Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

NOT: insan ve hayvan kanser hücreleri satın alınabilir veya lütfen bize sağlanan beri yok etik dikkate. Yapma 1. Kollajen Fiş (Sözde birincil Tümör) <ol><li> Bir kollajen dağılımını hazırlayın. Daha önce 17 ya da satın bildirdiği gibi ben de ayıklanır ve sterilize edilebilir sıçan kuyruğu tendonları (RTT) den kollajen yazın. (Beşi 2 dakika çalışır yüksek hız) üniform bir karışımı için dondurularak kurutulmuştur RTT kollajen (0.02 N, asetik asit içinde 3.25-3.50 mg / ml), bir karıştırıcı kullanılarak dağıtılır. </li><li> Hasat (tripsin-EDTA, genellikle) ve hemasitometre kullanarak canlı hücreleri saymak için tripan mavisi dışlama kullanın. İstenilen hücre yoğunluğu (fiş başına 5 x 10 4 hücreler) ayarlayın. </li><li> Steril koşullar altında tüm çözümler (NaOH, fetal sığır serumu, DMEM 5x, NaHCO3) Bileşen (Tablo 1) Hazırlama ve buz üzerinde soğutulmuş tutun. Not: çeşitli çözümler eklenme sırası hücreleri ozmotik veya asidik şokları önlemek için önemlidir. </li><li> Olabildiğince hızlı bir şekilde son kolajen çözeltisi (5 mi) içine hücre dispersiyonu (1.25 x 10 6 hücre) uygulayın. Hava kabarcıklarını önlemek ve daha sonra hızlı bir şekilde 96 oyuklu plakanın her bir hazır kullanımlı çözeltisi 200 ul dağıtma ise (aşağı yukarı pipetleme) tarafından iyice karıştırın. Yavaşça hava kabarcıklarını çıkarmak için hücre kültürü kaput çalışma alanı yüzeyinde çoklu plaka grev ve kuyuların içinde eşit çözüm yaymak için. </li><li> Tüm oyuklara doldurduktan sonra inkübatör içine saklamak, (bu adım 96 oyuklu plaka başına yaklaşık 15-20 dakika sürer). </li><li> gece boyunca 2 saat 37 ° C&#39;de inkübe plakası. Kollajen jelasyon (yani, fibrillogenesis), 30 dakika içinde meydana gelir. Bir gecelik inkübasyondan gerçekleştirmek için kültür kültür ortamı (100 uL / ​​oyuk) ilave edin. </li></ol> Fibrin Gel 2. İlk Katman <ol><li> Fibrinojen solüsyonu hazırlanması. Not: fibrin jel aynı parti, ideal daha tekrarlanabilir Resul için kullanılmalıdırts, fibrin jel oluşumu, ticari dondurularak kurutulmuş fibrinojen farklı yükleri arasında değişebilir. <ol><li> Her zaman taze hazırlanmış fibrinojen çözümü kullanın. Hidrat kristal oluşumunu önlemek için, şişeyi açarken, oda sıcaklığına kadar dondurularak kurutuldu fibrinojen getirin. </li><li> Kademeli önceden ısıtılmış (37 ° C), Ca2 + ile Hank&#39;in dengeli tuz çözeltisi (HBSS) / 3 mg / ml çalışma konsantrasyonunda Mg2 + (en az son hacminin% 15 fazlalık hazırlanması düşünülebilir gerekli fibrinojeni çözülür : örneğin, 5 ml çözelti için 5.75 ml 17.25 mg) verdi. <ol><li> fibrinojen parçaları çözmek için ilk başta önceden ısıtılmış HBSS damla damla ekleyin. beher bir spatula ile büyük parçaları yıkmak. zaman ve karıştırma işleminin kolaylaştırılması için zaman beher çalkalayın. İşlem sırasında bir karıştırıcı plaka kullanmayın. yukarı ve aşağı süspansiyon pipetleme kalan tozu eritin. </li></ol></li><li> Steri ise ılık fibrinojen çözüm tutun0.22 um&#39;lik bir filtreden geçirilerek çözelti lizing. Not: HBSS yeterince sıcak değil veya fibrinojen tamamen çözülmüş değilse, çözüm filtreyi tıkayabilir. Varsa, fibrinojen konsantrasyonunu azaltmak ve böylece fibrin pıhtı sertliği olabilir, bir veya iki kez filtreyi değiştirin. </li><li> Bir alternatif prosedür olarak, son hacmi ayarlarken hazır kullanım fibrinojen çözeltisi içine ilgi hücreleri (örneğin, endotel hücreleri) süspanse edin. </li></ol></li><li> Trombin Çözümleri hazırlanması. <ol><li> GKD 2 O (50 NIH birim / mi) içinde bir stok çözelti hazırlayın, daha sonra bir 0.22 mikron filtre kullanılarak sterilize edin. </li><li> bir fibrinojen / trombin oranı ≥1 kullanın: 0.0075 (h / h), fibrin jel oluşturmak için. </li></ol></li><li> Fibrin Jel oluşturuluyor. <ol><li> Steril fibrinojen tutmak ve tüm sonraki adımlar boyunca buz üzerinde stok solüsyonları trombin. fibrin jel 24 oyuklu plakalar içinde oluşturmak üzere izin verilir. </li><li> Derhal bindirme inciHer bir E yüzeyi de hava kabarcığı oluşumunu engelleyerek fibrinojen solüsyonu (200 ul / oyuk). Bir seferde 6 kuyu işleyin. </li><li> fibrinojen çözüm tamamen kuyu yüzeyini kaplayan sonra, bir 45 ° açıyla plaka yatırmak ve iyi merkezi haline trombini bırakarak ilk kuyuya trombin çözeltisi 1.5 ul ekleyin ve sonra yavaşça için yatay plaka girdap 1-2 sn. <ol><li> Laminer akış kaputu (5-10 dk) jelleşme / pıhtılaşma işlemi tamamlanıncaya kadar altında stabil pozisyonda plaka bırakın (Not: polimerizasyon işlemi inkübatör plaka taşıyarak, örneğin, rahatsız edilmemelidir). </li></ol></li><li> İlk altı kuyu polimerize sonra tüm kuyular işlendikten kadar, önümüzdeki altı kuyuları için aynı diziyi (yani 3 Önceki adımlar) tekrarlayın. </li></ol></li></ol> Fibrin Gel ve sandviç Kollajen Plug 3. İkinci Kat <ol><li> seçenekA: (hemen Kollajen Tak Kullanarak). <ol><li> fibrin jel ilk kat ince plaka eğerek bütün kuyularda polimerize emin olun. kolajen fişleri transferini kolaylaştırmak için (fibrin jeller ihtiva eden), 24 oyuklu bir plaka ile yan kollajen jel fişler yan ihtiva eden 96 oyuklu plaka koyun. </li><li> kolajen fişleri ihtiva eden plakanın her oyuğuna HBSS bir damla ekleyin. </li><li> İnce (bir kolu olarak kullanılır) bir şırınga üzerine monte iğne veya (video) bir mikro-kaşık kullanarak iyi her kollajen fişini çıkartın. kollajen fiş de kuyuya merkezli ve kısırlık bakımlı olduğundan emin yaparken, bir veya iki mikro-kaşık kullanarak ilk fibrin jel tabakası üzerine her kollajen fiş aktarın. </li><li> fibrinojen çözeltisi, ikinci tabaka ile daha önce oluşan fibrin jel Yerleşimi (300 ul / oyuk) ve 2.3 de tarif edildiği gibi en az 1 tutarak, trombin tanıtmak. seferinde 0.0075 oranı altı kuyu bir sekans &lt;/ Li&gt; </ol></li><li> (Büyüme Faktörü-azaltılmış Bodrum Membran (GFRBM) ince bir tabaka ile Kollajen Tak Kaplama) Seçenek B. <ol><li> Serin, tüm Hazırlanan çözümler ve aletleri önceden ve GFRBM donmuş alikotları beri işlenmesi sırasında (örneğin, pipet, ipuçları, test tüpleri), 4 ° C&#39;de veya buz üzerinde saklayın (üreticinin talimatlarına uyun) çözülme sırasında aşırı ısınma oranına çok duyarlı . </li><li> plaka oyuklarından ayrılmasından sonra, saf GRFBM çözeltisi 100 ul buz ihtiva eden bir 1.5-ml santrifüj tüpü içinde 2 dakika boyunca her kollajen fiş ıslatın. </li><li> Daha önce açıklandığı gibi sağlanması, iyi merkezli iken ilk fibrin tabakası üzerine her kaplanmış fişi aktarın. 5 dakika GRFBM bir jel oluşturmak için izin vermek için 37 ° C&#39;de eklenti ihtiva inkübe edin. Adım 3.1.4 olarak ikinci fibrin katmanı ekleyin. </li></ol></li></ol> 4. Hücre Kültürü Ortamı koşullar <ol><li> (Kültür ortamı ile de 4 her dolum00 ul). Kültür ortamları ve takviyeleri hücre çizgisi ve deneysel koşullara bağlı olarak seçilir. </li><li> 100 kallikrein önleyici birim (KIU) / ml&#39;lik bir son konsantrasyonda kültür ortamına aprotinin bir antifibrinolitik ekleyin. NOT: test edilen hücre hattı için kullanılan şartlar altında, bir hücre kültürü inkübatöründe plakaları saklayın. </li><li> Her gün taze ortam ile kültürler doldurun veya deneysel şemasına göre ve aprotinin ekleyin. Taze orta eklemeden önce biraz (30-35 ° açıyla) plaka yatırın ve dikkatle sürekli gözlem altında klimalı ortam aspirasyon sırasında kuyunun kenarına pipet eğin. </li></ol>

