This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

Bazal Membran Matrix Meme Kanseri Hücreleri 1. Üç boyutlu Kültür (gömme Tekniği) <ol><li> Taşıma Matrigel bazal membran matrisi: 4 ° C&#39;de gece boyunca buz üzerinde Çözülme Taban membran matrisi düşük bir sıcaklıkta sıvı olan, ancak oda sıcaklığında katılaşır. Buz (Şekil 1A-B) ile ilgili bazal membran matrisi tutun. </li><li> Spiral bir desen (Şekil 1C) ve P-200 Pipetman ucunu kullanarak matris yayma vasıtasıyla taban membran matrisi 50 ul Konfokal No.1 cam alt çanak örtün. Hava kabarcıklarının oluşumunu önlemek için bazal membran matrisi yayılan dikkatli kullanın. Aynı şekilde, menisküs oluşmasını önlemek için, cam alt-çanak sınırlarına yakın matris yayılmasını önlemek. Deneyimsiz işleme bazal membran matris, daha sonra buz üzerinde olabilir çanak, P-200 Pipetman ve pipetler soğutulmuş halinde (alternatif olarak 4 ° C &#39;de bir buzdolabında gece boyunca bırakılmıştır.) Bu adım tekliflerkatılaşmadan önce bazal membran matrisi yaymak için ek süre. (Şekil 1D) katılaşması için temel zar matris sağlamak için en az 30 dakika boyunca (% 5 CO2 ile 37 ° C &#39;de) bir hücre kültürü inkübatöründe çanak (ler) yerleştirin. </li><li> Matris katılaşma geçirmekte iken, hücre% 70-80 konfluent 100 mm plaka trypsinize. Hücreler, plakanın kapalı kaldırma bir kez başladıktan sonra, tripsin (Şekil 1 E) etkisiz hale getirmek için,% 10 (v / v) cenin sığır serumu (FBS) içeren RPMI 1640 ortamında 10 ml bunları tekrar süspansiyon. Daha sonra 15 ml konik bir tüp (Şekil 1F) içine yeniden süspansiyona hücreleri aktarın. </li><li> Özel bir hücre kültürü santrifüj (Şekil 1G-H), 3 dakika boyunca 100 x g (konik bir tüp içinde mevcut olan) hücreleri dönerler. </li><li> Hücreleri aşağı bükülmüş edilirken, bir 1 ml mikrosantrifüj tüp (not içine kısım matris 50 ul: Her cam dipli çanak bir mikrosantrifüj tüpü tahsis edilir, bu nedenle, eğerDeney, 3 yemekler gerektirir o) 3 mikrosantrifüj tüpleri de gerekli olduğu takip eder ve daha sonra buz (Şekil 1 B) tüpü yerleştirin. </li><li> (Şekil 1 H) rahatsız pelet terk ederken, aşama 1.4 &#39;dan konik tüp arasındaki orta aspire. Hücreler yeniden süspanse FBS ile desteklenmiş RPMI ortamı (Şekil 1J) 1 ml (aşağı bükülmüş olması). </li><li> Hücreler mikrosantrifüj tüpü içine, bir hemasitometre (Şekil 1 K), veya bir hücre partikül sayacı kısım 2.5 x 10 4 hücreleri kullanılarak sayılmıştır ve 50 ul (Şekil 1 L) toplam hacim elde etmek üzere, uygun ortam kullanılarak kapalı üst kez. </li><li> 1:1 oranında 1.5 adım matris içeren mikrosantrifüj tüpü ile adım 1,7 (50 ul içinde 25,000 hücre) gelen hücreleri karıştırın; Nihai hacim 100 ul (Şekil 1 M) olacaktır. </li><li> S üzerine aşama 1.8 &#39;den hücre karışımı: yavaşça matrisin 100 ul plakaadım 1.2 (Şekil 1N) den olidified bazal membran matris kaplı çanak. Hücreler bazal membran matris içine gömülü olması için bu sağlar. </li><li> (% 5 CO2 ile 37 ° C) hücre kültür inkübatörü içine çanak (ler) aktarın ve matris sağlar: Hücre karışımı en az 30 dakika boyunca katılaşmaya. </li><li> Matris kez: hücre karışımı katılaşmış, çanak (Şekil 1O) için FBS-desteklenmiş RPMI medya 2 ml ekleyin ve deney (Şekil 1P) geri kalanı için saklanır inkübatör çanak geri