Yazarlar ifşa hiçbir şey yok.

1. clonogenic Deneyi <ol><li> Adımları kullanarak MCF-7 ve T47D hücreleri aşağıda belirtilen estrojene bağlı meme kanseri hücre çizgilerinin bir tek hücre süspansiyonlarının hazırlanması <ol><li> MCF-7 ya da T-47D hücreleri ile en fazla% 50 konfluent bir 10 cm&#39;lik bir doku kültürü kabı kültür ortamı (DMEM /% 10 ısı ile inaktive edilmiş fetal dana serumu / glutamin ve pen / strep) aspire. PBS ile durulayın. 1-4 dakika boyunca, 37 ° C&#39;de% 0.25 / 2.21 mM EDTA DMEM yüksek glukoz içinde çözüldü tripsin kuluçkalayın. </li><li> Tek bir hücre dağılımı sağlamak için faz kontrast mikroskop altında 1 dakika aralıklarla hücreleri kontrol edin. Hemen hemen değişmez, tek hücre durumunu elde etmek için, hücre-hücre teması bozmak için birkaç kez pipetleme ve aşağı 2 mi pipet ile tekrar süspansiyon hücreleri. </li><li> Inkübasyon sadece 2 dakika sonra, hücre kümeleri gözlemlemek halinde en fazla 4 dakika boyunca 37 ° C&#39;de tripsin hücreleri inkübe devam edin. Onlar e yapışık kalırsa clonogenic çalışmalar için bu hücreleri kullanmayıntrypsinization 4 dakika sonra diğer ach hata koloni sayısı veriminde tanıtılacak çünkü. NOT: Hücreler clumped ise, oluşan kolonilerin sayısı kuluçkaya sayısından daha az hücre ürünü yansıtacaktır. Hücreler aşırı tripsinize edilir ise, bunların klonojenik potansiyeli azalabilir. </li><li> Için gerekli olan tüm birimi ihtiva eden bir ana tüp içinde seri seyreltmeler ile, (hücre türü ya da geçiş sayısına bağlı olarak, + 500 hücre / ml) 24 yuvalı plakalar ya da daha az, / ml kültür ortamı 1500 hücrelerinin tek bir hücre süspansiyonu hazırlamak deneyde tüm değişkenler. Not: Amaç 800 hücre / cm2 (yaklaşık 500 1.100 hücre / cm2 aralığında) bir nihai hücre yoğunluğu. amacı, nispeten kolay sayma izin veren, yaklaşık 100 ± 50 koloniler elde etmek için olan engellerse ve koloniler artan ya da deneysel perturba azalmıştır zaman istatistiksel olarak anlamlı farklar ile sonuçlanması için yeterli koloniler izin verirleri. </li></ol></li><li> Adımlar Kullanma Clonogenic Yoğunluk inkübe Hücreler Aşağıda Outlined <ol><li> 1500 hücre / ml&#39;lik bir ana tek bir hücre süspansiyonu tüp / oyuk 1500 hücre klonojenik yoğunlukta 24 yuvalı fibronektin kaplı plakalar üzerine dört kere ölçülen kuyucuklardaki hücreleri inkübe edin. 3 ml hazırlanması ve 2 kuyu her 1 ml ortam dağıtma ile 5 ml pipet olan hücreleri içeren bir ortam Karışım. Not: fibronektin kaplı plakalar önceden kaplanmış satın alınması gereken bir ticari bir satıcıdan. Kontrollü bir kalite dışında Kaplama tabaklar, pürüzlü bir yüzeyde otomatik işlem sonuçları bu deney için elverişsiz. </li><li> , 5 ml pipet ile pipetleme ve aşağı süspansiyon karıştırın hücre süspansiyonu 3 ml hazırlamak ve 1 ml her biri 2 kuyu doldurun. Tek pipet herhangi bir anda sadece 2 kuyu doldurun. Aşağı yukarı pipetleme ve tekrar ana tüp içinde hücre süspansiyonu pipet tekrar süspansiyon hücreleri kalan hacim dönün ve başka bir 2 wel doldurmak için başka 3 ml hazırlamak1 ml her biri ls. NOT: Hücreler sürekli tortu çünkü ana borusunun sürekli karıştırma gereklidir. Sadece 2 kuyu