We thank Dr. Jordi Ochando from the Flow Cytometry Shared Resources at the Mount Sinai Medical Center for their assistance in flow cytometry analysis. We thank Dr. Rumana Huq from the Microscopy Shared Resource Facility at the Mount Sinai Medical Center for their imaging assistance. The authors thank the TJ Martell Foundation for its support in this project.

Bu protokol kurumsal araştırma etik kurulu onayı ile kurumumuza yürütülen ve insan refahı için tüm kurumsal, ulusal ve uluslararası kurallar ile uyumlu değil. Gelişmiş Prostat Kanseri Hastalarında gelen Periferik Kan 1. Koleksiyonu Not: metastatik prostat kanserli hasta seçin. Yazılı hasta rızasını ve izolasyon yaşı ve önceki kemoterapi ve hormonal tedaviler de dahil olmak üzere hastaların klinik özelliklerini, kaydedin. Metastatik hastalığı olan hastalar potansiyel periferik kanda kapalı zaman eğrilerinin en yüksek konsantrasyonuna sahip olacaktır. <ol><li> Aydınlatılmış onam sonra uygun bir Kurumsal İnceleme Kurulu protokolü onayladı altında kan örnekleri toplamak. Lateks eldiven ve prosedür boyunca laboratuvar önlüğü giyin. </li><li> Iğne tutucu 25 G kelebek iğne ve tüp bağlayın. Alanını temizlemek için alkol bez kullanınpazı ve önkol arasında antekübital ven, daha sonra hastanın üst koluna turnike uygulanır. </li><li> Antekübital ven içine kelebek iğne takın ve toplama başlatmak için iğne tutucu içine kan toplama tüpü itin. Pıhtılaşmasını önlemek için etilendiamintetraasetik asit (EDTA) içeren toplama tüplerinde kan yaklaşık 10 ml toplayın. </li><li> Hastadan kelebek iğne çıkarın ve 1 dakika boyunca damar delinme sitesi üzerinden steril gazlı bezle basınç uygulayın. Steril bandaj ile Kapak sitesi. </li></ol> Mononükleer Hücreleri 2. İzolasyon Not: 1 aşamasından gelen toplanan kan mononükleer kapalı zaman eğrilerinin ilave olarak çevresel kan mononükleer hücreleri (PBMC) (örneğin, lenfositler ve monositler) ihtiva edecektir. <ol><li> : 1 oranında Pipet bütün 5 ml serolojik pipetle 50 ml polistiren konik tüp içine, kan ve 1 Hanks Dengeli Tuz Solüsyonu (HBSS) ekleyin. Yavaşça pipet karışımı homojenize etmek. </li><li> Boş 50 ml polistiren konik tüp düşük hızlı yoğunluk gradyan santrifüjü ile kan bileşiklerinin ayrılması için bir ticari olarak hazırlanmış solüsyonu (Ficoll-Paque) 15 ml ilave edilir. </li><li> Yavaşça farklı üst tabaka oluşturmak üzere, aşama 2,2 polistiren tüp içine, Aşama 2.1, tam kan ve HBSS karışımı pipetle. Çözümlerin karıştırma en aza indirmek için en düşük ayara ayarlayın pipet, bu verimli ayrılmasını sağlayacaktır. </li><li> Oda sıcaklığında 30 dakika boyunca 400 x g&#39;de (25 ° C) santrifüje tabi tutulur. En düşük ayara yavaşlama ayrıldıktan sonra çözümlerin karıştırma önlemek için. Hızlanma herhangi hıza ayarlanmış olabilir. </li><li> Santrifüj işleminden sonra tüpün alt kısmında üst ve ayırma çözüm üzerinde plazma arasındaki PBMC&#39;Ier ve KTC sandviç tabakasının ince beyaz-gri şerit tespit. </li><li> Plastik transfer pipet kullanarak 50 ml polistiren tüp içine adım 2.5 beyaz-gri şerit hücreleri toplayın. </li><li> PBMC&#39;leri ve kapalı zaman eğrilerinin ve c 50 ml polistiren konik tüp HBSS ekle10 dk RT yıkamak için 400 xg&#39;de tekrar entrifuge. </li><li> Yıkamak için oda sıcaklığında 3 dakika boyunca 400 x g hızında tekrar santrifüj HBSS ve 50 ml sıvı ve tekrar süspansiyon pelet boşaltacaktır. </li><li> Süpernatant atılır, kırmızı kan hücre lisis tamponu, 5 ml kalan pelet tekrar süspansiyon ve oda sıcaklığında 5 dakika boyunca çözelti inkübe edin. </li><li> Oda sıcaklığında 3 dakika boyunca 400 x g&#39;de santrifüj ile kırmızı kan hücre parçalama tamponu çıkarın. </li><li> Süpernatant atılır ve önceden antikor boyama engellemek için,% 10 fetal sığır serumu (FBS) ile desteklenmiş 1 ml PBS 1X pelletini. </li></ol> Kontrol THP için CD45-FITC antikoru 3. boyanması Tek Çekirdekli Hücreleri (FACS) <ol><li> Hemasitometre