almak. </li><li> (Bir tahlil için gerekli başına ya da, MDA-MB-231 hücreleri için, tahlilin süresi uzunluğu 5 gündür), 5 gün süreyle her gün ortam kullanın. </li><li> Bir ışık mikroskobu kullanılarak, diferansiyel girişim kontrast taban membran matrisi (Şekil 3A) içinde süspansiyon haline MDA-MB-231 koloni (DIC) görüntüleri alır. Görüntü 20 temsili alanlar 10X objektif seferde bir günBeş gün koloni morfolojilerinden (Şekil 3C) belirlemek için. </li><li> Hücre kolonisi Yıldızsı oluşumu (Şekil 3B) belirlemek için körü körüne görüntüleri analiz. A koloni hücrelerin sfero bir veya daha fazla çıkıntılar algılandığı takdirde yıldız şeklinde olduğu düşünülmektedir. Invazif stellate kolonilerin yüzdesini belirlemek elde edilen görüntünün başına hücre kolonilerinin toplam sayısına göre Stellate kolonilerin sayısını bölmek ve sonra her gün için 20 görüntülerin stellate kolonileri yüzdeleri ortalama için. </li></ol><img alt="Şekil 1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> Şekil 1. Taban membran matrisi (gömme teknik), MDA-MB-231 göğüs kanseri hücrelerinin üç boyutlu bir kültür. AB) çeker ocak içinde gerçekleştirilen deney kurulumu () kaplanmış cam tabanlı bir çanak. ° C) iyi bir şematiktaban membran matrisi 50 ul, en az 30 dakika boyunca katılaşması için matris izin vermek üzere% 5 CO2) 37 ° C &#39;de (bir hücre kültür inkübatörü içine yerleştirildi. D) Çanak (lar) ı ile. E) Hücreler, tripsinize edilmiştir. F ) Hücreler konik bir tüp içinde mevcut. GH) Hücreler (), bir doku kültürü santrifüj içinde 3 dakika boyunca 100 x g hızında döndürüldü edildi, 15 ml konik bir tüp içine yeniden süspanse edildi. I) Hücre topağı, aşağı döndürülmüştür edilmiştir. J) Hücreler () FBS ile desteklenmiş RPMI ortamı içinde, 1 ml olarak yeniden süspansiyon haline getirildi. K) Hücreler bir hemasitometre. L kullanılarak sayıldı) mikrosantrifüj tüpüne halinde bölünerek, 50 ul toplam hacim elde etmek üzere, uygun ortam kullanılarak tepesinde 2.5 x 10 4 hücreleri. M ), bir 1:1 oranında 1.5 adım matris içeren mikrosantrifüj tüpü ile adım 1.7 (50 ul içinde 25,000 hücre) hücreleri karıştırın; Nihai hacim 100 μ olacak.. Hücre karışımı katılaştırılır, hiç FBS ile desteklenmiş RPMI ortamı 2 ml ekleyin: matris kez) aşama 1.2 &#39;den katılaşmış matris ile kaplanmış yemek O üzerine adım 8 hücre karışımı:, l H) yavaşça, matrisin 100 ul plaka çanak. P) bu deneyin geri kalan saklanacak inkübatör çanak yerleştirin. İmmünofloresan 3D Kültürleri morfojenık Özellikleri 2. Incelenmesi <ol><li> Çanak (ler) üzerinden geçmeden önce (Şekil 2A),% 80 metanol (tespit edici çözelti) ve% 3 sığır serum albümini (BSA bloke çözeltisi) bir buz kaseti, soğuk fosfat tamponlu çözelti (PBS),% 20 aseton ayarlama inkübatör. </li><li> Buzluk ve aspire ortama çanak (ler) koyun, daha sonra soğuk PBS ve 2 ml (Şekil 2B) ile 3 kez yıkayın. </li><li> Son yıkama PBS aspire edildikten sonra,% 20 aseton içinde 2 ml ekleyin: çanak (ler) (Şekil 2C halinde% 80 metanol çözeltisi </st)&gt; rong 4 ° C&#39;de (buz üzerinde, ya da bir buzdolabında ya da) (Şekil 2B) &#39;de 20 dakika boyunca hücreleri düzeltmek için. </li><li> Fiksasyon 20 dakika sonra sona erer, oda sıcaklığında yemekleri geri getirmek fiksatif havalandırın, ve daha sonra 3 kez PBS