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The authors have no disclosure.

önemli bir teknik dipnot olarak, bir boşluk, merkezi ve periferik jeller arasındaki ara yüzeyde mevcut olması gereklidir. Aksi takdirde, / göç fibrin jel işgal hücrelerin kapasitesini azaltabilir. Trombin uygun seyreltilmiş edilmemiş ise, kollajen ve fibrin jel arasındaki boşluk kültür ilk 24 saat içinde meydana gelebilir. Kollajen jel neden olabilir test edilen hücre hattı, böylece, her iki jeller arasındaki oluşturmak için göreceli olarak büyük bir alan neden kültür sırasında sözleşme ol...

Kanser hücre işgali / göç ve sonraki metastaz kurulması temel ve biyomedikal özelliklerini inceleyen yoğun araştırma 1,2 konusudur. Metastaz kanser nihai aşamasıdır ve klinik yönetim belirsizliğini koruyor. Hücresel ve moleküler düzeyde metastaz daha iyi anlaşılması daha etkin tedavilerin 3 gelişmesini sağlayacaktır. Metastatik hücrelerin çeşitli özellikleri kendi stemness ve göç içinde ve primer tümörün 5&#39;ten işgal etmeye bir geçiş durumu (örneğin, epiteloid-mezenkimal geçiş) elde etmek için potansiyeli de dahil olmak üzere in vitro 4 keşfedilebilir. Neredeyse kan / lenfatik dolaşımı katkısını hariç çünkü Ancak, işgali / metastaz süreçlerinde in vitro değerlendirilmesi bir meydan okuma olmuştur. kollajen jel tümör fragmanları gömmek organotipik kültürlerinin Previou bilgisisinsi kanser saldırganlık izlemek için kullanılmaktadır. Tümörlerin karmaşıklığı korunmuş olsa da (örneğin, kanserli olmayan hücrelerin varlığı), tümör fragmanları örnekleme varyasyonuna, sınırlı orta difüzyon maruz ve stromal hücrelerinin 6 aşırı büyümesi için vardır. Alternatif bir yöntem, üç boyutlu (3D) hücre ortamı taklit eden hücre dışı matris (ECM) bileşenleri içinde mevcut kanser hücrelerinin büyüyen oluşur. kollajen jel ve / veya bir bazal membran türetilen matriks içinde meme kanseri hücre çizgilerinin proliferasyonu 3D hücre kültürünün en iyi karakterize örnekleri arasındadır. Belirli bir 3D hücre kültür ortamları kullanılarak standart koşullar altında yetiştirilen göğüs kanseri hücreleri için gözlemlenen dağınık düzeneği meme asinüs ve boru şeklindeki yapılar 7-10 kendiliğinden oluşumuna ters çevrilebilir. Ayrıca, adenokarsinom kanser hücrelerinden türetilen çok-hücreli tümör parçacıklarının oluşumunun (başka teknikler kullanılarak toplanmışÖrneğin, şu anda en yaygın olarak kullanılan 3D hücre kültürü tahlil 11-13 oluşturmaktadır) gömme ağar, sferoidler yüzen, damla asılı. Bununla birlikte, bu deney, parçacıklarının oluşturabilen kanseri hücre çizgilerinin sınırlı dizi ve bu koşullarda hücrelerin çalışma süresinin kısalığı