Bu çalışma cömertçe Çocuk Kanseri Vakfı (Baltimore, MD), Brandon Carrington Lee Vakfı (Silver Spring, MD), DOD (W81XWH-10-1-0137) ve NIH (R01CA108641) fon tarafından desteklenmiştir. Biz Dr Anton Wellstein ve deneyim paylaşımı ve sunulan yöntemleri optimize etmek için bize yardımcı olduğunuz için onun laboratuvar üyelerine teşekkür etmek istiyorum.

1. Hazırlık <ol><li> Tüm adımlar, bir doku kültürü kaputu steril koşullar altında gerçekleştirilmelidir. </li><li> XCELLigence istasyonu% 5 CO2 varlığında, 37 ° C inkübatör içine yerleştirilir. </li><li> Geçit 6 daha tercihen en fazla düşük geçiş HUVEC hücreleri, kullanın. Ayrıca, hücreleri daha fazla hasat sırasında birleşik% 75 daha olduğundan emin olun. </li><li> HUVEC hücreleri, büyüme faktörleri, ek ve% 5 cenin sığır serumu içeren EGM-2 mermi kiti (Lonza&#39;dan Biosciences) ile sulandırılır EGM-2 ortamında yetiştirilir. </li><li> Tripsin tüm izleri, 200xg de hücre iplik bir kez PBS ile yıkama ve belirtilen hücre yoğunlukları bunları tekrar süspanse ederek hücre süspansiyonları çıkarılmalıdır. </li><li> 37 ° C&#39;de 1 saat süreyle% 0.1 jelatin ile kaplayın XCELLigence e-levhası 16 ° C ya da gece boyunca 4 ° C&#39;de PBS ile plaka yıkayın ve 100μL sulandırılmış EGM-2 medya ekleyin. Arka plan ölçmek için XCELLigence sistemde boş bir okuma yapınhücrelerin yokluğunda empedansı. </li></ol> 2. HUVEC tek tabaka oluşturma <ol><li> Tripsinizasyon ile HUVEC hücreleri bir alt konfluent şişeye hasat. 2.5 x 10 5 hücre / ml &#39;lik bir nihai yoğunluğa sulandırılmış EGM-2 ortamı içinde yeniden süspanse hücreleri. </li><li> 100μL EGM-2 medya içeren e-plaka kalibre bir arka plan her kuyuya, HUVEC hücre süspansiyonu (2.5 x 10 4 HUVEC hücreleri) 100μL ekleyin. </li><li> Hemen XCELLigence aracı E-Plaka yerleştirin. </li><li> Program XCELLigence yazılım 10 dakikalık aralıklarla empedans okuma gerçekleştirmek için. </li><li> Bir tek tabaka oluşturacak kadar endotel hücreleri gibi (Şekil 2) hücre indeksi bir düzleşme kanıtladığı, 18-21 saat boyunca büyümesine izin. </li></ol> 3. Hücre ve veri normalleştirme işgal eklenmesi. <ol><li> Bir kez istikrarlı bir HUVEC tek tabaka bir son dens için trypsinization ve tekrar süspansiyon tarafından hasat tümör hücreleri, kurdu1 x 10 5 hücre / tümör hücreleri (RPMI ortam ya da DMEM% 10 FBS içeren gibi) yetiştirmek için kullanılan ortam içinde ml Sığ </li><li> Pause empedans XCELLigence yazılım okuma ve istasyondan E-Plaka çıkarın. </li><li> Aspirasyonu ile EGM-2 ortamı çıkarın. 