ile yıkayın. Nihai yıkama PBS aspire edildikten sonra, oda sıcaklığında (Şekil 2E) en az 30 dakika boyunca bloke etmek üzere çanak (ler) için% 3 BSA 2 ml ekleyin. </li><li> Bloke dönemde blokaj tamponu% 3 sığır serum albümini (BSA) içinde çözülmüş (uygun dilüsyonda) birincil (laminin V 1:100) ve ikincil antikor seyreltileri hazırlanır. Hücresi karışımı: &#39;BSA + primer antikor&#39; çözüm ul 400-500 matrisi doğrudan eklenmesi gerektiğini unutmayın. </li><li> Tıkanması süresi 30 dakika sonra, birincil antikor ekleyin ve oda sıcaklığında (Şekil 2F) en az 1 saat boyunca inkübe edilir. Hiçbir PBS yıkar 30 dk Blockin aşağıdaki yapılması gerektiğini unutmayıng dönem. </li><li> Aşama 2.6 tamamlanmasından sonra, &#39;BSA + birincil antikor &quot;solüsyon çıkarın ve 3 kez PBS ile yıkayın. </li><li> Doğrudan bir matris üzerine (uygun seyreltmede)% 3 BSA içinde çözülmüş ikincil antikor ekleyin: Hücre karışımı, yemekler kapak ve oda sıcaklığında (Şekil 2G) 1 saat boyunca inkübe edilir. </li><li> &#39;BSA + ikincil antikor&#39; çözüm çıkarın ve PBS ile 3 kez yıkayın. </li><li> Çekirdekler boyama için PBS içinde çözüldü, Hoechst 33258 (1:10,000) 2 ml ilave edilir, ve alüminyum folyo altında 5 dakika boyunca inkübe (veya photobleaching sınırlamak amacıyla ışık geçirmeyen bir kap ile kapak) (Şekil 2H). </li><li> Hoechst 33258t ile inkübasyon süresi 5 dakika sonra, PBS ile (ler) 5x çanak yıkayın. </li><li> Doğrudan matris üzerine montaj orta ekleyin: konfokal çanak hücre karışımı ve bazal membran Matri içinde kolonilerin bütünlüğüne herhangi aksaklıkları uyaran önlemek için dikkatli bir şekilde cam lamel ile kaplayınx: hücre karışımı (Şekil 2I). </li><li> Çanak (es) oda sıcaklığında (Şekil 2I) de gecede (veya 24 saat) kurumaya bırakın. Kuruduktan sonra, çanak (ler) -20 ° C&#39;de muhafaza edilebilir, ancak bunlar oldukça kısa sürede görüntülü gerektiği tavsiye edilir. </li><li> (Şekil 3B-E) kullanılan antikorların uygun lazer dalga boyları ile bir floresan mikroskop kullanılarak görüntü kazanır. </li></ol><img alt="Şekil 2," fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> Bağışıklık 3D kültürlerin Şekil 2.. Incelenmesi. . A) deney düzeneği şematik B) Çanak (es) buz tepsiye yerleştirilir ve medya aspire; . sonra hücreler soğuk PBS ve 2 ml C) 2 ml% 20 aseton ile üç kez yıkanmıştır:.% 80 metanol çözeltisi D) için, hücreler çanak (ler) ilave4 ° C&#39;de (ya da buz üzerinde, ya da bir buzdolabı içinde) 20 dakika. E)% 3 BSA içinde 2 ml, oda sıcaklığında en az 30 dakika boyunca bloke etmek üzere çanak (ler) ilave edildi. F) 30 dakika sonra, bloke sona ermiş, birincil antikor ekleyin ve oda sıcaklığında en az 1 saat süreyle inkübe G) () uygun seyreltme ile% 3 BSA içinde çözülmüş ikincil antikorlar taban membran matrisi üzerine direkt olarak ilave edildi:. hücre karışımı; . ardından yemekler üzeri örtüldü ve oda sıcaklığında 1 saat boyunca kuluçkaya H) Hoechst 2 ml (1:10000, PBS içinde çözülmüş) alüminyum folyo altında 5 dakika boyunca inkübe diShe (leriyle) ve hücrelere eklendi I) montaj ortamı, doğrudan ilave edildi. bir matris üzerine: konfokal çanak ve tabak içinde hücre karışımı cam lamel ile kaplıdır. Çanak (ler), oda sıcaklığında bir gece boyunca (veya 24 saat) kurutulmuştur.