ile sınırlıdır. Bu görsel teknik olarak, bu tarifnamede ilgi kanser hücrelerinin alternatif olarak bir bazal membran türetilmiş matris ile kaplanabilir sözde primer tümörün in vitro oluşumunu sağlamak için, bir kollajen jel gömülü gelişmiş bir 3D hücre kültürü analizinde getirmektedir. Oluştuktan sonra, psödo-primer tümör sonra kanser hücrelerinin, iki matris bölme arasında bir arayüz çapraz sağlayan bir hücresel olmayan matris (mevcut durumda fibrin jel) içinde sandviç (Şekil 1 e bakınız). İlginç bir şekilde, agresif kanser hücreleri ile sözde primer tümörden kaynaklanan sekonder tümör benzeri yapılar görünürfibrin jel. Bu 3 boyutlu bir kültür sistemi, örneğin, anti-kanser ilaçları, gen ifadesi ve hücre-hücre ve / veya hücre-ECM etkileşimleri 14-16 için araştırmak için gerekli esnekliği sunmaktadır. <img alt="Şekil 1" src="/files/ftp_upload/54322/54322fig1.jpg" /> Şekil 1:.. Yöntem bakış kanser çalışmaları için bir model olarak 3D hücre kültür sistemi oluşturmak için yöntemin şematik özeti <a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a>

<table><tbody><tr><td>Freeze-dried collagen</td><td>Sigma-Aldrich</td><td>C7661</td><td>from rat tail tendon (soluble dispersion) or home-made (see Rajan &lt;i&gt;et al&lt;/i&gt;., ref.#14)</td></tr><tr><td>Fibrinogen (freeze-dried)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F8630</td><td>Type I-S, 65 - 85% protein with &amp;ge; 75% of protein is clottable</td></tr><tr><td>Thrombin</td><td>EMD Chemicals Inc.</td><td>605157</td><td>&amp;nbsp;Gibbstown, NJ; NIH units/mg dry weight&amp;nbsp;</td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</td><td>Corning</td><td>356234</td><td>Previously from BD Biosciences</td></tr><tr><td>Aprotinin</td><td>Sigma-Aldrich</td><td>A6279&amp;nbsp;</td><td>&amp;nbsp;solution at 5 - 10T IU/ml (Trypsin Inhibitor Unit)&amp;nbsp;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Micro-spoons</td><td>Fisher Scientific</td><td>2140115</td><td>Fisherbrand Handi-Hold Microspatula</td></tr><tr><td>96 well plate, round base</td><td>Sarstedt</td><td>3925500</td><td></td></tr><tr><td>24 well plate</td><td>Sarstedt</td><td>3922</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s Medium</td><td>Sigma Chemical, Co.</td><td>D5546</td><td>DMEM</td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>CAA15-701</td><td>FBS, Canadian origin.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma Chemical, Co.</td><td>T4049</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>Sigma Chemical, Co.</td><td>H8264</td><td>HBSS</td></tr></tbody></table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

