Biz Harvard Tıp Okulu&#39;nda Nikon Görüntüleme Merkezi, özellikle Jennifer Waters, Lara Petrak ve Wendy Somon, eğitim ve timelapse mikroskopların kullanımı teşekkür etmek istiyorum. Biz de değerli tartışmalar için Rosa Ng ve Achim Besser teşekkür etmek istiyorum. Bu çalışma NIH hibe 5695837 (M. Iwanicki) ve JSB için GM064346 tarafından desteklenmiştir; Dr Miriam ve Sheldon Adelson G. Tıbbi Araştırma Vakfı (JSB için) bir hibe ile.

1. Yumurtalık Kanseri Hücre Sfero Oluşumu <ol><li> RFP-eksprese yumurtalık kanseri hücreleri% 10 baz ortamı (199 a ve MCDB105 ile 50:50 karışım,% 10 inaktive edilmiş fetal bovin serumu ile% 1 kalem Strep içeren bir özel hücre kültür ortamı) içinde kültürlenmiştir. Etiketsiz over kanseri hücrelerinde RFP ifade etmek için, bir plazmid içeren TTT ile hücreleri transfekte ve TÇD ifade hücreleri seçin. Alternatif olarak, viral vektörler geçici olarak floresan proteinleri ifade etmek için kullanılabilir, ya da hücre kırmızı bir floresan hücre izleyici boya (Invitrogen) ile ön-inkübe olabilir. </li><li> Önceden yumurtalık kanseri sferoidler oluşturulması için, bir düşük yapışma 96 yuvarlak dipli bir kültür tabaklarında hazırlamak için gereklidir. , Düşük yapışma kültür plakaları üretmek için 30μl poli-HEMA çözeltisi (1 ml% 95 EtOH içerisinde 6 mg polyhydroxyethylmethacrylate), bir 96 Corning hücre kültürü çanağı, her oyuğa ilave edilir. 96 kuyucuğu ° C olmayan nemli inkübatör etanol, l buharlaştırmak için kullanılan bir 37 inkübe edilirher bir kuyunun üzerine poli-HEMA bir film eaving. Bu poli-HEMA filmi hücreleri süspansiyon 18 büyümeye zorluyor, kuyunun dibinde takılarak gelen hücreleri engeller. [Alternatif olarak, ultra-düşük Eklenti kültür plakları (Corning) poli-HEMA kaplı yemekler yerine kullanılabilir.] </li><li> Düşük yapışma kültür plakaları hazırlandıktan sonra, yumurtalık kanseri hücreleri, pelet 3 dakika boyunca 900 RCF az bir masa üstü santrifüj (Heraeus) hücreleri, süpernatan aspire ve% 10 baz ortamı içinde tekrar askıya bir bord trypsinize. </li><li> Bir hemasitometre kullanarak hücreleri hesaplama. </li><li> Taban 10% Medium 50μl başına 100 hücreleri olduğu gibi hücrelerin konsantrasyonuna ayarlayın. </li><li> 96 poli-HEMA kaplı kültür kaplarına her kuyuya eşit şekilde süspanse seyreltilmiş hücre süspansiyonu 50μl ekleyin. </li><li> 16 saat boyunca 37 ° C&#39;de hücre kültürü inkübatöründe (zaman bu miktar için gereken zaman miktarına bağlı olarak artan ya da azaltılması gerekir içinde 96 kuyulu plakanın inkübeher bir yanı içerisinde tek bir hücreli steoid oluşturan birbirine kümeye yumurtalık kanseri hücrelerinin izin vermek için çok hücreli sferoidler ya da arzu edilen deneysel koşullar) oluşturmak üzere, belirli bir hücre dizisi. Bazı tümör hücreleri bu dönemde apoptoza, bu nedenle önceden apoptozis indüksiyonu için bir zamanı seçmek önemlidir. </li></ol> 2. Mezotelyal Hücre Monolayer Oluşumu <ol><li> Bir hücre kültür davlumbaz, ön-kat çanağı, her sıra için bir 5μg fibronektin / ml PBS çözeltisi 2mL ekleyerek ve 30 dakika boyunca oda sıcaklığında kuluçkaya fibronektin sahip bir 6-alt yanı cam tabağın MatTek Corp kuyuları. MatTek Corp yemekler cam altları ve optik kalitesi yüksek çözünürlüklü mikroskopik görüntüleme için izin verir. </li><li> GFP-salgılayan hücrelerin mezotel% 10 baz ortamı içerisinde kültüre edilir. Mezotel hücrelerinin bir tabak Trypsinize, 3 dakika için 900 RPM bir masaüstü