doldurmak için yeterli sesini Çizim hücrelerin yanı sıra pipet tortu, çünkü her bir kuyunun benzer hücre sayıları eklemek gerekir. </li><li> Izin hücreler clonogenic potansiyel (yayınlanmamış gözlemler) modüle edecek oda sıcaklığında ve CO 2 konsantrasyonunda süspansiyon oturup çünkü hücrelerin geniş hacimli dağıtmak için hızla çalışın. </li><li> Koloni deneyleri için kuyulara içine pipetleme eylemi sırasında hücrelerin mekansal dağılımını optimize edin. Yavaş yavaş kuyunun ortasına nihai hücre konsantrasyonu içeren süspansiyonu pipetleme bunu. Hücreler dibe önce hareket ilerletmek için plaka maruz bırakmayın. Dairesel karıştırma etkin bir 6 gün iyi yaratarak yüksek hücre yoğunlukları ve sayılamayan konfluen kolonilerinin çevresine hücreleri santrifüj çünkü plaka girdap etmeyin. NOT: Bu tüm süspansiyon hücreleri ile ses tanıttıktan sonra hiçbir karıştırma daha az tercih edilir çünkü gerekli olmadıkça karıştırmayın. Bu hücrelerin karışımı için gerekli ise, düz bir yüzeye duruyor ise dik yönlerde ileri geri hareket ettirerek plakayı bunu. </li><li> 6 gün süre ile ortam değişikliği olmadan, 37 ° C&#39;de% 5 CO2 ile inkübe hücreleri. iyi 6 gün sonra hücrelerin sayısının az ölçüde besin veya sitokin kompozisyonu ne de orijinal ortamın pH etkisi olmayacaktır. </li><li> Analiz aşağıda belirtildiği gibi zaman sürecini Tasarım: 1 ml taze ortam ve taze ortam içeren ortam, mevcut yerine 1. günde fibronektin kaplı alt tabakalarının üzerinde hücrelerin inkübe FGF-2 aşağıdaki 6. günde 0. günde Leke hücreleri üzerinde 10 ng / ml 1.4). ) 1.3 olarak aşağıda, 3. günde herhangi bir deneysel tedirginlikler yürütün. </li></ol></li><li> Moleküler Sinyali veya Adezyon Molekülleri Kurma Deneysel pertürbasyonlarının <ol><li> 3 gün, 100 eklemek56; kuyularda 1 mi ortamına bozucu maddenin nihai amaçlanan konsantrasyonu 10x ihtiva eden bir çözeltiye, l. Karıştırma. İlave bir 3 gün için 37 ° C,% 5 CO2 inkübe hücreleri devam edin. Not: maddeler rahatsızlık vermesini atıl durumunu desteklemek rol oynayabilir bir önleyicileri çeşitli ve yapışma moleküllerinin, reseptör veya diğer yüzey proteinleri, hücre içi sinyal yolları, moleküller, faktör, kofaktörler veya yapısal proteinlerin inhibitörleri bloke edici ajanlar içerebilir. </li><li> 6. günde leke koloniler, aşağıdaki gibi. </li></ol></li><li> Leke Koloniler <ol><li> % 2 etanol / 10 mM sodyum borat taze yapılmış bir% 0.1 kristal violet (pH 9.0) çözeltisi ile kültür içinde 6 gün sonra leke hücreleri. Aspire ortam ve 20 dakika boyunca her bir göze bir ml kristal viyole çözeltisi ilave edilir. </li><li> Bir buz kovası içine dar bir açıyla aşağı bakacak kuyu açıklıklar sürekli çalışan musluk ile taşan onları daldırarak plakaları yıkayınlavabo su. Kez sualtı yatay açı plaka yatırın kuyuya açarak ve bir yumuşak akan hareket çıkarırken daha sonra bir dar açıda geri yatırın. </li><li> Kuyuların dibinde su artık mavi olana kadar daldırma 2 ya da 3 kez tekrarlayın. Güçlü yıkama sayma ve veri hata önemli ölçüde katkı, deneysel müdahale daha az yapışkan olan hücreler ya da koloniler kaldırabilir. </li><li> Baş aşağı bunlara karşılık