mono tabakaları memeli hücre kültürü çok çeşitli fizyolojik ve moleküler süreçleri incelemek için kullanılır. Ancak, artan hücrelerin incelemek için bu yaklaşım, genellikle istenmeyen eserler üretir. Bu nedenle, genellikle hücre-dışı matris bileşenlerini kullanarak üç boyutlu (3D) bir ortamda, hücre kültürü, in vivo bağlı doku veya organ nativ yakından benzerlik için ilginç bir alternatif olarak ortaya çıkmıştır. Bir fibrin jel yapılmış bir sözde primer macrospherical tümör ve (ii) bir çevresel hücresiz bölme bir kollajen jel hareket gömülü kanser hücrelerini ihtiva eden bir merkez bölme iki bölme, yani: (i) ile 3 boyutlu bir hücre kültür sistemi geliştirmiştir örneğin, merkezi kullanılandan farklı bir hücre dışı matris bileşeni, burada kanser hücrelerinin göç (yayılma ön) ve / veya ikincil veya uydu tümörler temsil mikroküresel tümörler oluşturabilir. periferal bölme uydu tümör oluşumuoldukça bu 3D kültür sistemi benzersiz kılan doğal tümör hücrelerinin bilinen saldırganlık veya metastatik kökenli korelasyon. Bu hücre kültürü Kanserin hücre istilası ve motilite, hücre-hücre üstü matris etkileşimlerini ve anti-kanser ilaç özelliklerini değerlendirmek için bir yöntem olarak değerlendirilmesi için kabul edilebilir.

Daha önce de belirtildiği gibi, bu 3D hücre kültürü deneyinde ilginç bir özelliği, kanser hücreleri sadece bitişik fibrin jel kolajen fiş göç değil, aynı zamanda, ikincil tümörlerin (örneğin, uydu, tümör benzeri yapılar) tesis olup olmasıdır. Bu, doğrudan doğruya, özellikle uzun bir çalışma mesafesi kondansatör (Şekil 2) ile birlikte, jel kalınlığı boyunca düşük ve yüksek büyütmelerde bir ters faz kontrast mikroskop ile...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Kanser hücre istilası ve Uydu Tümör oluşumu keşfedin Kültür Testi Üç Boyutlu

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

Research

JoVE Journal

Medicine

10.4K Görüntüleme.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Video: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-β-kaynaklı Invasion Tedbir Sfero Testi

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

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3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

3-b hücre kültür sistemi istila eğitim ve Mesane kanserinde tedavi değerlendirmek için

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Kanser Araştırmaları

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

3D Kültürde Kanser Hücre İstilası ve T-Hücre Sitotoksisitesi İzleme

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

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 ...

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

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

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

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

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 ...

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 ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

İstilayı Yönlendirmek İçin Epitelyal Kanser Hücrelerinin İskelesiz 3D Kültürlerinde Heteromultihücreli Stromal Hücreler

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Bir Kanseri Hücre Sfero Testi 3D ortamda Invasion Değerlendirmek için

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

Source: Cvetkovi&#263;, D., et al. <a href="https://www.jove.com/v/51341/quantification-breast-cancer-cell-invasiveness-using-three">Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model.</a> J. Vis. Exp. (2014).
This video provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment.

3D Kültür Bazal Membran Deneyi: Hücre İstilasını İncelemek için 3D Bazal Membran Protein Matrisi Üzerinde Kanser Hücrelerinin Kültürlenmesi

Source: Cvetkovi&#263;, D., et al. Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model. J. Vis. Exp. (2014).
This video ...