santrifüj (Heraeus) aşağı spin süpernatan aspire, ve% 10 Base Orta yeniden askıya.Burada kullanılan mezotel hücreler elde edildi sırasında önceden GFP ifade edilmiştir, ancak etiketsiz mezotel hücreleri cDNA içeren bir plazmid GFP ile transfekte etmek ya da bir yeşil flöresanlı hücre izleyici boya (Invitrogen) içinde hücreleri preincubating tarafından üretilebilir. </li><li> 30 dakikalık fibronektin inkübasyon (adım 2.1) sonra, 2 mL PBS ile MatTek Corp çanak kuyuları yıkayın. </li><li> 6 sıra MatTek Corp çanak her iyi yanı başına PBS ve plaka 6 x10 5 mezotel hücre aspire. Mezotel hücre çanak takmak ve bir tek tabaka oluşturacak şekilde izin vermek için bir gecede 37 ° C hücre kültürü inkübatörde MatTek Corp çanak inkübe edin. </li></ol> 3. Mezotelyal Hücre Gümrükleme Testi <ol><li> 96 poli-HEMA kaplı plaka yumurtalık kanseri sferoidler toplamak için bir pipet kullanın. </li><li> Bir mezotel hücre monolayer içeren 6 sıra MatTek Corp çanak iyi birinden orta aspire. 2mL PBS ile bir kez yıkayın. 96 p gelen parçacıklarının bütün ekleMatTek Corp çanak (yansıma edilemez çanak parçası üzerinde sferoidler iniş hesaba yansıması olacak parçacıklarının ~ 3x sayısı) iyi birine geç. </li><li> En az 8 saat boyunca zaman atlamalı görüntüleme yapabilen bir ters Geniş açılı, floresan mikroskop sahnede MatTek Corp tabak yerleştirin. Tek bir deneyde, birden sfero ardalanması olaylar, çanak görüntü birden fazla pozisyon için motorlu bir sahne kullanın. Biz entegre Mükemmel odak sistemi ve düşük [20x-0.75 Sayısal açıklık (NA)] büyütme / NA diferansiyel müdahale kontrast (DIC) optik ile Nikon Ti-E Ters Motorlu Geniş açılı, Floresans time-lapse mikroskop kullanımı, 0.52 NA olan Nikon halojen transilluminator&#39;de uzun çalışma mesafesi (LWD) kondenser, Nikon hızlı (&lt;100 milisaniye anahtarlama zamanı) uyarılma ve emisyon filtreleri (GFP Ex 480/40, EM 525/50, TÇ-mCherry Ex 575/50 Em 640/50), Sutter hızlı bulaşan ve Epifloresans ışık yolu Akıllı Shutmektuplar, Nikon lineer kodlu motorlu sahne, bir Hamamatsu ORCA-AG şarj bağlı cihaz (CCD) kamera, sıcaklık ve CO 2 kontrolü ile özel-yerleşik mikroskop inkübasyon odası, Nikon NIS-Elements AR yazılım sürümü 3 adet soğutmalı ve TMC titreşim izolasyonu tablo. </li><li> Yumurtalık kanseri hücre sferoidler yemeğin dibe ve mezotel hücre monolayer eklenecektir. 8 saat GFP, RFP ve 20 faz görüntüleri + sfero / monolayer etkileşimleri, her 10 dakikada, toplayın. </li><li> TTT-ifade yumurtalık kanseri hücresi sferoidler tek tabakalı bir delik oluştururken GFP-ifade mezotel hücre tek tabaka halinde istila edecek. 8 saat sonra, Elements yazılımı (ya da örneğin, resim J gibi başka uygun bir yazılım) kullanılarak GFP resimlerde karadeliklerin izleme tarafından deliklerin boyutları ölçülür. Sıfır anındaki ilgili RFP görüntüde sfero büyüklüğü ile 8 saat delik boyutu bölerek ilk sfero boyutu delik boyutu Normale. Bu exbol, delik büyüklüğü sadece bir kez ölçüldü, ancak daha iyi ardalanması dinamiklerini anlamak için sekiz saatlik deney boyunca birden çok kez ölçülebilir. </li></ol> 4. Temsilcisi Sonuçlar Bu örnekte, OVCA433 sferoidler kontrol etmek Talin-1 zayıflatılmış ifade var OVCA433 yumurtalık kanseri hücre parçacıklarının mezotel klirensi yeteneği karşılaştırıldı. Her bir gruptan OVCA433 sferoidler ZT mezotel hücre mono tabakaları içeren bir MatTek Corp çanağı ilave edildi. Her gruptan Altı sferoidler sekiz saat boyunca her 10 