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta4+integrin-dependent+formation+of+polarized+three-dimensional+architecture+confers+resistance+to+apoptosis+in+normal+and+malignant+mammary+epithelium.">beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.</a> Cancer Cell. 2, 205-216 (2002).</li><li>Cvetkovic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KISS1R+Induces+Invasiveness+of+Estrogen+Receptor-Negative+Human+Mammary+Epithelial+and+Breast+Cancer+Cells.">KISS1R Induces Invasiveness of Estrogen Receptor-Negative Human Mammary Epithelial and Breast Cancer Cells.</a> Endocrinology. , 1999-2014 (2013).</li><li>Alemayehu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Arrestin2+regulates+lysophosphatidic+acid-induced+human+breast+tumor+cell+migration+and+invasion+via+Rap1+and+IQGAP1.">beta-Arrestin2 regulates lysophosphatidic acid-induced human breast tumor cell migration and invasion via Rap1 and IQGAP1.</a> PLoS One. , (2013).</li></ol>

Biz ifşa hiçbir şey yok.

3D hücre kültürü teknikleri geliştirilmesi araştırmacılar bize dramatik morfolojik değişikliklere görselleştirmek için izin göğüs epitel hücrelerinin dönüşümünü incelemek için izin verdi. Hücre istilasını analiz Bunun yanı sıra, tek ya da çok-hücreli küreler, meme epitelyal hücre yapışması, proliferasyon, boyut ve bazal-apikal polariteli değişiklikleri değerlendirmek için kullanılabilir. Hücreler ECM 8 ile kaplanmış olan daha önce bildirilen yöntemlere de, bizim s...

