Bu araştırmanın finansman kaynakları: Tel Aviv Üniversitesi teknoloji transferi şirketi, Ramot ve Sheba-Tıp Merkezi (M. CA ve SI.), ICRF ortak bir fon - İsrail Kanser Araştırma Vakfı (M. CA.) Ve İsrail bilim Vakfı ( SI).

1. Hücre Kültürü hazırlanması MDA-MB-231 hücreleri, ATCC (American Type Culture Collection) satın alındı ​​ve sıvı azot içinde saklandı. <ol><li> Dulbeco Modifiye Edilmiş Eagle Ortamı (DMEM),% 10 at serumu,% 1 L-glutamin ve% 1 Penstrep-Amfoterisin B içeren 10 ml tam ortam içinde 92 mm çapında Petri kabındaki Tohum 10 6, MDA-MB-231 hücrelerinin hücre Veren 80-100 yaklaşık% izdiham çoğalmaya. </li><li> Çanak kültür ortamı çıkarın ve atın. </li><li> % 0.25 &#39;lik serum tüm izlerini silmek için (w / v) Tripsin-EDTA çözeltisi ile kısa bir hücre tabakası yıkayın. </li><li> Hücre tabakası (genellikle 5-15 dakika içinde) dağılana kadar ters mikroskop hücreleri çanak ve gözlemlemek için tripsin-EDTA çözeltisi 2.0 ml ilave edilir. </li><li> 18 ml tam büyüme ortamı ekleyin ve yavaşça pipet hücreleri tarafından aspire. Bir tüp aktarın. </li><li> 1.200 rpm hücre süspansiyonu santrifüj. </li><li> Cu 24 ml hücre pelet yeniden askıyaorta LTURE. </li><li> 35 mm cam alt yemekleri (25% izdiham yaklaşık) ve inkübatör yer (% 5 CO 2, 37 ° C) hücre süspansiyonu 2 ml ekleyin. </li><li> Çözümler: <ol><li> Hücre proliferasyonu için tam ortam:% 10 FBS DMEM,% 1 antibiyotik (100 birim / ml penisilin G, 100 ug / ml streptomisin, Pen-Strep-Ampho çözeltisi) ve 2 mM L-glutamin. </li><li> % 0.25 tripsin-EDTA ihtiva Tripsin-EDTA solüsyonu,. </li></ol></li><li> Yemekleri: 92 mm çapında Petri kapları. 35 mm çapında poli-D-lizin kaplı cam alt kültür tabaklarında. </li></ol> 2. Canlı Konfokal Görüntüleme için Hücreleri hazırlanması <ol><li> Tohum 2 x 10 1 &#39;de belirtildiği gibi 2 ml komple ortam içinde cam alt kültür kapları içinde 5 MDA-MB-231 hücreleri. Hücre kültürü% 60-70 izdiham (çanak başına 3-4 x 10 5 hücre yaklaşık) ulaştığında, transfeksiyon devam. </li><li> Iki plazmit dekoderi ile hücrelerin transfekteüretim protokolü takip, lipozomal transfeksiyon reaktif Jet-PI kullanarak füzyon proteinleri γ-tubulin-GFP (centrosomes floresan tespiti için) ve Histon-KIRMIZI (H2b-KIRMIZI, kromozom floresan tespiti için) ing. Kısaca, 100 ul NaCl (150 mM) ile bir boru her bir plazmit 2 ug karıştırın. Ikinci bir tüp içinde 100 ul NaCl (150 mM) ile transfeksiyon reaktifi (100 ul) karıştırın ve oda sıcaklığında (RT) 5 dakika inkübe edin. Daha sonra iki çözüm, karışım (hafif vorteks kullanarak) birleştirmek ve çevirme özelliği. Oda sıcaklığında 30 dakika süreyle inkübe edilir. </li><li> Transfeksiyon karışımı İnkübasyon sırasında, bir kez PBS ile yıkayın ve hücrelerin herhangi bir ek ile sıcak DMEM, 2 ml (37 ° C) hücre orta yerine. </li><li> Yavaşça DMEM hücrelere transfeksiyon karışımı eklenir ve daha sonra 8 saat boyunca (37 ° C,% 5 CO2) inkübatör hücreleri döndürür. </li><li> 24 inkübasyondan 8 saat sonra, 2 ml komple ortam ile DMEM değiştirmek ve inkübatör hücrelerin inkübesa. </li><li> 24 saat transfeksiyondan sonra, 2 ml komple ortam 20 uM PJ-34 içeren hücrelerin orta yerine. </li><li> İlave 18 saat (37 ° C,% 5 CO2) için hücreler inkübe edin. </li><li> % 5 CO 2 ve 37 ° C&#39;de hücreleri tutarak görüntüleme odasında en az 16 saat süreyle konfokal görüntüleme yaşamak Konu hücreleri </li><li> Buna paralel olarak, aşağıdaki gibi floresan mikroskopi kullanılarak transfeksiyon etkinliği 36 saat sonrası transfeksiyon inceleyin: <ol><li> Tohum 2 x 10 1 lamel çukur başına 2 ml komple ortam içinde içeren 6 oyuklu plaka 5, MDA-MB-231 hücreleri. </li><li> 2.2-2.5 bölümlerde belirtildiği gibi hücrelere transfekte. </li><li> 36 saat transfeksiyondan soğuk metanol içinde inkübe edilerek, bir lamel üzerine monte edilmiş, transfekte edilmiş hücreleri sabitleştirmek: aseton (1:1) çözeltisi, 7 dakika, -20 ° C </li><li> Sabitleme çözüm aspire ve bir kimyasal kaputu kurumaya monte hücreleri ile lamel sağlar. </li><li> DAPI Altın antifade reaktif uzatmak ve inci izin uygulayınE lamel 6 saat boyunca karanlıkta kurumasını bekleyin. </li><li> Floresan mikroskop altında slayt inceleyin ve hücrelerin toplam nüfusun (DAPI DNA boyama) gelen transfekte hücreleri (kırmızı ve yeşil sinyalleri) yüzdesini hesaplar. 