veya otomatik hücre sayacı kullanılarak (adım 2.11 itibaren) canlı (tripan mavi-negatif) hücrelerin sayısını ölçmek. (% 10 FBS ile PBS 1x), 1 x 10 6 hücre / ml&#39;lik bir süspansiyon hazırlayın ve 1 saat boyunca buz üzerine yerleştirin. </li><li> Sayısal Dağıtiki tüp içine, önceki aşamada elde edilen hücre süspansiyonu. Kontrol ve CD45 boyanma olarak Etiket tüpleri. </li><li> 250: kontrol tüpüne 1, bir seyreltme IgG1κ-FITC (2,5 ug / ml) ekleyin. CD45 boyama tüpü, 1 bir seyreltme CD45 FITC (2,5 ug / ml) konjüge birincil antikor ekleyin: 250 ve 30 dakika boyunca buz üzerinde inkübe hücre süspansiyonları. CD45 antijeni markalama hematopoietik hücreleri için de kullanılır. </li><li> 4 ° C&#39;de 3 dakika boyunca 400 x g&#39;de santrifüj hücreleri ve süpernatant atılır. </li><li> Steril 1 x PBS ile her pelet süspanse edilmesi suretiyle, hücreler iki kez yıkanır, 4 ° C&#39;de 3 dakika boyunca 400 x g&#39;de santrifüj ile ve ardından,% 10 FBS ile takviye edilmiştir. </li><li> 10 ug / ml&#39;lik bir konsantrasyonda 4 &#39;, 6-diamidino-2-fenilindol (DAPI) içeren PBS 1x 1 ml çözelti içinde tekrar süspansiyon hücreleri. </li><li> 12 mm x 75 mm polistiren tüpler içine 35 mikron süzgeç kapaklar sayesinde nihai çözüm Filtre. </li></ol> Prostat kapalı zaman eğrilerinin 4. İzolasyon tarafındanFACS Not: Canlı CD45 negatif CTCS toplamak için bir akış sitometresinin yararlanın. <ol><li> Etiketli tüpten hücreleri kullanılarak Set tazminat kontrolleri &quot;Denetim&quot;. </li><li> Enkaz ve uygun ileriye dağılım ve yan dağılım parametreleri kullanılarak hücrelerin kümelenmeleri hariç kapıları oluşturun. </li><li> Etiketli tüp, hücre süspansiyonları kullanılarak FITC kapıları kurulması &quot;CD45-FITC.&quot; </li><li> Pasifik mavi-A kanalı kullanılarak Kapısı canlı (DAPI-negatif) hücreleri. </li><li> % 10 FBS ile takviye edilmiş Roswell Park Memorial Institute (RPMI) 1640 2 ml ihtiva eden steril bir 15 ml polistiren konik tüp içine CD45 negatif popülasyonu toplayın. </li><li> % 10 FBS ile takviye edilmiş RPMI 500 ul - santrifüj 3 dakika boyunca 400 x g&#39;de kriteri prostat tümörü hücre süspansiyonu daha sonra 200 pelet tekrar süspansiyon. </li></ol> Xenograftlarında Büyüme Fareler ve İzleme içine kapalı zaman eğrilerinin 5. Enjeksiyon Not:Kurumsal hayvan bakım komitesi tarafından onaylanmış protokoller uyarınca tüm hayvan prosedürleri yürütün. Bu protokol tüm ilgili düzenleyici ve kurumsal kurumlar, yönetmelik ve yönergelere uygun olarak ve uygun bizim Hayvan Bakım Komitesi tarafından onaylanan belirli Hayvan Kullanımı Protokolü kapsamındaki bizim kurumda yapılmıştır. <ol><li> Buz üzerinde 1 oranında ve yeri: 1 hücre dışı matriks ile sıralanmış prostat tümör hücre süspansiyonu karıştırın. </li><li> Oksijen / dak, 1 L% 5 (h / h) inhale izofluran sağlayan bir oda içinde deri altından implante edilmesinden önce fare anestezisi. Fare kornea ve ayak refleks kaybı için kontrol ederek uygun anestezi sağlamak. </li><li> Bir 25 G iğne ve 1 ml şırınga kullanarak, 8-10 haftalık erkek bağışıklık yetersizliği olan fare hem üst yanları içine prostat tümörü hücre ve hücre-dışı matris hücre süspansiyonu deri altından 250 ul enjekte edilir. Enjeksiyon hacmi i olarak 250 ul bakılmaksızın CTC konsantrasyonu enjektemportant değer. Farede maksimum SQ uygulanması en fazla 1 ml&#39;dir. &#160;</li><li> Subkutan nodüler yoğunlukları büyümesi için fare enjeksiyon siteleri haftalık palpasyonu yaparak ve haftada iki kez fareler ağırlık ölçerek fareler izleyin. </li><li> Tümör büyüklüğü, moleküler analiz ve seri pasaj için yeterli tümör dokusu sağlamak için en fazla 0.5 cm ve en büyük çapı en az 1 cm servikal dislokasyon ile hasat deri altı hümen prostat kanseri ksenograftları ardından karbon dioksit ile boğarak fareler öldürülür. Hiçbir tümör rağmen sıkıntı ya da% 15 kilo kaybı belirtileri ile fareler Euthanize hissediliyor. </li></ol>