Göç ve bireysel ya da kolektif hücrelerin işgali kanseri iki özellikleridir ve kanser hücrelerinin 1-4 metastatik yayılması için gereklidir. Metastaz başlatmak için kanser hücrelerinin yeteneği göç ve hücrelerin, temel zara inerek için invadopodia kullanarak komşu doku içine işgal etme yeteneğine bağlıdır. Invadopodia matris-bozucu proteazlar 5 salınmasıyla hücre dışı matrisin bozulmasını sağlayacak dinamik aktin zengin matris bozulması çıkıntılar bulunmaktadır. Kanser hücre istilası kanser hücrelerinin taşınması takip matrisin bozulmasını içerir ve bu, üç boyutlu (3D) matris ortamı 2 bir yeniden eşlik eder. Bu durumda, matris boyunca nüfuz bir hücre şekli dönüştürmek ve hücre dışı matrisin (ECM) 2 ile etkileşim gerekir. Meme dokusu bütünlüğünün bakım sıkı controlle bağlıdırhücre-ECM ve hücre-hücre yapışma bağlantıları gen ekspresyonu ve epitel kutupluluğun bozulması etkilediğinden d, doku mimarisi, kanser 6-10 başlamasından yol açabilir. Ancak, bu tür transwell oda tahlillerde ya da yara tırmığı tahlilleri gibi çok in vitro göçü ve istila deneyleri, iki boyutlu (2B) ve bu nedenle, bu hücreleri ve bunların bitişik çevre 3,6,8,11-14 arasındaki karmaşık etkileşimler ihmal. Hücresel morfoloji varyasyonlar, hücre farklılaşması, hücre-matris adezyonlar ve gen ekspresyonu dahil olmak üzere önemli morfolojik ve fonksiyonel farklılıklar genel olarak 2,6,8,11 deneyleri, 2 boyutlu olarak eksik 3B kültürlerinde hücrelerin kültürlenmesi ile tespit edilmiştir. Böylece, kliniğe 6-10 temel araştırma çığır açan bulgularının daha iyi çeviri açan, 3D tahlillerin in vivo durumda bir daha fizyolojik yansıtan anlamlı yararlı edilir kullanır. Bununla birlikte, dikkat edilmelidir3D kültürlerin kullanımı ile kazanılan pek çok avantajı olmasına rağmen, bu model çeşitli hücre tiplerini içeren in vivo tümör mikro tüm karmaşıklığı yakalamak değildir. Ancak, bu 3 boyutlu modellere stromal hücreleri dahil etmek mümkündür (örneğin, fibroblastlar, lökositler ve makrofajlar) kanser hücre yapışması ve yayılması 15-17 tümör stromal etkileşimlerin etkilerini incelemek için. Örneğin laminin ve kolajen gibi ECM proteinlerinin mevcut olduğu zaman kültür meme epitel hücrelerinin en etkili şekilde büyür. Bu bilinen ile, ticari olarak temin edilebilir matris karışımı Engelbreth-Holm-Swarm (EHS) murin tümör elde edilmiştir ve Matrigel taban membran matrisi 2,8 olarak bilinir. Bir takım teknikler taban membran matrisi 2,8 3D koloniler olarak epitel hücreleri büyütmek için kurulmuştur. 3D bazal membran matrisi modeli habis ve habis olmayan meme hem hücre kurulması için etkilidirbüyümesi, in vivo ortamda 18,19 meydana ne benzer. MCF10A hücreleri habis olmayan meme epitel hücreleridir. Temel zar matris içinde yetiştirildiğinde, bu hücrelerin normal meme hücrelerinin in vivo özellikleri sergiler ve hücre proliferasyonu, hücre polarizasyon ve lümen alanı 8,12,20 kurmak için kontrol apoptoza uğrarlar. Ayrıca, 3D kültürlerde asinüsü oluşturan MCF10A hücrelerinin hücre çekirdeklerinin görünümünü daha yakından dokuda, meme epitelyal hücrelerinin kişilerce tek tabaka 21 içinde kültüre daha benzemektedir. Bissell&#39;e ve arkadaşları tarafından yürütülen çalışmalar, habis hücreler, bir çok düzensiz fenotipi gösterdiğinden, bir laminin zengin bir ortamda yetiştirilen proliferasyon artan azaldığı habis meme hücreleri habis olmayan meme hücrelerinden ayırt edilebilir olduğunu ortaya koymaktadır için ilk hücreden hücre yapışması, artan mezenkimal markerlerin ekspresyonu ve invazif yapının sayısında bir artış meydana 3,6,22. Hücre ortamı anormallikleri tümör oluşumunu 20 etkileyebilir. 3D kültür yöntemi etkili tümör hücreleri ve bunların çevreleyen çevre arasında meydana iletişimi çalışma ve ne kadar protein sentezleme etkenler, iletişim 14,20,21,23 belirlemek için kullanılabilir. Bu makalede, analiz invaziv 3D kültürlerde MDA-MB-231 göğüs kanseri hücrelerinin büyümesini ve bir epitel işaretleyici laminin, hücre bazal membran 18,19,24 bir bileşenini kullanarak epitel morfolojisi kaybını incelemek için detaylı bir yöntem sağlar 25. Detaylı prosedürler, doğru ve tekrarlanabilir bir invazif kanser hücresi tarafından yıldız şeklinde (istilacı) yapısının oluşumunu ölçmek için ve bu MDA-MB-231, Hs578T, MCF-7 ya da T47D gibi ortak meme kanseri hücre hatları (sınırlayıcı değildir olanağı sağlar .) Bu nedenle, bu deney ile nasıl protein hücrelerde ekspresyonu ya da tedavi değerlendirmek için bir platform olarak hizmet edebilir pro-ya da anti-invazif bileşikler, bir ya da birden çok hücre tarafından, hücre dışı matris bozulması düzenler.

