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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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><tbody><tr><td>MCF-7 cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>T47D cells&amp;nbsp;</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&amp;nbsp;basic</td><td>R&amp;amp;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&amp;nbsp;</td><td>CalBiochem</td><td>19-142</td><td>Chenical PI3K inhibitor</td></tr><tr><td>C3 transferase&amp;nbsp;</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&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>129830-28-2</td><td></td></tr><tr><td>BODIPY FL-Phallacidin (green)&amp;nbsp;</td><td>Molecular Probes</td><td>B607</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>BODIPY FL-Rhodamine phalloidin (red)&amp;nbsp;</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&amp;nbsp;</td><td>Molecular Probes</td><td>R37114</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI&amp;nbsp;</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...

Watch this Scientific Journal Video about An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation at JoVE.co

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

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Osteoclastogenesis periferik kan monosit kültürlü meme kanseri hücre hatları ile birlikte gelen bir insan preklinik modelinin geliştirilmesi

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Kanser Araştırmaları

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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Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Üç boyutlu kemik hücre dışı matriks modeli osteosarkom için

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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 ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Meme Kanseri için Preklinik Modeller Olarak Meme Tümörlerinin Ortopedik Transplantasyonu

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

İnsan Meme Kanseri Kök Hücrelerinin Hücre ve Doku Örneklerinden İzolasyonu ve Fonksiyonel Değerlendirmesi

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Meme Kanseri Araştırmaları için Potansiyel Fonksiyonel In vitro Model Olarak Meme Epitel ve Endotel Hücre Sferoidleri

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Meme Kanseri Hücrelerinde östrojen düzenlenmiş microRNA profil

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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 ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Kültürleme İnsan Femur Doku Eksplantlarından yöntemleri Metastatik Niche Meme Kanseri Hücre Kolonizasyon Eğitim için

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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