Name: isolation and functional assessment of human breast cancer stem cells from cell and tissue samples
Uploaded: 2020-10-02T14:00:00.000+00:00
Duration: 7 min 3 s
Description: Watch this Scientific Journal Video about Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples at JoVE.com

Laboratuvar üyelerimize yararlı tartışmaları ve destekleri için teşekkür ederiz. Meme kanseri kök hücreleri ve tümör mikro ortamı üzerine yaptığımız araştırmalar, Kanada Kanser Araştırma Derneği Araştırma Enstitüsü ve ABD Ordusu Savunma Bakanlığı Meme Kanseri Programı'ndan (Hibe # BC160912) gelen hibelerle finanse edilmektedir. V.B., Batı Doktora Sonrası Bursu (Western University) tarafından desteklenmektedir ve hem A.L.A. hem de V.B. Kanada Meme Kanseri Derneği tarafından desteklenmektedir. C.L., Kanada Hükümeti'nden Vanier Canada Yüksek Lisans Bursu ile desteklenmektedir.

Yazarların açıklayacak hiçbir şeyleri yoktur.

Meme kanseri metastazı ve tedaviye direnç tüm dünyada kadınlarda mortalitenin önemli nedeni haline gelmiştir. Meme kanseri kök hücrelerinin (BCSC) bir alt popülasyonunun varlığı, metastaz 26,43,44,45,46 ve tedavi direncinin21,47,48 artmasına katkıda bulunur....