100-200 hücre sayılmıştır zaman arzu edilen transfeksiyon yüzdesi% 20-40 kadardır. </li></ol></li></ol> 3. Canlı Konfokal Görüntüleme Tarayıcı Ayarları Teknik Parametreler <ol><li> Tarama modu XYZT; İğne [havadar] 1.00; Zoom 3.5, Çözünürlük 8 bit; Lazer DPSS 561 nm, Argon, görünür lazer 488 nm, Lazer O / Ne görünür 633 nm; Amaç HCX PL APO CS 63X 1.40 YAĞ UV; Sayısal diyafram 1.4; Kırılma endeksi 1.52; hızı 700 Hz tarama. </li><li> Görüntü 3 boyutlu sunum IMARIS görüntüleme yazılımı 7.0 ile hazırlanmıştır. </li></ol> 4. Sabit Hücreler Mitoz konfokal Görüntüleme <ol><li> Tohum 2 x 10 5, MDA-MB-231 göğüs kanseri hücreleri (ATCC), normal fare embriyonik fibroblastlar (MEF) veya eksik PARP1 ME2 ml tam orta 6-plaka cam lamelleri F (, Dr Francoise Dantzer tarafından hazırlanan PARP - - /). Lameller, 2 saat boyunca kurutuldu ve 6-çanak her iyi yerleştirilen steril DD su ile yıkama ile takip edilerek,% 96 etanol ile yıkanmıştır. </li><li> Orta PJ-34 (10-30 mcM) ekleyin ve gerekli süre (genellikle en fazla 96 saat) için hücreler kuluçkaya yatmaktadır. </li><li> Bir kez PBS ile yıkayın lamelleri (fosfat tamponlu tuz), ve buz gibi soğuk metanol ile inkübasyon kullanarak hücreleri gidermek: aseton (1:1) çözeltisi, 7 dakika, -20 ° C </li><li> Sabitleme çözüm aspire ve lamelleri kimyasal kaputu kurumaya izin (bu aşamada, lamelleri birkaç hafta -20 ° C tutulabilir). </li><li> Hücre zarının geçirgen hale getirilmekte ve oda sıcaklığında 1 saat boyunca PBST içinde% 10, NDS (normal eşek serumu) (&#39;engelleyici bir çözüm&#39;) ile hücrelerin engellemek için PBST (PBS% 0.1 Tween-20 ile takviye edilmiş) ile bir kere lamelleri yıkayın. </li><li> Birincil antibodi ile permeabilize sabit hücreleri inkübeoda sıcaklığında 2 saat için ES (iğ ve sentrozom boyama için). Anti-α tübülin (1:250 seyreltme) ve anti-γ tübülin (1:200 seyreltme) antikorları takip olarak bloke çözelti içinde seyreltilir. Birincil antikorlar, şöyle uygulanır: 6-plaka kapağı (kapağın baş aşağı) üzerinde her bir lamel için çözüm engelleme antikorların bir karışımı, 100 ul (bir damla) uygulanmaktadır. Yavaşça antikorlar damla lamel koymak, numaralı seribaşı hücreleri açılan karşı karşıya. Oda sıcaklığında 2 saat için de antikorların bakan lamelleri inkübe edin. </li><li> Kuyularda geri lamelleri yerleştirin ve PBST ile hücreleri 3 kez yıkayın. Daha sonra, floresan ikincil antikorlarla lamelleri hücrelerin etiketlenmesi için 4.6 &#39;de tarif edilen aynı prosedürü kullanmak. , Karanlıkta, 1 saat, RT, ikincil antikorlar ile lamelleri hücreleri inkübe edin. Antikorlar aşağıdaki gibi engelleme çözümü seyreltilir: ve Alexa Fluor 568 (1:1,000 seyreltme Alexa Fluor 488 (yeşil 1:1,000 seyreltme);kırmızı). </li><li> Kullanarak lamelleri DAPI (kromozomlar boyama için) ile Altın antifade reaktif uzatmak ve kuruması için karanlıkta oda sıcaklığında gece inkübe monte edin. </li><li> Konfokal mikroskobu ile kapak fişleri inceleyin. </li></ol> 5. Hücre Canlılık ATP üretimi ile ölçülür ATP üretimi bir lüminesan ATP tespit kiti kullanılarak ölçülür. <ol><li> 96 oyuklu plaka, her bir 800 ul ortam içinde yaklaşık olarak 20.000 hücre tohum hücreleri. Üç boş bir kuyu ortamı arka lüminesans belirlenmesi için kullanılmalıdır. </li><li> Yaklaşık 10 mcM 100 mcM ATP standart seyreltme serisi hazırlayın ve buz üzerinde tutmak. </li><li> Her bir kuyucuğa 50 ul deterjan ekleyin ve orbital çalkalayıcı, 700 rpm&#39;de 5 dakika boyunca plaka sallayın. </li><li> Kit &#39;substrat tampon&#39; 5 ml &#39;liyofilize yüzey&#39; bir sulandırın her flakon. </li><li> Oyuklara sulandırılmış substrat çözeltisinin 50 ul ekle ve sallayınorbital çalkalayıcı, 700 rpm, 5 dakika süreyle plaka. </li><li> 10 dakika karanlıkta plaka tutun. </li><li> ELISA mikro okuyucu tarafından her kuyunun lüminesans ölçün. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Konfokal görüntüleme mitoz sırasında canlı çok centrosomal hücreleri (Şekil 3 ve Ek bilgileri) PJ-34 sitotoksik etkisinin gerçek zamanlı belgeler temin yaşıyor. Bu PJ-34 5-9 Mitotik Afet hücre ölümünün indüksiyon düşündüren, ekstra centrosomes de-kümeleme ve hücre ölümüne insan kanser hücrelerinde PJ-34 sitotoksisite atfederek ilk canlı belgelerine oldu. Bunun aksine, süper numerary sentrozom iki odak kümeleme iki focali kümelenmiş ilave sentrozom (Ş...