dakikada bir (Şekil 4, Movie 3, Movie 4) görüntülendi. Yayılma sferoidler tarafından üretilen tek tabakalı delikler ölçüldü ve her gruptan altı pozisyonları ortalaması alınmıştır. Talin 1 Knockdown sferoidler tarafından yaratılan ortalama açıklık kontrolü sferoidler tarafından yaratılan ortalama alan çok daha küçük olduğu Şekil 4, Talin O tarafından mezotel izni için gerekli olduğunu düşündürmektedirVCA433 yumurtalık kanseri sferoidler. <img alt="Şekil 1" src="/files/ftp_upload/3888/3888fig1.jpg" /> Şekil 1. Yumurtalık Kanseri Metastaz. Primer over tümörleri over yüzey epiteli veya fallop tüplerinden birini geliştirmek. Tümör hücreleri / kümeler primer tümörden kopar ve periton boşluğuna toplamak. Tümör hücreleri daha sonra çok hücreli sferoidler oluşturmak için toplamak olabilir. Spheroidlerinde periton boşluğuna döşeyen mezotel hücre mono tabakaları iliştirin. Mezotel hücre sferoidler alttaki bazal membran erişmek için izin ekli yumurtalık kanseri sfero altından dışındadır. Film 1. Yumurtalık Kanseri Metastaz. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">filmi görmek için buraya tıklayın</a> . <img alt="Şekil 2" src="/files/ftp_upload/3888/3888fig2.jpg" /> Şekil 2. </strong&gt; Mezotelyal hücrelerin satır insan periton dokusunun yüzey ve yumurtalık kanseri hücre implantları altından dışındadır. <img alt="Şekil 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> Şekil 3. Mezotelyal Boşluk Assay. Yumurtalık kanseri sferoidler 100 kuluçkaya tarafından oluşturulan RFP-eksprese 16 saat boyunca 37 ° C&#39;de, bir poli-HEMA kaplanmış 96 tabanı yuvarlak bir kültür tabağına oyuk başına yumurtalık kanseri hücrelerinin. Poli-HEMA hücreleri süspansiyon kalır ve başı da tek bir küme oluşturacak şekilde birbirine yapışması sağlayan kültür kaplarına takılarak gelen hücreleri engeller. Mezotel hücre mono tabakaları bir fibronektin kaplı 6 sıra MatTek Corp tabağına oyuk başına 6x10 5 mezotel hücreleri kaplama ve 16 saat süreyle 37 ° C de kuluçkaya levha hazırlanır. Spheroidlerinde mezotel tek tabaka ile MatTek Corp kaba aktarılır ve iki tip hücre topluluğu bir Nikon Ti-E kullanıyorum 8 saat boyunca her 10 dakikada bir yansıması olannverted Motorlu Geniş açılı, Floresans time-lapse mikroskop ve Elements yazılımı. Film 2. Mezotelyal Gümrükleme Assay. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">filmi görmek için buraya tıklayın</a> . <img alt="Şekil 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> OVCA433 sferoidler yılında Talin 1 ifade Şekil 4. Zayıflama mezotel klirensi yeteneğini azaltır. Ve Talin 1 zayıflatılmış ifadesi olmadan OVCA433 sferoidler (kırmızı) için ve her bir ZT mezotel tek tabakalı (yeşil) içine işgal için izin verildi. İki hücre popülasyonlarının bir Nikon Ti-E Ters Motorlu Geniş açılı, Floresans time-lapse mikroskop ve elemanları yazılımı kullanarak 8 saat boyunca her 10 dakika görüntülendi. Grafiği, Talin 1 zayıflama gösterirönemli mezotel hücresi klirens (anlama geldiğine yeşil çubuklar ile Dilim arsa) azalır. Film 3. Kontrol bir mezotel tek tabakalı (yeşil) içine işgalci OVCA433 sferoidler (kırmızı). <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">filmi görmek için buraya tıklayın</a> . OVCA433 sferoidler yılında Talin 1 ekspresyonu (kırmızı) Film 4. Zayıflama (yeşil) temizleme yeteneği mezotel azalır. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">filmi görmek için buraya tıklayın</a> .