İnsan meme kanseri kök hücrelerinin (BCSC'ler) hücresel ve moleküler mekanizmalarını anlamak, meme kanseri tedavisinde karşılaşılan zorlukları ele almak için çok önemlidir. BCSC kavramının ortaya çıkışı, CD44 + CD24 / düşük meme kanseri hücrelerinin küçük bir popülasyonunun farelerde heterojen tümörler üretebildiği 1,2 bulunduğu21. yüzyılın başlarına kadar uzanmaktadır. Daha sonra, aldehit dehidrogenazın (ALDH yüksek) enzimatik aktivitesiyüksek olan insan meme kanseri hücrelerinin de benzer kök hücre benzeri özellikler gösterdiği gözlenmiştir3. Bu BCSC'ler, kendini yenileme ve farklılaşma yeteneğine sahip küçük bir hücre popülasyonunu temsil eder ve toplu tümörlerin heterojen doğasına katkıda bulunur 1,2,3. Biriken kanıtlar, evrimsel olarak korunmuş sinyal yollarındaki değişikliklerin BCSC'nin hayatta kalmasını ve bakımını sağladığını göstermektedir 4,5,6,7,8,9,10,11,12,13,14 . Ek olarak, hücre dışsal mikro ortamının farklı BCSC fonksiyonlarının dikte edilmesinde çok önemli bir rol oynadığı gösterilmiştir15,16,17. Bu moleküler yollar ve BCSC fonksiyonunu düzenleyen dış faktörler meme kanseri nüksetmesine, metastaza18 ve tedavilere direnç gelişimine katkıda bulunur 19,20,21, tedavi sonrası BCSC'lerin kalıntı varlığı meme kanseri hastalarının genel sağkalımında büyük bir zorluk oluşturmaktadır22,23 . Bu nedenle, bu faktörlerin klinik öncesi değerlendirilmesi, meme kanseri hastalarında daha iyi tedavi sonuçları elde etmek ve genel sağkalımı iyileştirmek için yararlı olabilecek BCSC hedefleme tedavilerinin belirlenmesinde çok önemlidir.
 BCSC'leri 24,25,26,27,28,29 karakterize etmek için çeşitli in vitro insan meme kanseri hücre hattı modelleri ve in vivo insan ksenograft modelleri kullanılmıştır. Hücre hatlarının her ardışık geçişten sonra sürekli olarak yeniden doldurulma yeteneği, bunları omik tabanlı ve farmakogenomik çalışmalar yapmak için ideal bir model sistemi haline getirir. Bununla birlikte, hücre hatları genellikle hasta örneklerinde gözlenen heterojenliği özetlemekte başarısız olur. Bu nedenle, hücre hattı verilerini hasta kaynaklı örneklerle tamamlamak önemlidir. BCSC'lerin en saf haliyle izolasyonu, BCSC'lerin ayrıntılı karakterizasyonunu sağlamak için önemlidir. Bu saflığın elde edilmesi, BCSC'lere özgü fenotipik belirteçlerin seçimine bağlıdır. Şu anda, ALDHyüksekCD44 + CD24- hücre fenotipi, maksimum saflık için floresan aktive hücre sıralama (FACS) kullanarak insan BCSC'lerini toplu meme kanseri hücre popülasyonlarından ayırt etmek ve izole etmek için en yaygın olarak kullanılmaktadır1, 3,26. Ayrıca, izole BCSC'lerin kendini yenileme, proliferasyon ve farklılaşma gibi özellikleri in vitro ve in vivo teknikler kullanılarak değerlendirilebilir.
Örneğin, in vitro koloni oluşturma testleri, farklı tedavi koşullarının varlığında tek bir hücrenin 50 veya daha fazla hücreden oluşan bir koloni oluşturmak için kendini yenileme yeteneğini değerlendirmek için kullanılabilir30. Mamosfer testleri, ankrajdan bağımsız koşullar altında meme kanseri hücrelerinin kendini yenileme potansiyelini değerlendirmek için de kullanılabilir. Bu tahlil, tek hücrelerin serumsuz yapışkan olmayan kültür koşullarındaki her ardışık geçişte küreler (BCSC'lerin ve BCSC'lerin karışımı) olarak üretme ve büyüme yeteneğini ölçer31. Ek olarak, in vivo mikro çevreyi yakından özetleyen ve potansiyel BCSC hedefli tedavilerin aktivitesinin araştırılmasına izin veren hücre-hücre ve hücre-matris etkileşimleri de dahil olmak üzere BCSC fonksiyonunu değerlendirmek için 3 Boyutlu (3D) kültür modelleri kullanılabilir32. İn vitro modellerin çeşitli uygulamalarına rağmen, in vivo koşulların karmaşıklığını sadece in vitro tahlilleri kullanarak modellemek zordur. Bu zorluk, BCSC davranışını in vivo olarak değerlendirmek için fare ksenograft modellerinin kullanılmasıyla aşılabilir. Özellikle, bu tür modeller meme kanseri metastazı33'ü değerlendirmek, hastalık progresyonu sırasında mikroçevre ile etkileşimleri araştırmak 34, in vivo görüntüleme35 ve antitümör ajanların hastaya özgü toksisitesini ve etkinliğini tahmin etmek için ideal bir sistem olarak hizmet eder34.
Bu protokol, insan ALDHyüksekCD44 + CD24- BCSC'lerinin heterojen meme kanseri hücrelerinin toplu popülasyonlarından maksimum saflıkta izolasyonu için ayrıntılı bir açıklama sağlar. Ayrıca, üç in vitro tekniğin (koloni oluşturma testi, mamosfer testi ve 3D kültür modeli) ve BCSC'lerin farklı fonksiyonlarını değerlendirmek için kullanılabilecek bir in vivo tümör ksenograft testinin ayrıntılı bir tanımını sunuyoruz. Bu yöntemler, BCSC biyolojisini anlamak ve / veya yeni BCSC hedefleme tedavilerini araştırmak amacıyla BCSC'leri insan meme kanseri hücre hatlarından veya birincil hasta kaynaklı meme kanseri hücrelerinden ve tümör dokusundan izole etmek ve karakterize etmek isteyen araştırmacılar tarafından kullanılmak üzere uygun olacaktır.