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The authors declare that they have no competing financial interests.

Bu yazıda kapalı zaman eğrilerinin prostat kanseri PDX modellerin üretimi için bir yöntem tarif eder. PDX üretimi için kapalı zaman eğrilerinin kullanılması Mevcut yöntemlerle karşılaştırıldığında modelleri birçok potansiyel önemli avantajlara sahiptir. İlk olarak, periferal kan, kapalı zaman eğrilerinin erişilebilir toplanması, farklı hastalık safhalarında aynı hastadan alınan deney modellerinde üretilmesini sağlar. İkinci olarak, kan toplama tümör hücrelerinin ...

A correction was made to <a href="http://www.jove.com/video/53182">Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells</a>. There was a spelling error in one of the authors' surname. The author's name was corrected from:
Veronica Rodriquez-Bravo
to:
Veronica Rodriguez-Bravo

A correction was made to Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells.

Erratum: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Hasta kaynaklı ksenogratflardır kanser araştırmaları için kullanılan giderek daha popüler deneysel modellerdir. Onlar biyomarkerların ve biyolojik yollar, klinik öncesi ilaç etkinliğinin değerlendirilmesi ve kişiselleştirilmiş kanser tedavilerinin 1,2 avatars oluşturulması karakterizasyonu için kullanılabilir. Daha önce, diğer araştırma grupları implante veya immün sistemi baskılanmış farelerin 1 içine tek tümör hücre süspansiyonları veya tüm tümör eksplantlar enjekte edilerek ya PDX modeller geliştirdik. Bu PDX Modeller taze katı tümörün cerrahi koleksiyonu, malign asiti veya masraflı hem de ve iatrojenik morbidite riski hastayı gösterir bir cerrahi prosedür geçiren bir hastanın plevral sıvı gerektirir. Kanser araştırmalarında çok belirgin bir gelişme dolaşan tümör hücrelerinin tespit, izolasyon ve karakterizasyonudur. Bu tümör hücreleri primer tümör kitlesi kaçmak ve dolaşıma gireronlar metastaz ve nüks önemli bir rol oynar nerede, kanserin en sık nedeni mortalite 3 ile ilgili. Birkaç solid tümör türlerinden kapalı zaman eğrilerinin değerlendirilmesi ve karakterizasyonu tanısı, prognozu ve rezidüel hastalık 3 izlemek için klinik bilgiler sağlamıştır. Ya da fiziksel özellikleri, biyo-ifadesi veya kapalı zaman eğrilerinin fonksiyonel özelliklerine dayanan şu anda kullanılan çeşitli yaklaşımlar verimli kapalı zaman eğrilerinin 4 izole etmek için kullanılabilir. Mevcut macroscale CTC izolasyon yöntemleri yüzey moleküllerine karşı yoğunluk gradyan santrifüj filtre gözenekleri fiziksel filtrasyon ve ayırma bulunmaktadır. En yaygın olarak kullanılan CTC izolasyon yöntemleri kapalı zaman eğrilerinin antikor bazlı yakalama dayanmaktadır. Her iki hücre yüzeyi markerlerinin pozitif ve negatif seçim periferal kandan izole edilmesi için kapalı zaman eğrilerinin kullanılabilir. Periferik dolaşımdaki kapalı zaman eğrilerinin için pozitif seçim yaygın epitel belirteçleri (örneğin, EpCAM) kullandığı birkapalı zaman eğrilerinin ancak hematopoietik olmayan hücreler üzerinde eksprese edilen yeniden. Bu yöntemin dezavantajı, metastatik potansiyele sahip kapalı zaman eğrilerinin genellikle epitelyal to-mezenkimal 3 epitel yüzey belirteçleri aşağı doğru düzenleyen bir geçiş (EMT) geçirmiş olması. Metastatik potansiyele sahip CTCS izole etmek amacıyla, hematopoietik bir yüzey işaretleyici CD45 kullanan bir negatif seçim yöntemi, lökositlerin normal hücre popülasyonu 5 kullanılabilir tüketmek için. Prostat kanseri, en sık görülen kanserdir ve erkeklerde 6 kansere bağlı ölümlerin önemli bir nedenidir. Tümör ilerlemesi ve saldırganlık mekanizmaları tam olarak anlaşılamamıştır ve bu nedenle üretim ve prostat kanserinin moleküler heterojenite özetlemek deneysel modellerin karakterizasyonu önemli ilgi konusudur. Prostat kanserinin PDX model olmuştur immunocom, insan prostat kanseri hücrelerinin nakli yoluyla daha önce oluşturulansöz fareler 7,8. Ancak bu tür modellerin üretimi öncelikle hastalığın tembel doğası atfedilen immün sistemi baskılanmış farelere prostat kanserinin düşük engraftmant oranı, engel olmuştur. Son zamanlarda, kapalı zaman eğrilerinin meme kanseri 9, akciğer kanseri ve prostat kanseri 10 11 PDX modelini oluşturmak için kullanıldı. Bunlar proof-of-concept çalışmalar cerrahi numune toplama ihtiyacı PDX modelleri bağımsız üretme olasılığını tanıttı. Bu yazıda ayrıntılı olarak yeni bir deneysel model üretimi için bir yöntem açıklanmaktadır.