Fenantren, PJ-34 de dahil olmak üzere PARP1 inhibitörleri, elde edilen stres koşulları (inme ya da miyokard infarktüsü) 1 altında enerji tüketen PARP1 aracılı DNA-onarım ile oluşturulan apoptotik hücre ölüm pasif hücreleri korumak için tasarlanmıştır. Ancak, son zamanlarda biz PJ-34, bu uyaran PARP1 inhibe göre iki kat daha yüksek konsantrasyonda, sadece insan kanser hücrelerinin 2,3 hücre ölümüne neden olabilir keşfetti. Hücrenin daha hızlı yayılması olduğu, hücrelerin daha verimli ortadan kaldırılması oldu. PJ-34 sitotoksik aktivite ekstra centrosomes mitoz 2 de-kümeleme bağlandı. Birçok insan kanser hücreleri liman 4,5 multicentrosomes. Etkili bir mitoz iki sentrozom barındıran hareketsiz hücreler ya da iyi huylu proliferatif hücrelerin zarar vermeden 72-96 saat içinde, bu hücrelerin ortadan 20 uM PJ-34 ile supernümere sentrozom liman insan meme kanseri hücreleri, MDA-MB-231, inkübasyonu <sup&gt; 2,3. İyi huylu hücrelerin insan timus hazırlanan MCF-10, insan meme epitelyal hücreleri, insan endotelyal hücreleri (HUVEC) ve primer mezenkimal hücreler dahildir. Bu hücreler, PJ-34 sitotoksik aktivitesi dirençli bulunmuştur. PJ-34 96 saat inkübasyon sırasında kendi cep döngüsü müdahale ya da centrosomes ve iki odak milli oluşumu 2,3 etkilemedi. Bipolar centrosome montaj mitoz 4,5 bipolar mili oluşumu için çok önemlidir. Bu nedenle, ikiden fazla centrosomes ile hücre iki kutup 4-9 kendi ekstra centrosomes kümeleme, bir güçlükle anlaşılan moleküler mekanizması geliştirdik. Kendi centrosomes bipolar montaj hatası tutuklamalar G2 / M tutuklama hücre döngüsü ve hücre ölümüne yol açan 4,5 mitotik başarısızlığı bu çok kutuplu bozuk iğ ve anormal kromozomlar ayrışmaya neden olabilir. Ekstra centrosomes de-kümeleme altında yatan moleküler mekanizmalar yoğun incelenmiştir &lt;&gt; 10 destek. Sağlıklı dokuları 5,10 korurken bu ölüm mekanizması anlamak kanser hücrelerinin özel ortadan kaldırılması sağlayacaktır. Bu nedenle, mitotik felaket hücre ölümü geçiren bileşiklerin konfokal görüntüleme İlave sentrozom kümeleme etkileyen molekülleri tanımlamak için kullanılabileceğini göstermektedir, insan katı cancers.Our sonuç geniş etkili olabileceği seçici bir kanser tedavisinde yeni bir mod, teklif mitoz 2,3, ilaç adayları hedef bu bileşiklerin kanser render. Biz sabit ve canlı insan kanser hücreleri (mitoz ekstra-centrosomes yüksek çıkması ile) karşı normal hücreler tarayarak fenantridin PJ-34 sitotoksik aktivite belgeledi. Bir adım insan kanser hücrelerinde PJ-34 sitotoksik aktivitesini tanımlamak için kullanılan görüntüleme yöntemleri adım açıklama aşağıda yer almaktadır.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