1 x 10 hücre ihtiva eden 4 tümör hücre süspansiyonu 100μL ekleyin. Genel olarak, tümör hücre oranı 1:2.5: endotel hücreleri ekilmiş, tahlil için iyi çalışır. Bununla birlikte oran, tek bir hücre hatları için optimize edilebilir. </li><li> Inkübatör XCELLigence sistemine E-plaka koyun ve 10 dakika aralıklarla empedans okumaları devam ediyor. </li><li> Invasion tümör hücreleri işgal ederek endotel kavşaklar ve penetrasyon geri çekme nedeniyle hücre indeksi bir düşüş olarak önümüzdeki 6-12 saat üzerinden gerçek zamanlı olarak izlenebilir. </li><li> Tümör hücreleri istila ilavesi zaman sonuç normalize. XCELLigence yazılım, herhangi bir zaman noktasında ve sonuçlarına normalleşme izindoğrudan yazılım penceresinde görülebilir. </li></ol> 4. Temsilcisi Sonuçlar: Metastatik ve metastatik olmayan hücreler tarafından HUVEC tek tabaka işgali bir örneği, Şekil 3&#39;te gösterilmiştir. K7M2 metastatik osteosarkoma hücre hattıdır. Bu hücreler bu hücre hattı 13,14 invaziv özellikleri oluşturan, sitoskeletal bağlayıcı proteinin Ezrin yüksek düzeyde ifade eder. HUVEC hücreleri K7M2 hücreleri ile meydan olduğunda, 6 saat içinde elektrik direnci bir düşüş vardır. Elektrik direnci bu düşüş işgalci tümör hücreleri tarafından HUVEC tek tabaka işgali temsil eder. K12 hücre hattı ile, diğer taraftan, Ezrin çok daha düşük seviyelerde ifade eden ve 13 ve sonuçta daha az metastatik. Hücrelere bağlı veya HUVEC tek tabaka (Şekil 3) ile işgal edemiyoruz gibi direnci daha az dik açılan K12 hücreleri sonuçları ile HUVEC tek tabaka Zorlu. <imgsrc = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot; alt = &quot;Şekil 1&quot; /&gt; Şekil 1. Tümör hücreleri tarafından endotel hücreleri işgali için iş akış süreci şematik diyagramı. HUVEC hücreleri jelatin kaplı E-plakaları üzerine kaplanmış ve konfluent tek tabaka oluşturmak için izin verilir. Bir tek tabaka oluşturmuştur ve hücre indeksi endotel tek tabaka işgali nedeniyle değiştirir gibi işgali gerçek zamanlı olarak takip edilmektedir kez malign hücre eklenir. <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="Şekil 2,"/> Şekil 2. HUVEC tek tabaka oluşumu. HUVEC hücrelere tutunur ve jelatin kaplı altın elektrotlar üzerinde yayılmış gibi hücre indeksi değişiklikler. Konfluent tek tabaka oluşumu, 18 saat sonra hücre indeksi bir sabitleme ile temsil edilir. <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="Şekil 3,"/> Şekil 3,. K7M2 ve K12 osteosarkom hücreleri tarafından HUVEC hücrelerinin Invasion. Cel bir dik düşüşl-indeksi yüksek metastatik K7M2 hücrelerinin giriş birkaç saat içinde ortaya çıkar. K12 hücreleri, diğer yandan daha az metastatik ve verimli olarak K7M2 hücreleri gibi endotel tek tabaka nüfuz etmek mümkün değildir. Bu HUVEC tek tabaka K12 hücreleri ile meydan hücre endeksinde daha az dik düşüş ile temsil edilmektedir. Kontrol kanser hücrelerinin yokluğunda HUVEC tek tabaka empedans temsil eder. Deneyler, üç kopya halinde gerçekleştirildi.