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+cell+movement+in+tumours.">Intravital imaging of cell movement in tumours.</a> Nat. Rev. Cancer. 3, 921-930 (2003).</li><li>Dai, J., Ting-Beall, H. P., Hochmuth, R. M., Sheetz, M. P., Titus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+I+contributes+to+the+generation+of+resting+cortical+tension.">Myosin I contributes to the generation of resting cortical tension.</a> Biophys. J. 77, 1168-1176 (1999).</li><li>Laferriere, J., Houle, F., Taher, M. M., Valerie, K., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J. Biol. Chem. 276, 33762-33772 (2001).</li><li>Dong, C., Slattery, M. J., Rank, B. M., You, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+and+micromechanics+of+tumor+cell+chemotactic+protrusion,+locomotion,+and+extravasation.">In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation.</a> Ann. Biomed. Eng. 30, 344-355 (2002).</li></ol>

Biz ifşa hiçbir şey yok.

Burada sunulan &quot;Mezotelyal Gümrükleme Assay&quot; büyük mekansal ve zamansal ayrıntılı olarak, yumurtalık kanseri çok hücreli sferoidler ve mezotel hücre tek katmanlarının etkileşimleri izlemek için time-lapse mikroskop kullanır. Daha önce, 8-14 çeşitli gruplar olduğu yumurtalık kanseri hücrelerine tutunur ve mezotel hücre mono tabakaları içine işgal göstermek için son nokta deneyleri kullandı. Bu tahlil benzersiz olduğunu da bu iki hücre popülasyonlarının dinamiklerini ...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> OVCA433 Yumurtalık Kanseri Hücreleri </td><td></td><td></td><td> Dr Dennis Slamon gelen Hediye </td></tr><tr><td> Mezotelyal Hücreler ZT </td><td></td><td></td><td> Dr Tan dan Hediye Ince </td></tr><tr><td> Orta 199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> Hücre Uygulamaları A.Ş. </td><td> 117-500 </td><td></td></tr><tr><td> FBS-ısı inaktive </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> Kalem-Strep </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> 96 oyuklu plakalar </td><td> Corning Costar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polyhydroxyethylmethacrylate (poli-HEMA) </td><td> Sigma Aldrich </td><td> 192.066-25G </td><td> Poli-HEMA çözümü için% 95 EtOH 1ml içinde 6 mg poli-HEMA tozun </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> DH 2 0% 95&#39;e sulandırınız </td></tr><tr><td> Hücre kültürü kaput </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> Doku kültürü inkübatör </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> poli-HEMA plakaları için inkübatör </td><td> Labline Aletleri </td><td> Imperial III 305 </td><td></td></tr><tr><td> Masaüstü santrifüj </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 sıra cam-alt tabak </td><td> MatTek Corp corp. </td><td> P06G-1,5-20-F </td><td></td></tr><tr><td> Fibronektin </td><td> Sigma </td><td> F1141-1MG </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td> Timelapse Mikroskop: </td><td></td><td></td><td></td></tr><tr><td> Mikroskop </td><td> Nikon </td><td></td><td> Entegre Mükemmel odak sistemi ile Ti-E Ters Motorlu Floresans time-lapse mikroskop </td></tr><tr><td> Lens </td><td> Nikon </td><td></td><td> 20X-0.75 sayısal Aperture </td></tr><tr><td> Halojen transilluminator&#39;de </td><td> Nikon </td><td></td><td> 0.52 NA uzun çalışma mesafesi kondenser </td></tr><tr><td> Uyarma ve emisyon filtreleri </td><td> Nikon konut Chroma tek geçiş filtreleri </td><td></td><td> GFP Ex 480/40, Em 525/50 TÇ-mCherry Ex 575/50 Em 640/50 </td></tr><tr><td> Bulaşan ve ışık yolu Epifluoresce </td><td> Sutter </td&gt; <td></td><td> Akıllı Kepenkler </td></tr><tr><td> Doğrusal kodlanmış motorlu sahne </td><td> Nikon </td><td></td><td></td></tr><tr><td> Soğutmalı şarj çiftli aygıt kamera </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Sıcaklık ve CO 2 kontrolü ile Mikroskop inkübasyon odası </td><td> isteğe göre yapılmış </td><td></td><td></td></tr><tr><td> Titreşim izolasyonu tablosu </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elements yazılımı </td><td> Nikon </td><td></td><td> Sürüm 3 </td></tr></tbody></table>