<table border="0" cellpadding="0" cellspacing="0" width="1062">
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			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
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			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
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			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

Şimdi de hücre ve doku mikro çevre tümör başlangıcı ve ilerlemesini etkileyen kritik düzenleyiciler olduğu bilinmektedir. Ayrıca, hücre dışı matris (ECM) in vivo kültür ve homeostazında hücre davranışını önemli bir düzenleyici olduğu gösterilmiştir. Iki boyutlu (2D) üzerindeki hücrelerin kültüre mevcut yaklaşım, hücreler ve bunların mikro arasındaki karmaşık etkileşimlerin rahatsızlık ve zarar plastik yüzeyler sonuçları. Üç boyutlu (3D) kültür tahlillerin kullanımı sayesinde, hücreye mikro-ortam etkileşimi için koşullar, in vivo benzer mikro kurulmuştur. Bu makalede, etrafındaki çevreye hücre istilası değerlendirilmesinde 3D kültür potansiyelini örnekleme, 3 boyutlu bir bazal membran protein matrisi içinde meme kanseri hücrelerinin büyümesini detaylı bir yöntem sağlar. Buna ek olarak, bu deneyler, 3D molekül ağırlığına işaret kaybını incelemek için potansiyele sahip görüşmekprosedürleri immün epitel morfolojisi Modüller düzenler. Bu çalışmalar meme kanseri yayılması için gerekli istilasını düzenleyen işlemler, içine önemli mekanik bilgilerini tespit etmek yardım.

3 boyutlu bir matris içinde işgal eden MDA-MB-231 hücrelerin bir örneği, Şekil 3C &#39;de gösterilmiştir. Hücreler, matris (Gün 1) gömülü ve 3. gün göre invaziv (yıldız biçimi) yapılar oluşturan başlar ve tam olarak 5. günde (Şekil 3C) ile matrise işgal edilir. Oluşan yıldız şeklinde kolonilerin sayısı sayılır ve (invaziv ve non-invazif) çanak başına koloni toplam sayısının yüzdesi olarak ifade edilmiştir. Ölçümler beş gün boyunca her gün ya...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

Bu makalede, meme kanseri hücre istilasını ölçmek için üç boyutlu (3D) tahlillerin kullanımı için ayrıntılı yöntemler sağlar. Özellikle, biz hücreleri istila oluşur membran bütünlüğünün kaybı incelemek için bu tür deneyleri kurmak için gerekli prosedürleri, ölçümlerini ve veri analizi gibi yöntemleri tartışmak.

Üç boyutlu (3D) Modeli Kullanılarak Meme Kanseri Hücre invazivliğinin Kantitasyonu

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

quantification-breast-cancer-cell-invasiveness-using-three

Research

JoVE Journal

Medicine

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

15.7K Görüntüleme.  University of Western Ontario.  Bu makalede, meme kanseri hücre istilasını ölçmek için üç boyutlu (3D) tahlillerin kullanımı için ayrıntılı yöntemler sağlar. Özellikle, biz hücreleri istila oluşur membran bütünlüğünün kaybı incelemek için bu tür deneyleri kurmak için gerekli prosedürleri, ölçümlerini ve veri analizi gibi yöntemleri tartışmak. 

Video: Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Meme Kanseri Beyin metastaz tümör Hipoksi Dynamics in vivo Biyoparlaklık Görüntüleme Fare Modeli

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Tıp

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

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

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

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

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

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

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

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

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

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

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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

İnsan Nöroendokrin Tümör Terapi Çalışmaları Üç Boyutlu Model İnsan nöroendokrin tümör hücre hatlarında

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

İlköğretim İnsan Kemik İliği ve Maligniteleri Eğitim Bir Üç boyutlu Doku Kültürü Modeli

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Meme Kanseri Bir Roman Transgenik Fare Modeli: Adenovirüs-Kretasedekine ile epiteli Hedefleme ile Metastatik Meme Kanseri Başlatma

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

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 ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Üç Boyutlu Kültürler: Meme hücrelerinin malign Dönüşüm vs Normal Asiner Mimari Eğitim için Bir Aracı

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tümör-stromal Etkileşim için üç boyutlu Co-kültür Modeli

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

Üç boyutlu (3D) Tümör Sfero Yayılması deneyindeki

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