<ol>
	<li>Klein, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer.+The+metastasis+cascade.">Cancer. The metastasis cascade.</a> Science. 321, 1785-1787 (2008).</li><li>Chiang, A. C., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+metastasis.">Molecular basis of metastasis.</a> N Engl J Med. 359, 2814-2823 (2008).</li><li>Orr, F. W., Wang, H. H., Lafrenie, R. M., Scherbarth, S., Nance, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+cancer+cells+and+the+endothelium+in+metastasis.">Interactions between cancer cells and the endothelium in metastasis.</a> J Pathol. 190, 310-329 (2000).</li><li>Bacac, M., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+cancer+cell.">Metastatic cancer cell.</a> Annu Rev Pathol. 3, 221-247 (2008).</li><li>Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+signals+from+the+invasive+front.">New signals from the invasive front.</a> Nature. 441, 444-450 (2006).</li><li>Kramer, R. H., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+tumor+cells+with+vascular+endothelial+cell+monolayers:+a+model+for+metastatic+invasion.">Interactions of tumor cells with vascular endothelial cell monolayers: a model for metastatic invasion.</a> Proc Natl Acad Sci U S A. 76, 5704-5708 (1979).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proc Natl Acad Sci U S A. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromotion+of+mammalian+cells+measured+electrically.">Micromotion of mammalian cells measured electrically.</a> Proc Natl Acad Sci U S A. 88, 7896-7900 (1991).</li><li>Tiruppathi, C., Malik, A. B., Del Vecchio, P. J., Keese, C. R., Giaever, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+method+for+detection+of+endothelial+cell+shape+change+in+real+time:+assessment+of+endothelial+barrier+function.">Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.</a> Proc Natl Acad Sci U S A. 89, 7919-7923 (1992).</li><li>Whipple, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+promotes+tubulin+Detyrosination+and+Microtentacles+that+enhance+endothelial+engagement.">Epithelial-to-mesenchymal transition promotes tubulin Detyrosination and Microtentacles that enhance endothelial engagement.</a> Cancer Res. 70, 8127-8137 (2010).</li><li>Boyd, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-microelectronic+sensing+technique+for+profiling+cytotoxicity+of+chemicals.">A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.</a> Anal Chim Acta. 615, 80-87 (2008).</li><li>Khanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane-cytoskeleton+linker+ezrin+is+necessary+for+osteosarcoma+metastasis.">The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.</a> Nat Med. 10, 182-186 (2004).</li><li>Ren, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin-cytoskeleton+linker+protein+ezrin+is+regulated+during+osteosarcoma+metastasis+by+PKC.">The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.</a> Oncogene. 28, 792-802 (2009).</li></ol>