gelen etiketli kapakları bitişik tezgah üstünde havlu yerleştirerek gecede Kuru plakalar. </li></ol></li><li> Koloni Sayısı <ol><li> İnkübasyon, boyama ve kurutma her iyi sonra 6 gün büyüyen ve uykuda kolonilerin sayısını. Bir ters faz kontrast mikroskop optimal 40X büyütmede kolonileri sayın. Hücreler büyüdükten büyük expa kıyasla çok büyük boyutta morfolojik görünüşü ile, büyüyen ve 12 ya da daha az hücre koloniler olarak&gt; 30 hücre kolonileri sayınBüyük sitoplazmalı NDED sitoplazma Şekil 1&#39;de 7,15 gösterilen oranları, çekirdeğe. NOT: basit uyuşukluk tayinlerde çok sık değil gibi 13-29 hücre kümeleri normal sayılmaz. Tedirginlikler ya büyüyen ya da durağan hücrelerin büyüme potansiyelini vardiya halinde Onlar sayılabilir. Bu sonuçlar, daha sonra biyolojik öneme sahip ilişki gerekecektir. </li></ol></li></ol> İmmünofloresan Çalışmaları 2. clonogenic Kuluçka <ol><li> Sayılar / 24 yuvalı deneylere benzer bir yüzey alanı hücre karşılık, 6 oyuklu plakalarda / oyuk yaklaşık 7,500-8,000 hücrelere hücre sayısını ayarlar. </li><li> Hücre eklenmesinden önce görüntüleme çalışmaları için 6 oyuklu plakaların her bir oyuğa tabanına yuvarlak steril, fibronektin kaplı kapak kayma yerleştirin. ) 1.2 yukarıda clonogenic deneyler için tarif edildiği gibi, her kuyuya 3 ml hacminde Pipetleyin hücreleri konsantrasyonlarda bir anda 2 kuyu, ana hatlarıyla. </li><li> Inkübe hücreleriYukarıdaki 1.2.4 ve 1.2.5, 6 gün için. 1,3 koloni deneyi prosedürleri de tarif edildiği gibi), 300 ul hacminde 10 x konsantrasyonda günde 3 bozucu etkenler ekleyin. </li><li> Antikorlarla 6. günde Leke hücreleri, örneğin integrinler α4, α5, α6, β1, β3, örneğin fokal yapışma karmaşık moleküllerdir FAK, paxillin ve vinkülin olarak hücre yapıştırma moleküllerine, örneğin α-tubulin motilitesinde karışan proteinler, örneğin, fosfo-Akt, fosfo-ERK, fosfo-p38, fosfo-JNK, örneğin, ya da uyuşukluk rolü için araştırmanın hedefi, doğrudan ya da dolaylı olarak ilişkin standart teknikler kullanılarak, herhangi bir başka protein gibi sinyal yolu üyeleri immünofloresan boyama. <ol><li> Aseton forseps gün 6. Fix slaytları çıkarın / metanol 1: 1 -20 ° C&#39;de 20 dakika ve kuru hava için ° C. Alternatif bir sabitleyici, gerektiğinde paraformaldehid gibi, kullanılabilmektedir. 2 dakika f% 0.1 Triton X-100,% 0.1 sodyum sitrat ile Permeabilize hücrelerihücre içi antijenlere ya da tespiti. Hücrelerin PBS ile yıkayın. </li><li> Dolaylı immunofluorecence boyama,% 5 BSA ile oda sıcaklığında 1 saat süre ile ya da ikincil bir antikor elde edildiği türe% 10 preimmün serum blok slaytlar için. </li><li> Yol üyeleri veya PBS içinde üretici tarafından tavsiye edilen özel dilüsyonları seyreltilmiş uyuşukluk rolü için araştırmanın hedefi olan herhangi bir başka protein sinyalizasyon, yapışma molekülleri, fokal moleküllerin hücre primer antikorlar ile 4 ° C&#39;de gece boyunca inkübe% 0.1 TRİTON X- -100. </li><li> 3 kez PBS ile yıkayın. 