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

Over kanseri Amerika Birleşik Devletleri 1 kansere bağlı ölümlerin beşinci önde gelen nedenidir. Tedavilere olumlu bir ilk yanıt rağmen, yumurtalık kanseri olan kadınların 70 ila 90 oranında yeni metastaz geliştirmek ve nüks genellikle 2 ölümcüldür. Bu, orta ve geç evre yumurtalık kanseri için daha iyi tedaviler geliştirmek için ikincil metastazlar ortaya nasıl anlamak için, bu nedenle gereklidir. Malign hücrelerin primer tümörün sitesinden ayırmak ve periton boşluğuna genelinde yaygınlaştırmak zaman yumurtalık kanseri metastazı oluşur. Dissemine hücrelerin periton boşluğuna 3 (Şekil 1, Movie 1) içindeki organlar üzerine çok hücreli kümeleri, ya da küresel cisimler, ya kalır ayrıldığı edecek, ya da implant oluşturabilir. Periton boşluğu içinde organlarının Tüm mezotel hücreleri 4-6 (Şekil 2) bir tek, sürekli, tabaka ile kaplıdır. Ancak, mezotel hücre altından bulunmadığınaolarak eksize insan tümör doku kesitlerinde 3,5-7 (Şekil 2) elektron mikroskobu çalışmalarla ortaya peritoneal tümör kitleleri,. Bu mezotel hücreler bilinmeyen bir işlem tarafından tümör kitlesi altında dışında olduğunu göstermektedir. In vitro deneylerde Önceki primer over kanseri hücreleri 8 mezotel hücrelere daha ekstraselüler matriks daha verimli takın ve daha yeni çalışmalar primer periton mezotel hücreleri aslında yumurtalık kanseri hücre adezyon ve invazyon (as adhezyon ve invazyon göre bir bariyer sağlamak gösterdi gösterdi ) mezotelyal hücrelerle 9,10 kapsamı dışında kalan yüzeylerde. Bu mezotel hücreleri yumurtalık kanseri metastazı karşı bir bariyer görevi öneririm. Yumurtalık kanseri hücreleri bu engeli ihlal ve sahip mesothelium hariç hangi hücresel ve moleküler mekanizmalar, yakın zamana kadar bilinmeyen kaldı. İşte biz th tarifin vitro bir e metodolojisi bu modeller in vivo (Şekil 3, Movie 2) yumurtalık kanseri hücre sferoidler ve mezotel hücreleri arasındaki etkileşim. Bizim protokolü mezotel monotabaka 8-16 ile over tümör hücre etkileşimleri analiz etmek için daha önce açıklanan yöntemlerden adapte edildi ve önce o over tümör hücreleri hariç teşvik etmek miyozin ve çekiş kuvveti bir integrin bağımlı aktivasyon kullanmak gösteren bir rapor açıklanmıştır bir tümör sfero 17 altından mezotel hücreleri. Bu model etkileşime zamansal ve mekansal bilgi sağlayarak, gerçek zamanlı iki hücre popülasyonlarının izlenmesi için time-lapse floresan mikroskobu yararlanır. Mezotel hücreleri yeşil floresan protein (GFP) ifade ederken yumurtalık kanseri hücreleri kırmızı floresan proteini (TÇ) ifade eder. TTT-ifade yumurtalık kanseri hücresi sferoidler GFP-ifade mezotel monolayer iliştirin. Sferoidler yayılması, işgal vekenara tek tabakalı bir delik oluştururken mezotel hücreleri zorlamak. Bu delik GFP görüntü negatif boşluk (siyah) olarak görüntülenmiştir. Delik alanı ardından nicel kontrolü ve yumurtalık kanseri ve / veya mezotel hücrelerinin deneysel popülasyonlar arasındaki boşluk aktivite farklılıkları analiz etmek için ölçülebilir. Bu test yumurtalık kanseri hücreleri (koşul başına sfero X 20-30 sferoidler başına 100), hücrelerin sadece az sayıda gerektirir, bu yüzden değerli primer tümör hücre numuneleri kullanılarak bu deney yapmak mümkündür. Dahası, bu tahlilde kolayca yüksek verimli tarama teknikleri için adapte edilebilir.