Bu yazının için yayın maliyet nazik Roche Applied Science tarafından sağlandı.

Gerçek zamanlı HUVEC işgali tahlil izleme işgali için basit ama güçlü bir yöntemdir. Bu son nokta analizi kullanan diğer geleneksel teknikleri üzerinde önemli bir avantaj olan, gerçek zamanlı sonuçlar sağlar. Tahlilinde test ilaçlarının, antikorlar, ligandlar ve metastazının önleyicileri veya uyarıcıları gibi proteinler için modifiye edilebilir. Kanser / endotel hücre çapraz konuşma farklı ajanların etkisi de değerlendirilebilir. Bu kültür ortamı içinde büyüme faktörleri ve uyuşturucu gibi eksojen ajanlar, tanıtan, bu onlar HUVEC tek tabaka bütünlüğünü etkilemez sağlamak için önemlidir. HUVEC tek tabaka bozulması hücreleri istila yokluğunda eksojen ajan tanıtan ve hücre indeksi bir değişiklik yoktur sağlayarak test edilebilir. Bu nedenle, uygun kontrollerin deney tasarımının bir parçası olarak dahil edilmelidir. Bir conf biçimi olarak farklı hücre hatları farklı hücre-indeksi eğrileri göstermekluent tek tabaka. Eğri, hem de yüksek hücre-indeksi elde, şekli, hücre tipine bağlıdır. HUVEC hücreleri 16-18 saat sonra daha yüksek bir hücre dizindeki başka bir istikrar ardından, 4-6 saat tohumlama sonra hücre indeksi düzleştirme bir karakteristik geçici göstermektedir. Bu nedenle, bu hücrelerin, tümör hücrelerinin ilave edilmeden önce en az 18 saat boyunca konfluent tek tabaka oluşturacak izin vermek önemlidir. Hücre indeksi elektrot / hücre interfaz iyonik ortamına bağlıdır, bu tür hücre morfolojisi ve hücre-hücre adezyon kalitesi gibi çeşitli faktörlere de kapalı alt alanında ek olarak, hücre dizini etkiler. Bu nedenle, tümör hücre istilasını üzerine oluşur hücre indeksi azalma, endotel birleşme ve takip eden tümör hücre istilası geri çekme dahil çeşitli faktörlere, bir fonksiyonudur. XCELLigence araç üç farklı konfigürasyonlarda imal edilir: gerçek zamanlı hücre analiz(RTCA) SP, RTCA MP ve RTCA DP. RTCA MP alet 96-E-plakaları aynı anda altı tutabilir ise RTCA SP araçlar, bir 96-E-plaka tutar. Bu ilaçlar ve bileşiklerin yüksek verimli tarama için izin verir. Bu makaledeki deneyler üç 16-iyi E-plakaları tutabilir RTCA DP cihazı kullanılarak yapılmaktadır. Her E-plaka tutma istasyonu bağımsız olarak birden fazla kullanıcı aynı anda deneyler çalıştırmanıza olanak veren, çalıştırılabilir. RTCA DP araç aynı zamanda CIM-plakaları kullanarak göç incelemek için kullanılabilir. Bu 8um bir ortalama gözenek boyutu olan bir polietilen tereftalat (PET) membran ile ayrılan üst ve alt odaları ile 16 oyuklu plakalar vardır. Elektronik sensörler, üst bölmenin gözenekli zarın alt kısmında bulunur. Alt bölme odasının üst hücreleri için kemo-çekici için bir rezervuar olarak hizmet vermektedir. Bununla birlikte, CIM-plakalar üzerinde HUVEC işgali tahlil önemli bir avantajı ise, bu eski içinde tümör hücre istilasını iendotel hücre işgali tümör ve endotel hücreleri arasında çapraz konuşma bağlıdır gibi s, gözenek boyutu ile sınırlı değildir. HUVEC işgali tahlil da Uygulamalı Biyofizik (Troy, New York) tarafından üretilen, ECIS Z enstrüman üzerinde gerçekleştirilebilir. ECIS Z enstrüman dizi ve elektrot konfigürasyonları farklılıklar ile, XCELLigence araç benzer bir teknoloji kullanır. Biz başarıyla 8-iyi ECIS dizileri kullanarak HUVEC işgali deneyleri gerçekleştirdik. 2.5 x 10 5 ve 1 x 10 5 hücre sırası: Bu iyi alt yüzey alanı kaplama için endotelyal ve tümör hücrelerinin daha büyük bir sayı gerektirir ECIS dizileri, daha büyük olduğuna dikkat etmek önemlidir.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr> 
 <tr>
 	<td>xCELLigence RTCA DP station</td>
 <td>Roche	</td>
 <td>05469759001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>RTCA control unit</td>
 <td>Roche</td>
 <td>05454417001</td>
 <td>Notebook with preinstalled RTCA software</td>
 </tr>
 <tr>
 	<td>E-Plate 16</td>
 <td>Roche</td>
 <td>05469830001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HUVEC cells</td>
 <td>Lonza</td>
 <td>CC-2517</td>
 <td></td>
 </tr>
 <tr>
 	<td>EGM-2 media</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>EGM-2 bullet kit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>0.1% Gelatin in 
sterile H2O
</td>
 <td>Millipore</td>
 <td>MCPROTO045</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells

Malign hücrelerin metastatik yayılması konakçıda, uzak bölgelere bir taşıma aracı olarak kan dolaşımına endotel tabakasının temel membran, vasküler endotelyum, tümör hücrelerinin bağlanma, endotel birleşme geri çekilmesi ve tümör hücre istilası ve son olarak göç bozunma gerektiren 1-3. Bir kez dolaşım sisteminde, kanser hücrelerinin duvarları kapiller ve metastatik tümörler 4,5 oluşturmak için çevre dokuya extravasate uygun. Tümör hücre-endotelyal hücre etkileşiminin çeşitli bileşenleri, kanser hücreleri, insan göbek bağı damarı endotel hücrelerinin (HUVEC) tek tabakalı bir zorlu tarafından in vitro olarak çoğaltılabilir. Elektron ve faz-kontrast mikroskobu ile yapılan çalışmalar in vitro etkinlikleri sırayla oldukça in vivo metastaz 6&#39;da temsil etmektedir. Burada, izler ve gerçek zamanlı invas içinde rakamlarla bir elektrik-empedans tabanlı teknik açıklarhabis tümör hücrelerinin endotel hücrelerinin iyon. Giaever ve Keese ilk hücre popülasyonunun elektrotlar 7,8 yüzeyi üzerinde büyümeye zaman empedans dalgalanmalar ölçmek için bir yöntem tanımlanmıştır. Roche tarafından üretilen XCELLigence enstrüman,, hücrelerin iyi altındaki alanın yaklaşık% 80 kapsayan bir altın mikroelektrot dizi ile kaplı bir kültür çanak eklemek ve yaymak gibi elektrikli empedans değişiklikleri ölçmek için benzer bir teknik kullanır. Hücreleri eklemek ve elektrot yüzeyine yayılmış olarak, 9-12 elektrik empedans bir artışa yol açar. Empedans hücreleri ile kaplıdır de doku kültürü toplam alanı ile doğrudan orantılı olan bir boyutsuz olarak adlandırılan hücre-göstergesi olarak gösterilir. Bu nedenle, hücre dizini, hücre yapışması, yayma, morfolojisi ve hücre yoğunluğunu izlemek için de kullanılabilir. Bu makalede açıklanan işgali tahlil değişikliği esas alınmaktadırelektrot / hücre interfaz de elektrik empedans, malign hücrelerin bir nüfus olarak bir HUVEC tek tabaka (Şekil 1) ile işgal. Endotel kavşak bozulması, tümör hücreleri tarafından endotel tek tabaka ve yedek geri çekilmesi empedans büyük değişikliklere yol. Bu değişiklikler doğrudan tümör hücrelerinin invaziv kapasitesi ile ilişkili, yani, son derece agresif hücreleri tarafından işgali hücre empedansı ve tersi büyük değişikliklere yol. Endotel hücre-tümör hücresi etkileşim daha yakından taklit eden in vivo sürecinde, ve 2), veri gerçek elde edilir: 1) Bu teknik, Boyden odasının ve Matrigel tahlilleri gibi istila ölçme mevcut yöntemleri, üzerinde bir iki kat avantaj sağlar zaman ve diğer yöntemler için son nokta analizi aksine, daha kolay ölçülebilir.

Watch this Scientific Journal Video about A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells at JoVE.com

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Bu makalede, bir açıklar<em&gt; In vitro</emEndotel hücrelerinin bir tek tabaka ile istila izleme kanser hücreleri&gt; tekniği. Bu veriler de alt elektrotların yüzeyini empedansın değişikliklerin bir fonksiyonu olarak gerçek zamanlı olarak elde edilir.

Kanser Hücreleri Endotel Hücre Tek tabakalı Invasion Tedbir Gerçek zamanlı Elektrik Empedans Tabanlı Tekniği

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

a-real-time-electrical-impedance-based-technique-to-measure-invasion

Research

JoVE Journal

Medicine

27.2K Görüntüleme.  Georgetown University. Bu makalede, bir açıklar In vitro tekniği. Bu veriler de alt elektrotların yüzeyini empedansın değişikliklerin bir fonksiyonu olarak gerçek zamanlı olarak elde edilir.

Video: A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital Görüntüleme Gerçek Zamanlı tümörler Nanopartikül Alımı Değerlendirme

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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

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

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

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

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

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.

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

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 ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelleme ve Görüntüleme 3-Boyutlu Toplu Hücre Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Kültür Hücreler Kanser ile ilişkili Sinyal İletimi sorgulanması için bir Multiplexed Lusiferaz tabanlı Tarama Platformu

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

Kantitatif Real Time PCR kullanarak Beyin Omurilik Sıvısı MicroRNA Profil

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Bir Modifiye<em&gt; In vitro</em&gt; Invasion Deneyi Hormonlar, Açan Kanser Hücre Invasion Sitokinlerin ve / veya Büyüme Faktörleri Potansiyel Rolü belirlemek için

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

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

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

Real Time Mezotelyal Hücrelerinin Yumurtalık Kanseri Sferoid Ek ve Invasion Değerlendirilmesi

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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 ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Çoklu-parametre floresans ile aktive edilen hücre sınıflandırma İnsan lenfatik endotel hücreleri İzolasyonu

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...