<table><tbody><tr><td>7-Aminoactinomycin D (7AAD)</td><td>BD</td><td>51-68981E</td><td>suggested: 0.25 &amp;micro;g/1x10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Acetone</td><td>Fisher</td><td>A18-1</td><td></td></tr><tr><td>Aldehyde dehydrogenase (ALDH) substrate</td><td>Stemcell Technologies</td><td>1700</td><td>Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer&#039;s instructions for ALDH substrate preparation</td></tr><tr><td>Basement membrane extract (BME)</td><td>Corning</td><td>354234</td><td>Sold under the commercial name Matrigel</td></tr><tr><td>Cell culture plates: 6 well</td><td>Corning</td><td>877218</td><td></td></tr><tr><td>Cell culture plates: 60mm</td><td>Corning</td><td>353002</td><td></td></tr><tr><td>Cell culture plates: 96-well ultra low attachment</td><td>Corning</td><td>3474</td><td></td></tr><tr><td>Cell strainer: 40 micron</td><td>BD</td><td>352340</td><td></td></tr><tr><td>Collagen</td><td>Stemcell Technologies</td><td>7001</td><td>Prepare 1:30 dilution of 3 mg/mL collagen in PBS</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>11088807001</td><td>1x</td></tr><tr><td>Conical tubes: 50 mL</td><td>Fisher scientific</td><td>05-539-7</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma</td><td>C6158</td><td>Use 0.05% crystal violet solution in water for staining</td></tr><tr><td>Dispase</td><td>Stemcell Technologies</td><td>7913</td><td>5U/mL</td></tr><tr><td>DMEM:F12</td><td>Gibco</td><td>11330-032</td><td>1x, With L-glutamine and 15 mM HEPES</td></tr><tr><td>DNAse</td><td>Sigma</td><td>D5052</td><td>0.1 mg/mL final concentration</td></tr><tr><td>FBS</td><td>Avantor Seradigm Lifescience</td><td>97068-085</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Flow tubes: 5ml</td><td>BD</td><td>352063</td><td>Polypropylene round-bottom tubes</td></tr><tr><td>Methanol</td><td>Fisher</td><td>84124</td><td></td></tr><tr><td>mouse anti-Human CD24 antibody</td><td>BD</td><td>561646</td><td>R-phycoerythrin and Cyanine dye conjugated Clone: ML5</td></tr><tr><td>mouse anti-Human CD44 antibody</td><td>BD</td><td>555479</td><td>R-phycoerythrin conjugated, Clone: G44-26</td></tr><tr><td>N,N-diethylaminobenzaldehyde (DEAB)</td><td>Stemcell Technologies</td><td>1700</td><td>Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer&#039;s instructions DEAB preparation</td></tr><tr><td>PBS</td><td>Wisent Inc</td><td>311-425-CL</td><td>1x, Without calcium and magnesium</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Mammosphere Media Composition&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504-44</td><td>1x</td></tr><tr><td>bFGF</td><td>Sigma</td><td>F2006</td><td>10 ng/mL</td></tr><tr><td>BSA</td><td>Bioshop</td><td>ALB003</td><td>04%</td></tr><tr><td>DMEM:F12</td><td>Gibco</td><td>11330-032</td><td>1x, With L-glutamine and 15 mM HEPES</td></tr><tr><td>EGF</td><td>Sigma</td><td>E9644</td><td>20 ng/mL</td></tr><tr><td>Insulin</td><td>Sigma</td><td>16634</td><td>5 &amp;micro;g/mL</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;3D Organoid Media Composition&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A8301</td><td>Tocris</td><td>2939</td><td>500 nM</td></tr><tr><td>B27</td><td>Gibco</td><td>17504-44</td><td>1x</td></tr><tr><td>DMEM:F12</td><td>Gibco</td><td>11330-032</td><td>1x, With L-glutamine and 15 mM HEPES</td></tr><tr><td>EGF</td><td>Sigma</td><td>E9644</td><td>5 ng/mL</td></tr><tr><td>FGF10</td><td>Peprotech</td><td>100-26</td><td>20 ng/mL</td></tr><tr><td>FGF7</td><td>Peprotech</td><td>100-19</td><td>5 ng/mL</td></tr><tr><td>GlutaMax</td><td>Invitrogen</td><td>35050-061</td><td>1x</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td>10 mM</td></tr><tr><td>N-acetylcysteine</td><td>Sigma</td><td>A9165</td><td>1.25 mM</td></tr><tr><td>Neuregulin &amp;beta;1</td><td>Peprotech</td><td>100-03</td><td>5 nM</td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td>5 mM</td></tr><tr><td>Noggin</td><td>Peprotech</td><td>120-10C</td><td>100 ng/mL</td></tr><tr><td>R-spondin3</td><td>R&amp;amp;D</td><td>3500</td><td>250 ng/mL</td></tr><tr><td>SB202190</td><td>Sigma</td><td>S7067</td><td>500 nM</td></tr><tr><td>Y-27632</td><td>Tocris</td><td>1254</td><td>5 &amp;micro;M</td></tr></tbody></table>

isolation and functional assessment of human breast cancer stem cells from cell and tissue samples