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

Burada anlatılan mezotel klirensi tayini floresan ile işaretlenmiş hücreler ve zaman atlamalı video mikroskopi görselleştirmek ve kantitatif yumurtalık kanseri çok hücreli sferoidler ve mezotel hücre tek katmanlarının etkileşimleri ölçmek için yararlanır. Bu test yumurtalık kanseri metastaz erken adımları modeller.

In vitro Mezotelyal Gümrükleme Assay o Yumurtalık Kanseri Metastaz Erken Adımlar modeller

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

14.4K Görüntüleme.  Harvard Medical School. Burada anlatılan mezotel klirensi tayini floresan ile işaretlenmiş hücreler ve zaman atlamalı video mikroskopi görselleştirmek ve kantitatif yumurtalık kanseri çok hücreli sferoidler ve mezotel hücre tek katmanlarının etkileşimleri ölçmek için yararlanır. Bu test yumurtalık kanseri metastaz erken adımları modeller.

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Deneysel metastaz Testi

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

Tıp

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

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Tümör 4D Canlı hücre Görüntüleme için MAME Modeller: Mikroçevre etkileşimler olduğunu Etki Malign İlerlemesi

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

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Manyetik Rezonans Görüntüleme, Volumetrik Bilgisayarlı Tomografi ve Ultrason Kullanımı Meme Kanseri Kemik metastazı bir Nude Rat Model Angiogenezis Multi-modal Görüntüleme

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

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

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

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Üç boyutlu heterotipik<em&gt; In Vitro</emYumurtalık Kanseri Başlatma ve İlerleme sırasında Stromal-Epitel Etkileşimleri&gt; Modelleme

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Kemik Metastaz Modelleri

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

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Real Time Mezotelyal Hücrelerinin Yumurtalık Kanseri Sferoid Ek ve Invasion Değerlendirilmesi

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

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Yumurtalık Kanseri Tümör başlatan Hücrelerinin in vitro Zenginleşmeden içinde

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

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Üç boyutlu (3D) Tümör Sfero Yayılması deneyindeki

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

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

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

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.

Vivo ve Ex Vivo Periton yağ içinde Samanyolu Nokta Yapıların Yumurtalık Kanseri Metastatik Kolonizasyon Eğitim Yaklaşımlar içinde

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

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 women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

İçi periton Yumurtalık Kanseri Metastaz kantitasyonu

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 ...