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640</td>
			<td>Gibco Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning Cell Gro</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#181;m Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml polystyrene conical tube</td>
			<td>Crystalgen</td>
			<td>23-2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>d3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm Polystyrene tubes with cell strainer cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Lavender Blood Collection Tubes with EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Winged Blood Collection Set with Push Button Retract Needle 23 gauge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer One Use Needle Holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Latex Tourniquet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Latex or non-latex gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 cotton gauze pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive bandage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of prostate cancer patient derived xenograft models from circulating tumor cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

Bu protokol izole CD45 negatif prostat kanseri kapalı zaman eğrilerinin gelen PDX modellerinin oluşmasına yol açacaktır. Protokolde kullanılan negatif seçim yöntemine dayanarak DAPI leke kullanılarak ölü hücreleri hariç gereklidir. Akış sitometrisi ile tespit CD45 negatif hücrelerin yüzdesi değişkendir ve hasta (Şekil 1A) tümör yükünün bağlıdır. Beyaz oklar (Şekil 1B) ile belirtildiği gibi (hücre çekirdekleri belirlemek için) CD45 ve DAPI kullanılarak ...

Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Prostat Kanseri Hasta Üretimi Tümör Hücreleri Sirkülasyon gelen Xenograftlarında Modeller Türetilmiş

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

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14.0K Görüntüleme.  Icahn School of Medicine at Mount Sinai. This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Video: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Birincil Preneoplastic Meme Epitel Hücrelerinin Time-lapse görüntüleme Meme Kanseri Genetik Fare Modelleri türetilen

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Klinik ve Klinik Öncesi Araştırma Uygulamaları Tümör Hücre (CTC) Tahliller Sirkülasyon semiautomated adaptasyon

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Hastalar ve İnsan endotel hücrelerinden türetildiği Prostat dolaşımdaki tümör hücrelerinin arasında E-selektin aracılı Etkileşimleri gözlemleyin vitro Yöntemi

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Musküler Distrofi Hastalarda gelen uyarılmış pluripotent kök hücrelerin oluşturulması: İdrar Türetilmiş Hücre Verimli Entegrasyonu-ücretsiz yeniden programlanması

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Hastalık Modelleme için kas biyopsisi Hasta türetilmiş Hücre Hatları İzolasyonu ve Ölümsüzleştirme

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Yumurtalık Kanseri Tümör başlatan Hücrelerinin in vitro Zenginleşmeden içinde

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Chicken İnsan Multipl Miyelom Xenograftlarında Modeli Kurulması Tümör Büyüme, Invasion ve Anjiogenez Eğitim için

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.