2 st için oda sıcaklığında florofor-konjuge antikor ile kapak slipi inkübe edin. Bir örnek olarak, Alexa Fluor 488 eşek anti-fare IgG antikoru murin monoklonal primer antikorları tespit etmek için kullanılabilir. Dağı Dapi ile antifade madde kullanılarak cam slaytlar aşağı hücre tarafı lamelleri. Oje ile çevrelerinin Seal. </li><li> Doğrudan Immünofloresan için, BSA yürütmekYukarıdaki 2.4.2) bloke 4 ° C&#39;de gece boyunca florofor ile konjüge primer antikor ile inkübe edin. 3 kez PBS ile yıkayın. Oje ile bir antifade ajan ve Dapi ve mühür ile slaytlar kuluçkaya yatmaktadır. </li><li> 4 ° C&#39;de alüminyum folyo ve mağaza ile Kapak slayt tepsiler Görüntüleme ve fotoğrafçılık zaman kadar birkaç hafta sonra C °. 1,000x büyütmede bir kamera ile donatılmış herhangi bir floresan mikroskobik görüntüleme sistemlerini kullanarak görüntüleme ve fotoğraf hücreleri. </li><li> Fibril aktin boyama için,% 1 sığır serum albümini (BSA), blok slaytlar, 30 dakika ve 20 dakika boyunca oda sıcaklığında BODIPY FL-Phallacidin (yeşil) ya da Rhodamine phalloidin (kırmızı) inkübe edilir. Bir antifade maddesi ekleyin ve yukarıdaki gibi mühür. </li></ol></li></ol> Moleküler Çalışmaları 3. clonogenic Kuluçka <ol><li> Western blot <ol><li> Fibronec 20.000 hücre / 60 mm levha ve 50,000 hücre / 100 mm plaka klonojenik yoğunluklarda ER + meme kanseri hücreleri, MCF-7 ya da T47D inkübekalay kaplı% 5 CO2 içinde 37 ° C&#39;de plakalar. </li><li> Lizatlanndan protein mg miktarlarda gerektiren moleküler çalışmalar için 75.000 hücre / 100 mm plaka biraz daha yüksek yoğunluklarda hücreleri inkübe. Western lekeleri kullanılarak molekül çalışmalar ya da Northern blotlar için RNA izolasyonu için yeterli protein toplamak için deney noktası için on plakalara kullanın. </li><li> Hücre sayısı göre jel yükleme çok farklı büyüklükte büyüyen ve durağan hücrelerde protein ifadesini karşılaştırmak için gerekli bir ilave maddedir. Trypsinizing hücreleri toplamak ve yerine tek kenar bıçak ile hücrelerin kapalı kazıma Western lekeleriyle için lizatları hazırlanması için% 0.2 Tripan Mavi bir hemositometrede bir kısım saymak. </li><li> Santrifüj hücreler 2 dakika için 10,000 x g&#39;de, aspirasyon ile ortamını çıkarın 200 ul liziz tamponu ilave lizis tampon hücreleri sonikasyon ve protein konsantrasyonu belirlenir. </li><li> Hücrelerin sayısına göre protein verimi bölünmesiyle hücre başına protein miktarını hesaplayıno miktarda üretilir. Eşdeğer hücre sayıları temsil protein) eşdeğer miktarlarda ve B) protein miktarları hem de temsil eden ayrı bir poliakrilamid jeller her kuyuya lizat yükleyin. En az 25 ug protein, her bir kuyunun içine yüklenmiş olması gerekir. NOT: Bu boyut ve protein içeriği (şekil 1) önemli ölçüde farklı olan büyüyen ve uykuda hücreleri arasında karşılaştırmalar izin verir. </li></ol></li><li> Akış sitometrisi <ol><li> 3.1 gibi, tripsinizasyon ile 100 mm tabak hücreleri toplamak ve 2.4 de tarif edilen hücre içi ya da hücre dışı antijenlere ya birincil veya ikincil immünofloresan boyama ile (FACS) protokollerini ayırma, standart floresan aktive hücre tarafından analiz eder. Not: Antikor etiketleme ile belirlendiği gibi tripsinizasyon ile doku kültürü plakalarından hücrelerin ayrılması membran proteinlerinin konsantrasyonu etkilemez. </li></ol></li></ol>

