Name: evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays
Uploaded: 2015-01-03T15:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

This study was supported by a grant from the Baden-W&#252;rttemberg Stiftung (Adult Stem Cells Program II) awarded to C.L. We thank Dr. Martina Konantz for critical input and review of the manuscript. We thank Emmanuel Traunecker and Toni Krebs from the DBM FACS Facility (University Hospital Basel) for assistance with FACS sorting.

OVCAR-3 insan yumurtalık karsinoma hücrelerinde 1. Üretim Stabil Lentivirüsler Sox2 düzenleyici bölge, haberci yapı İçeren kalıt <ol><li> Tarif 10,21 bir Sox2 düzenleyici bölgesini tanıyan bir raportör yapısı ile HEK 293T paketleme hücre hattı transfekte edilmesiyle lentiviral parçacıkları oluşturur. Not: raportör yapısının ayrıca ileride tdTomato floresan protein ProteoTuner kalkan sisteminin dengesizleşmesine alanı içerir. Shield1 böylece floresan proteini 22 aşağılamak için proteasom engelleyen istikrarsızlaştırma etki bağlanır. </li><li> 24 saatlik bir süre boyunca, lentiviral parçacıklarla OVCAR-3 hücreleri nakletmek. Daha sonra, viral süpernatant kaldırmak ve tam ortamda fosfat tamponlu tuz (PBS) ve kültürlenmiş hücreleri yıkayın (RPMI, 100 U / ml penisilin / ml streptomisin, 100 ug,% 10 FBS ile takviye edilmiş). </li><li> 48 saat sonra, 10 ug / ml puromycin kültürlere ilave edildi ve uygun bir şekilde kalıt aktarımlı hücrelerin seçimine izin vermek için 5 gün için muhafaza edilmiştir. </li></ol> Hücre Sorting ve Kaplama 2. Hazırlık <ol><li> 1 at Shield1 ekleyin: hücre sıralama öncesinde 1,000 seyreltme 24 saat. Negatif kontrol olarak Shield1 tedavisi (Şekil 1) bir stably transduse OVCAR-3 hücreleri kullanır. Şişeden aspire ortam, 3 dakika boyunca% 0.05 tripsin-EDTA ile hücreler 1 x hücrelerin PBS ile yıkayın ve trypsinize. </li><li> 5 dakika - (25 ° C 15) oda sıcaklığında 300 xg&#39;de hücre sayıları, santrifüj hücreleri saymak, (yukarıya bakınız) tam orta kullanarak tripsin durdurun. </li><li> Durusu süpernatant ve tekrar süspansiyon hücreleri dikkatli bir 0,5-1 ml steril PBS. </li><li> Tek bir hücre süspansiyonu elde etmek için 40 mikron hücre süzgeç kapağı filtresi kullanın. </li><li> Ml başına 5000000 hücrelere hücre sayımı ayarlayın. </li><li> , MEGM büyüme faktörleri, sitokinler ve ek takviye (100 ul ortam ultra düşük bağlanma, 96 gözlü levhalar küreler hazırlanmasıB-27, heparin-sodyum; ya da büyüme faktörleri, sitokinler ve ek B-27, ya da% 1 metilselüloz eklenmeden heparin-sodyum ile desteklenmiş DMEM / F12), aynı zamanda Tablo 1 e bakınız. İsteğe bağlı olarak mümkün kontaminasyon riskini en aza indirmek için, 100 U / ml penisilin ve 100 ug / ml streptomisin bir konsantrasyonda ortam antibiyotik ekleyin. </li><li> Sıralama TTT + ve yukarıda hazırlanan 96 oyuklu plakalara RFP- hücreleri, oyuk başına 1 hücre (tek hücre bazlı küreler deneyi), sırasıyla oyuk başına 100 hücre (çoklu hücre bazlı küreler deneyi). 100 μ meme, kılıf basıncı 20 psi ve verim maskesi 0, saflık maskesi 32, faz maskesi 16: Tek hücreli modu, kurulum sırala kullanarak (Malzeme) sıralama ticari olarak temin hücre sıralayıcı gerçekleştirin. </li><li> Mikroskopik olarak (tek bir hücre bazlı deney için) hücreler içeren kuyu puanlama ile ve çoklu hücre bazlı tahlilde (bireysel çukurlarındaki hücre saymak suretiyle kaplama verimlilik değerlendirmek; Şekil 2, </strong&gt;). </li><li> Orta alanda standart koşullar altında kuluçkaya bırakılır, 37 ° C&#39;de ve% 5 CO2 (bileşim için adım 2.6). Günlük bFGF (20 ng / ml) ve EGF (20 ng / ml), Supplement. </li><li> Bir hafta sonra, entegre muhabiri sisteminden floresan sinyal tespit etmek 4X veya 10X büyütme ve floresan mikroskop ile standart mikroskop kullanılarak tümör küreler ortaya çıkan numaralarını saymak. &quot;Küçük&quot; küreler olarak 100 mikron (Şekil 3) - çapı 50 ile &quot;büyük&quot; küreler olarak 100 mikron aşan bir çapa sahip küreler ve küreler Kont. Gerçek küreler saymak ve kümeler hücre değil emin olun. NOT: Tek hücre bazlı deneylerde küre oluşumu kültür 10 (7 karşı) gün sonra mikroskopik olarak puan daha kolaydır. </li><li> TTT + küre-oluşturucu hücre ve tek bir hücre bazlı deneylerde, sırasıyla RFP- hücreleri (her bir deney için, bir 96-yuvalı plaka) ve çok hücre-ba oranını hesaplamakShaw ve arkadaşları tarafından sunulan sed küreler deneyler (her bir deney için, bir oyuk). 16 Not: küre oluşturan hücrelerin (%) = (küre sayısı) / (tohumlanmıştır hücre sayısı) oranı 100 x </li></ol> Kürelerin 3. Seri Pasajlanması <ol><li> Oda sıcaklığında 10 dakika boyunca 300 x g&#39;de uygun bir steril tüp ve santrifüj her bir oyuğa içeriğini yerleştirin. Çok hücre tabanlı küreler tahliller için, birlikte iyi biri küreler toplamak. PBS ve uzun santrifüj 2 dakika ile 5 kez - KUYU 3 yıkayın. Tek hücre tabanlı küreler tahliller için, bireysel küreler toplamak. Nedeniyle hücrelerin düşük sayıda, santrifüjleme ve yıkama adımları için 1.5 ml tüpler kullanmaktadır. </li><li> % 0.05 tripsin-EDTA, 200 ul süpernatan ve tekrar süspansiyon pelet çıkarın. </li><li> Optimal hücre ayrılmasını sağlamak amacıyla, yumuşak bir çalkalayıcı içinde 5 dakika için 37 ° C &#39;de bir hücre süspansiyonu inkübe edilir. Alt hücre ölümü oranı cep hattı için trypsinization zaman Optimize: hiçbir büyük olursaküreler yavaşça 100 ul pipet kullanarak görünür çiğnemek olduğunu. Durumunda büyük küreler, hala mevcut başka bir 3 dakika tripsin ile kuluçkaya ve daha sonra toz haline getirme adıma geçin. Tek durumunda hücre tabanlı deneyler trypsinization adımı sırasında canlı hücrelerin optimum hücre verimi için zaman optimize etmek emin olun. </li><li> Tripsin inaktive tek hücre tahlilleri, ilave bir 2 dakika boyunca santrifüj halinde, 10 dakika boyunca 300 x g&#39;de 500 ul komple ortam ve santrifüj ekleyin. </li><li> Küreler orta dikkatle süpernatant ve tekrar süspansiyon hücreleri çıkarın. </li><li> Bir tek hücre süspansiyonu elde etmek için 40 mikron hücre süzgeç kapağı filtresi kullanın. </li><li> TTT + ve çoklu hücre-bassed deneylerde RFP- hücrelerin kullanılması durumunda, analiz akış sitometresi aracılığı ile her bir göze daha sonra süspansiyon içinde flüoresan hücrelerin yüzdesini değerlendirmek. </li><li> Yukarıda ayrıntılı olarak açıklandığı gibi hazırlanan tek hücre seri şarjı tahlilleri, tohum oyuk başına 1 hücre, bir ultra düşük bağlanma, 96 oyuklu bir plakaya için. Tek bir kişi alandan yaklaşık 20 ayrı kuyu tohum. Çoklu hücre bazlı primer kürelerin şarjı tahlilleri için, tohum hücreleri yeni bir 96 oyuklu bir plakaya birinci küre bir kuyudan elde edilen ve mikroskopi ile hücre sayıları ertesi gün sayısı. </li><li> Adım 2.11 tarif edilen formüle göre ikincil küreler tahlillerinde küre oluşturan hücrelerin oranını değerlendirmek. </li></ol> 4. Sonuç Analizi <ol><li> Bağımsız üçlü olarak yapılan deneylerden elde edilen sonuçları analiz ve iki taraflı Student t-testi istatistiksel analiz için normal dağılıma değerleri ve aksi Mann-Whitney-Testleri analiz için kullanabilirsiniz. </li></ol>

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The authors have nothing to disclose.