Güçlü PARP1 inhibitörleri olarak hareket fenantren türevleri mitoz çoklu-centrosomal insan kanser hücrelerinde Sürnümerer centrosomes en iki odak kümeleme engelledi. Fenantridin PJ-34 molekülü en güçlü idi. İlave sentrozom Declustering çoklu centrosomal hücrelerindeki mitotik yetmezliği ve hücre ölümüne neden olur. En katı insan kanser ekstra centrosomes yüksek olay var. PJ-34 aktivitesi canlı bir insan meme kanseri sentrozom oldukça bol olduğu floresan γ-tubulin kodlayan vektörler ve floresan histon H2b mevcut için transfekte edilmiş MDA-MB-231 hücrelerinin konfokal görüntüleme ile gerçek zamanlı olarak belgelenmiştir kromozomlar. Anormal kromozomlar düzenlemeler ve declustered centrosomes temsil eden de-kümelenmiş γ-tubulin odakları PJ-34 ile tedavi sonrası transfekte MDA-MB-231 hücrelerinde tespit edildi. Iki mil kutuplarda un-kümelenmiş ekstra centrosomes kendi hücre ölümü öncesinde. Bu sonuçlar, FO bağlantılıR, ilk kez İlave sentrozom mitoz de-kümeleme ve hücre ölümüne yol açan mitotik yetmezliği olan insan kanser hücreleri, PJ-34 arasında en son tespit edilen özel sitotoksik aktivitesi. Iki centrosomes ve iki odak iğ ile mitoz geçiren normal hücrelere zarar vermeden sabit hücreler, çok centrosomes ile PJ-34 özel ortadan kanser hücrelerinin konfokal görüntüleme tarafından gözlemlenen önceki bulgulara göre. PJ-34 Bu sitotoksik aktivite diğer güçlü PARP1 inhibitörleri tarafından paylaşılan değildi ve PARP1 inhibisyon bağımsızlığını düşündüren, PARP1 eksik MEF barındıran extracentrosomes gözlendi. Konfokal görüntüleme Canlı mitoz sırasında hücrelerin ortadan kaldırılması yeni moleküller belirlenmesi için yararlı bir araç sundu.