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Cellular Physiology. 177, 411-425 (1998).</li><li>Brunner, G., Gabrilove, J., Rifkin, D. B., Wilson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+release+of+basic+fibroblast+growth+factor+from+human+bone+marrow+cultures+as+a+biologically+active+complex+with+a+phosphatidylinositol-anchored+heparan+sulfate+proteoglycan.">Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.</a> J Cell Biol. 114 (6), 1275-1283 (1991).</li><li>Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., Gabrilove, J. 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Savunma Hibeler DAMD17-01-C-0343 Bölümü ve DAMD17-03-1-0524, New Jersey Eyalet Komisyonu Kanser Araştırma 02-1140-CCR-E0 ve Kanser Araştırma Ruth Estrin Goldberg Memorial (RW) tarafından desteklenen

Bizim modeli kemik iliğindeki uyuşukluk birkaç anahtar unsurlarından oluşur. O fibronektin, kemik iliği önemli bir yapı elemanının, FGF-2, bir büyüme faktörü bol kemik iliği stroma ile sentezlenebilir oluşur, östrojen uzun süreler için 10 iliğinde atıl kalması muhtemel tip duyarlı hücreler, oluşmaktadır ve ağır etkileşimleri alt tabaka ile öncelikle klonojenik yoğunlukta hücrelerin kemik iliğinden 31,32 ve inkübasyon dışı matriks içinde yatırılır. Kemik ili?...