Meme kanseri kök hücreleri (BCSC'ler), kalıtsal veya edinilmiş kök hücre benzeri özelliklere sahip kanser hücreleridir. Düşük sıklıklarına rağmen meme kanserinin başlamasına, nüksetmesine, metastaza ve tedavi direncine önemli katkılarda bulunurlar. Meme kanseri tedavisinde yeni terapötik hedefler belirlemek için meme kanseri kök hücrelerinin biyolojisini anlamak zorunludur. Meme kanseri kök hücreleri, CD44, CD24 gibi benzersiz hücre yüzey belirteçlerinin ekspresyonuna ve aldehit dehidrojenazın (ALDH) enzimatik aktivitesine dayanarak izole edilir ve karakterize edilir. Bu ALDHyüksekCD44 + CD24- hücreleri BCSC popülasyonunu oluşturur ve aşağı akış fonksiyonel çalışmaları için floresan ile aktive edilmiş hücre sıralama (FACS) ile izole edilebilir. Bilimsel soruya bağlı olarak, BCSC'lerin fonksiyonel özelliklerini değerlendirmek için farklı in vitro ve in vivo yöntemler kullanılabilir. Burada, insan BCSC'lerinin hem meme kanseri hücrelerinin heterojen popülasyonlarından hem de meme kanseri hastalarından elde edilen primer tümör dokusundan izole edilmesi için ayrıntılı bir deneysel protokol sunuyoruz. Ek olarak, BCSC fonksiyonunu değerlendirmek için kullanılabilecek koloni oluşturma testleri, mamosfer testleri, 3D kültür modelleri ve tümör ksenogreft testleri dahil olmak üzere aşağı akış in vitro ve in vivo fonksiyonel tahlillerini vurguluyoruz.

Tanımlanan protokol, insan BCSC'lerinin heterojen bir meme kanseri hücresi popülasyonundan, hücre hatlarından veya ayrışmış tümör dokusundan izole edilmesine izin verir. Herhangi bir hücre hattı veya doku örneği için, BCSC'leri maksimum saflıkta izole etmek için tek tip bir tek hücreli süspansiyon oluşturmak çok önemlidir, çünkü BCSC olmayan popülasyonları kirletmek, özellikle de çalışmanın amacı BCSC'leri hedef alan terapötik ajanların etkinliğini değerlendirmekse, değişken hücre...

Watch this Scientific Journal Video about Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples at JoVE.com

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Bu deneysel protokol, BCSC'lerin meme kanseri hücre ve doku örneklerinden izolasyonunun yanı sıra BCSC fenotipini ve fonksiyonunu değerlendirmek için kullanılabilecek in vitro ve in vivo testleri tanımlamaktadır.

İnsan Meme Kanseri Kök Hücrelerinin Hücre ve Doku Örneklerinden İzolasyonu ve Fonksiyonel Değerlendirmesi

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

isolation-functional-assessment-human-breast-cancer-stem-cells-from

Research

JoVE Journal

Cancer Research

4.3K Görüntüleme.  London Regional Cancer Program. Bu deneysel protokol, BCSC'lerin meme kanseri hücre ve doku örneklerinden izolasyonunun yanı sıra BCSC fenotipini ve fonksiyonunu değerlendirmek için kullanılabilecek in vitro ve in vivo testleri tanımlamaktadır.

Video: Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

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

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

NFKB Aktivite ve Meme Kanseri Kök Hücreler önler Sentezi ve bir Aspirin-fumarat ön-ilacının Karakterizasyonu

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Sıralama Kök Hücre Tahliller ve Akış Primer Beyin Tümörü Doku İşleme

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Tıp

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 of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

3 Boyutlu Spheroid Kültürlerden Kök Benzeri Hücrelerin İzolasyon

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

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 ...

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 ...

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.

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 ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

İnsan Pankreas Kanseri Xenografts Kök Hücre İzolasyonu

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Biyoloji

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 ...