PJ-fenantridin 34 1 çözelti, kararlı suda (Şekil 1). Daha önceki sonuçlar hücre ölümünü ortaya ve PJ-34 ile tedavi edilen sabit çoklu centrosomal kanser hücrelerinin çeşitli ekstra-sentrozom de kümelenmiş. Aksine, normal hızla çoğalan hücrelerde 2,3 etkilenmedi. Centrosomes sabit ekstra centrosomal hücreleri 2 centrine1 ve γ-tubulin karşı antikor çift etiketleme tarafından tespit edilmiştir. Burada, PJ-34 sitotoksi...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Mitoz geçiren kanser hücrelerinde fenantridin PJ-34 sitotoksik aktivite canlı konfokal görüntüleme ile gerçek zamanlı olarak belgelenmiştir. PJ-34 eradike insan meme kanseri MDA-MB-231 mitoz İlave sentrozom barındıran hücreler. Normal iki odak mitoz farklı olarak, ekstra-sentrozom PJ-34 varlığında iki kutup mili kümelenmiş değildi.

Anormal Mitoz ile İlişkili Hücre Ölümü Canlı Kanser Hücreleri Konfokal Görüntüleme Uyacakları

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

cell-death-associated-with-abnormal-mitosis-observed-confocal-imaging

Research

JoVE Journal

Medicine

14.9K Görüntüleme.  Sheba Medical Center. Mitoz geçiren kanser hücrelerinde fenantridin PJ-34 sitotoksik aktivite canlı konfokal görüntüleme ile gerçek zamanlı olarak belgelenmiştir. PJ-34 eradike insan meme kanseri MDA-MB-231 mitoz İlave sentrozom barındıran hücreler. Normal iki odak mitoz farklı olarak, ekstra-sentrozom PJ-34 varlığında iki kutup mili kümelenmiş değildi.

Video: Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Rekombinant Adeno Associated Virus serotip 3 Vektörler Karaciğer Kanseri Hücreleri Yüksek Verimlilik İletimi

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Tıp

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 ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Bölümlere Mikroakışkan Cihazlar ve Canlı Hücre Görüntüleme kullanma Kanser Kök Hücre Göç Değerlendirilmesi

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Tümör 4D Canlı hücre Görüntüleme için MAME Modeller: Mikroçevre etkileşimler olduğunu Etki Malign İlerlemesi

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Azoxymethane (AOM) ve Dekstran Sülfat Sodyum (DSS) ile Modelleme Kolit-İlişkili Kanser

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Meme Kanseri Fare Modelleri Tümör Mikroçevre İlaç Yanıtlarının Görüntüleme Canlı

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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  ...

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

Ligatable Floresan Sondalar Deneysel İnme Hücre Ölümü Fagositik Gümrükleme Değerlendirilmesi

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

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 ...