Hastalık kısa sürede küçük tümörler kan damarlarını 2,3 geliştirmek olarak saptanabilir 1 önce meme kanseri hücrelerinin kemik iliğine metastaz. Metastatik sürecin hızlı ama verimsiz. Hücreler günde 4 başına milyonlarca hızla yeni kan damarları girmek ancak birkaç uzak organlara 5 gezi hayatta. Bununla beraber, bazı mikrometastazlar kemik hayatta ve tek hücre veya yeni tanı konmuş hastaların 1 kemik iliği aspiratlarında küçük hücreli kümeleri olarak bulunabilir. Bu hücreler, onları 6 ortadan kaldırmak çok amaç için uygulanır adjuvan kemoterapi, karşı. Bu direnç kemik iliği mikroçevresindeki 7,8 ile etkileşimler tarafından başlatılan sinyalizasyon canlılığında, esasen, sahip olduğunu. Mikrometastaz lokalize meme kanseri olan kadınların yaklaşık üçte birinde bulunabilir ve tek değişkenli analizde 9 tarafından bakıldığında sağkalım bağımsız göstergesi temsil edilebilir. Bazı micromeyıda metastaz yapmış büyüme başlatılan, ancak nüks hücre türüne bağlıdır. Üçlü negatif meme kanserli hastalar uykuda devlet üzerinde kötü kontrol düşündüren, 1 ila 4 yıl arasında tekrarlama eğilimindedir. ER / PR + hücreleri de dahil olmak üzere diğer hücre tipleri, nüks 10 sabit, sürekli oranı ile en fazla 20 yıl boyunca uykuda kalabilir. ER + ve ER- meme hücre hatları ve tümörler arasında dinlenme gen ifadesi imzaları farklılıklar farklı dinlenme potansiyelleri 11 yansıtmak iken, kemik iliği stroma ile etkileşimleri olası dinlenmesi önemli bir katkı oluşturmaktadır. mikrometastazlar nadirdir ve 10&#39;dan fazla 6 kat ile hematopoetik hücreler tarafından sayıca az, çünkü in vivo uyuşukluk çalışma son derece zordur. Bu nedenle, ilgili modelleri mekanizmaları önermek ve in vivo test edilebilir hipotezler üretebilir in vitro verilere sağladığı oluşturulmalıdır. MATHE içeren dinlenme modellerinin bir sayımatical modeller 12,13, in vitro modeller 7,8,14,15, in vivo ksenograft modelleri 16, in vitro kombinasyonları ve ksenograft modelleri 17,18 ve spontan tümör ve metastaz modelleri 19, kanser hücresi dinlenmesi 20 içine bazı bilgiler vermiştir . Bu modellerin her biri, kendi sınırlamaları vardır ve kendileri moleküler sinyalizasyon ve dormansi daha biyolojik ilgili modelleri test edilmesi yöneten etkileşimler ile ilgili hipotezler üretme için öncelikle yararlıdır. Dormansinin moleküler mekanizmaları, ER + hücreleri nüks neden döngüsü tutuklama, redifferentiation ve tedavi direnci ve mekanizmalar ile sonuçlanan mikroçevresinin ile etkileşimleri tanımlayan genel hedefi ile, ilgili unsurları seçilmiş sağlayan bir in vitro model geliştirdi stromal mikroçevresinin 7. Kendi bilefleninde ise nispeten seyrek Bu model,alınan parçaları, dormansi önemli fonksiyonlarını etkileyen spesifik moleküler mekanizmaları elde etmek için araştırmacılar izin yeterince sağlamdır. Bu deneyler, doğrudan in vivo test edilebilir hipotezler üretir. Model biz dormansi ilgili olduğu gösterilmiştir birkaç temel unsuru dayanır. Bunlar etkileşimi alt tabakası ve orta çözünür bileşenler, bir fibronektin alt tabaka ve bazik fibroblast büyüme faktörünün varlığı ile öncelikle bir klonojenik yoğunlukta estrojene bağlı meme kanseri hücrelerinin, hücre kültürünün kullanımı (FGF-2) ortam içinde. Biz, TGFβ 22 aracılık FGF-2 21 ile hücre döngüsü tutuklama indüksiyon dahil olmak üzere in vitro sistemi yöneten mekanizmalar, karakterize bağlıydı bir epitel fenotip için PI3 Kinaz 7,8 ve ERK 8 ve morfojenik farklılaşma yoluyla sinyal sağkalım RhoA inaktivasyonu integrin45; 5β1 upregülasyonu ve hayatta kalma için 7,15 stromal fibronektin ligasyonu (Şekil 1). MCF-7 hücreleri üzerinde FGF-2, in vitro hücre döngüsü etkileri konsantrasyonlarda 10 ng / ml 21,23 altında en az bir günlük başlar. mantık memeli sistemlerinde 24-27 bir dizi meme kanalı morfojenezi siklik genişleme ve durgunluk yöneten FGF-2 ifadesinin zamansal kontrol dayanıyordu. Biz FGF-2 duktal 3D kültür 28 morfogenez ve genellikle insan tümörlerinin 29 malign transformasyonu ile kaybolur FGF-2 ifadesi de dahil olmak üzere, farklılaşmasını uyardığı gösterilmiştir. FGR1 ifadesi, tüm 4 FGF alıcılarını 30 ifade etmeye devam 29 ve MCF-7 hücreleri incelenen göğüs karsinomalarında bozulmadan kalmıştır. Uyuşukluk bağlamında, FGF-2 tarafından verilen ve ağır hematopoetik kök hücrelerin 33 korunmasında fonksiyonları, kemik iliği stroma 31,32 üzerine bırakılır. Biz demFGF-2 o morfojenik farklılaşmasını 7 indükler kemik iliğinde, ayrıca bol fibronektin Tabakasındaki, kültüre ER + meme kanseri hücrelerinde sönmüş bir devlet uyardığı belirlenmiütir. Modelde, göğüs kanseri hücrelerinin büyümesi inhibe edilir, RhoGap GRAF ile Rho A inaktive epitelyal fenotipe redifferentiate ve habis ilerlemesi ile integrinleri α5β1 lost-ekspres yeniden. Bu integrin α5β1 ile fibronektin bağlanan ve bu sitotoksik tedavi 7,8,15 (Şekil 1), karşı dirençli hale sinyal hayatta aktive eder. Rho sınıfı GTPases inhibisyonu atıl bir fenotip 34 uyarılması için, daha önce ortaya konmuştur. Burada modelini kurmak ve ER + meme kanseri hücrelerinin dormansi düzenleyen spesifik moleküler ve hücresel mekanizmaları incelemek için araştırmacılar izin verecektir belirli prosedürleri açıklayacağım. Burada yer alan deneylerde modeli kullanımını göstermek içinBiz bir Akt inhibitörü ve bir PI3K inhibitörü ve bir pan-Rho inhibitörü ve bir Rho Kinaz (ROCK) inhibitörü ile Rho ailesi (Şekil 1B) tüm üyeleri ile PI3K yolu (Şekil 1B) hedef aldı.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MCF-7 cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>T47D cells&#160;</td>
			<td>ATCC</td>
			<td>HTB-123</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td>
			<td>Corning</td>
			<td>354411</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 60 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354403</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 100 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354451</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td>
			<td>Corning</td>
			<td>354088</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well tissue culture plate</td>
			<td>CellTreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td>
			<td>Corning Life Sciences</td>
			<td>50-013-PB</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated, Fetal Bovine Serum</td>
			<td>Serum Source International</td>
			<td>FB02-500HI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin/2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, 100x, Liquid</td>
			<td>Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF&#160;basic</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td>
			<td>CalBiochem</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr>
			<td>LY294002&#160;</td>
			<td>CalBiochem</td>
			<td>19-142</td>
			<td>Chenical PI3K inhibitor</td>
		</tr>
		<tr>
			<td>C3 transferase&#160;</td>
			<td>Cytoskeleton</td>
			<td>CTO3</td>
			<td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>129830-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY FL-Phallacidin (green)&#160;</td>
			<td>Molecular Probes</td>
			<td>B607</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>BODIPY FL-Rhodamine phalloidin (red)&#160;</td>
			<td>Molecular Probes</td>
			<td>R415</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&#160;</td>
			<td>Molecular Probes</td>
			<td>R37114</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI&#160;</td>
			<td>Molecular Probes</td>
			<td>P-36931</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

