مجموعات تستخدم 8-14 المقايسات نقطة ال...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> OVCA433 خلايا سرطان المبيض </td><td></td><td></td><td> هدية من الدكتور دنيس سلامون </td></tr><tr><td> ZT الخلايا الظهارية </td><td></td><td></td><td> هدية من الدكتور تان اينس </td></tr><tr><td> متوسط ​​199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> خلية تطبيقات شركة </td><td> 117-500 </td><td></td></tr><tr><td> FBS حرارة المعطل </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> القلم بكتيريا </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> 96 لوحات جيدة </td><td> كورنينج Costar </td><td> 3799 </td><td></td></tr><tص&gt; <td> هيدروكسي إيثيل ميثاكريليت (بولي HEMA) </td><td> سيجما ألدريتش </td><td> 192066-25G </td><td> لبولي HEMA حل حل 6mg بولي HEMA مسحوق في 1ml من EtOH 95٪ </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> تمييع إلى 95٪ في DH 2 0 </td></tr><tr><td> خلية ثقافة غطاء محرك السيارة </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> نسيج الثقافة الحاضنة </td><td> الحرارية العلمية </td><td> 3110 </td><td></td></tr><tr><td> حاضنة للبولي HEMA لوحات </td><td> آلات Labline </td><td> الامبراطورية الثالث 305 </td><td></td></tr><tr><td> منضدية الطرد المركزي </td><td> هيراوس </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 كذلك الزجاج قاع طبق </td><td> ماتيك كورب. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> فبرونيكتين </td><td> سيغما </td><td> F1141-1MG </td><td></td></tr><tr><td> برنامج تلفزيوني </td><td> Cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td> Timelapse المجهر: </td><td></td><td></td><td></td></tr><tr><td> مجهر </td><td> نيكون </td><td></td><td> TI-E بمحركات الإسفار معكوس الوقت الفاصل بين مجهر المتكاملة مع نظام التركيز مثالي </td></tr><tr><td> عدسة </td><td> نيكون </td><td></td><td> 20X-0.75 apeture العددية </td></tr><tr><td> الهالوجين transilluminator </td><td> نيكون </td><td></td><td> 0.52 NA مسافة العمل الطويلة مكثف </td></tr><tr><td> الإثارة وانبعاث مرشحات </td><td> صفاء مرشحات مرور واحد في السكن نيكون </td><td></td><td> GFP تحويلة 480/40، إم 525/50 RFP-mCherry تحويلة 575/50 إم 640/50 </td></tr><tr><td> أحال وEpifluoresce مسار الضوء </td><td> سوتر </tد&gt; <td></td><td> ذكي مصاريع </td></tr><tr><td> خطي ترميز مرحلة بمحركات </td><td> نيكون </td><td></td><td></td></tr><tr><td> كاميرا جهاز تبريد مشحونة، بالإضافة </td><td> هاماماتسو </td><td> ORCA-AG </td><td></td></tr><tr><td> مجهر غرفة حضانة مع تحكم في درجة الحرارة وثاني أكسيد الكربون 2 </td><td> مبنية خصيصا </td><td></td><td></td></tr><tr><td> اهتزاز العزلة طاولة </td><td> TMC </td><td></td><td></td></tr><tr><td> شيكل، عناصر البرنامج </td><td> نيكون </td><td></td><td> الإصدار 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

الفحص تخليص الظهارية وصفها هنا يستفيد من خلايا fluorescently المسمى والوقت الفاصل بين الفحص المجهري لتصور فيديو وكميا قياس التفاعلات بين الأجسام الشبه الكروية سرطان المبيض المتعددة الخلايا الظهارية وmonolayers الخلية. طرازا هذا الفحص الخطوات في وقت مبكر من ورم خبيث سرطان المبيض.

في الفحص المختبري التخليص الظهارية النماذج أن الخطوات في وقت مبكر من الإنبثاث سرطان المبيض

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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JoVE Journal

Medicine

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Experimental Metastasis Assay

الفحص التجريبي الإنبثاث

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

نماذج مامي لتصوير الخلايا الحية 4D من ورم: التفاعلات المكروية أن التقدم تأثير الخبيثة

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

ومتعددة الوسائط التصوير من الأوعية الدموية في نموذج الفئران عاري من سرطان الثدي الإنبثاث العظام باستخدام التصوير بالرنين المغناطيسي، الحجمي التصوير المقطعي والموجات فوق الصوتية

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

غيروي ثلاثي الأبعاد<em&gt; في المختبر</em&gt; نمذجة التفاعلات اللحمية-طلائي سرطان المبيض خلال بدء والتقدم

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Models of Bone Metastasis

نماذج من الانبثاث العظام

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

تقييم سرطان المبيض كروي مرفق وغزو الخلايا الظهارية في الوقت الحقيقي

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

في التخصيب في المختبر من خلايا سرطان المبيض-الشروع ورم

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

ثلاثي الأبعاد (3D) ورم الجسم الشبه الكروي الغزو الفحص

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

في فيفو وفيفو السابقين المداخل لدراسة سرطان المبيض النقيلي الاستعمار من الهياكل التبانة بقعة في البريتوني الدهنية

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

الكميات من البينية البريتوني ورم خبيث سرطان المبيض

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...