Küreler kültürleri kanser kök hücre potansiyeli tahlil, insan tümör hücrelerinin 15,25,26 geniş bir kök-benzeri hücreler için zenginleştirmek için yaygın olarak kullanılan bir yöntemdir. Bu kültür koşulları altında, kendini yenileme yeteneğinden yoksun kanser hücreleri ayırt etmek ve en sonunda hücre ölümü beklenmektedir. Başlangıçta hücre kümeleri veya özellikle ilköğretim tayinlerde bile tümör küre oluşturabilir rağmen, onlar yüzünden kendini yenileyen özellikler...

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.
A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable6. Consistently, in ovarian cancer, other than for example in breast carcinoma7, expression of ALDH1 associates with favorable outcome8 and expression of the proposed stem cell marker CD44 variant has no prognostic value9. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells10 and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.
By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice10,13.
An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells14. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast17, brain18, pancreas19 and colon20 tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models10. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells10. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo10 suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.
In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

<table><tbody><tr><td>Low-attachment plate</td><td>Corning</td><td>3474</td><td></td></tr><tr><td>MEGM</td><td>Lonza</td><td>CC-3151</td><td></td></tr><tr><td>Insulin</td><td>Lonza</td><td>CC-4136</td><td>SingleQuots&amp;trade; Kit</td></tr><tr><td>Hydrocortisone</td><td>Lonza</td><td>CC-4136</td><td>SingleQuots&amp;trade; Kit</td></tr><tr><td>EGF</td><td>Lonza</td><td>CC-4136</td><td>SingleQuots&amp;trade; Kit</td></tr><tr><td>EGF</td><td>Sigma</td><td>E9644</td><td>end concentration: 20 ng/ml</td></tr><tr><td>FGF</td><td>PeproTech</td><td>100-18B</td><td>end concentration: 20 ng/ml</td></tr><tr><td>B-27</td><td>Invitrogen/ Gibco</td><td>17504-044</td><td>end concentration: 1X</td></tr><tr><td>Heparin-Natrium-25000 IE</td><td>Ratiopharm</td><td>N68542.02</td><td>dilution 1:1,000</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>FCS</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>21875-034</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s PBS (1X)</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Shield1</td><td>Clontech</td><td>632189</td><td>dilution 1:1,000</td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>21041-025</td><td></td></tr><tr><td>DMEM/F12 (powder)</td><td>Gibco</td><td>42400-010</td><td></td></tr><tr><td>Methyl cellulose</td><td>Sigma</td><td>M0387</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Applichem</td><td>A2856</td><td></td></tr><tr><td>Cell sorter</td><td>BD</td><td></td><td>Aria III cell sorter</td></tr><tr><td>FACS analyser</td><td>BD</td><td></td><td>Accuri c6 flow cytometer</td></tr><tr><td>Microscope</td><td>Olympus</td><td></td><td>IX50 Osiris</td></tr></tbody></table>

evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

Geleneksel küreler deneylerinde, RFP + OVCAR-3 hücrelerinin genel RFP- hücrelerin% 20, yaklaşık% 40, birincil küreler deneyi (Şekil 4A) &#39;de tek bir tümör alanına yol açmıştır. Ayrıca, RFP + hücreleri tarafından oluşturulan küreleri RFP- hücrelerden oluşan daha büyük boyutta olmuştur. Tek bir hücre bazlı deneylerde kaplama olduğunda, TTT + hücreleri aynı zamanda, yukarıda sonuçları teyit RFP- hücrelerden daha fazla küreler oluşturulur. B...

Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Çoklu ve tek hücre bazlı küreler tahliller kullanılarak, insan yumurtalık karsinoma hücrelerinde Kök Hücre Özelliklerinin Değerlendirilmesi

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

evaluation-stem-cell-properties-human-ovarian-carcinoma-cells-using

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Medicine

9.5K Görüntüleme.  University Hospital Basel. In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Video: Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

Kanser kurulması İnsan Konvansiyonel Osteosarkom Hücre Kültürleri Kök

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Kanser Araştırmaları

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

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

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

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

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

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

Tıp

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

Fizyolojik hasta, kanser kök hücrelerini hedef alan anti-neoplastik Ilaç taraması için 3B kürler türetilmiştir

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Jinekolojik ve Meme Kanseri Tümörlerinden Kanser Kök Hücre Kürelerinin Alınması

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Neurosphere Testi İnsan Glioblastoma Multiforme Tümör Hücreleri İzolasyon ve Genişleme

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Nörobilim

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 ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

İnsan Hücre Lines Kemorezistans Yumurtalık Kanser Kök Hücreleri Zenginleştirme

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

İnsan Göğüs Kanseri dokuları ve hücre hatlarından gelen Mammosphere Oluşumu Testi

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Üç Boyutlu Eş-Kültür Sistemi Kullanılarak Yumurtalık Kanseri Kök Benzeri Hücrelerin Anjiyogenetik Özelliklerinin Değerlendirilmesi, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...