<html> Deneyler tahlili özetlemek için yürütülmüştür. Deney zaman süreci Şekil 2A&#39;da gösterilmiştir. Hücreler, gün -1 &#39;de klonojenik yoğunlukta inkübe edilir, FGF-2, taze ortam içinde 0. günde ilave edilir ve boyanmış ve koloniler sayılmıştır, hücreler 6 gün kadar kültürlenir. Sistem için herhangi bir pertürbasyonlar 10 X son konsantrasyonda 100 ul hacim içinde 3. günde tatbik edilmektedir, istenen. Şekil 2B büyüyen ve atıl kolonilerin tipik bi...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Bir<em&gt; In Vitro</em&gt; Kemik İliği östrojen duyarlı Meme Kanseri Dormansi Model: Moleküler Mekanizması Çalışmaları ve Hipotez Üretimi için A Takım

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

8.2K Görüntüleme.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

Video: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Bir In Vitro</emMetastatik Büyüme Tümör Dormansi ve Switch Eğitim> Sistem

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

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Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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 ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Bir Zebra balığı Otokton Tümör Modeli kullanılarak Melanom Değiştiricileri için Tarama

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Rekabetçi bir mürin Kemik İliği Transplantasyon Modelinde Donör Hücre Aşı belirleme Kantitatif Gerçek-zamanlı PCR kullanarak

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Canlı Farelerde Femoral Kemik İliği Aspirasyon

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Bir<em&gt; Ex vivo</em&gt; Model İnsan Meme Hormon Eylem Eğitim için

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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 ...

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