Bu çalışma Hilary Craft ve Sir Douglas Myers ile anılmayı İmparatorluk BRC, Sağlık Araştırmaları Ulusal Enstitüsü ve Eylem Karşı Kanser tarafından desteklenmektedir.

prosedürler aşağıda etik Imperial College, Londra tarafından onaylanmıştır. İnsan Göğüs Kanseri Hücre Hatlarında Primer Mammospheres 1. Üretim NOT: Bir steril kültür kaputu altında aşağıdaki adımları uygulayın. <ol><li> Mammosphere Ortam, 2 mM L-glutamin ile desteklenmiş DMEM / F12, 100 U / ml penisilin, 100 U / ml streptomisin ihtiva eden hazırlayın. 10 ng / ml yeniden birleştirici insana ait bazik fibroblast büyüme faktörü (bFGF R &amp; D Systems) ve 1 x B27 ek, 20 ng / ml yeniden birleştirici insan epidermal büyüme faktörü (EGF Sigma) ilave edilerek, kullanımdan hemen önce komple ortamı hazırlar. </li><li> (Ya da seçtiğiniz bir meme kanseri hücre hattı,% 70-80 izdiham) yapışık MCF-7 veya MDA-MB-231 hücreleri içeren bir şişeye aspire medya, 1x PBS içinde iki kez yıkayın ve hücreleri trypsinize. </li><li> 5 dakika boyunca oda sıcaklığında, 200 x g&#39;de santrifüje hücreleri. Mammosphere medya 1-5 ml Durusu süpernatant ve tekrar süspansiyon hücreleri. Pipet40 mikron hücre süzgeç kapağı filtresi yukarı ve aşağı ve gerekli kullanım halinde tek hücre süspansiyonu elde etmek. Tek bir hücre süspansiyonu (değilse, 3 kez hücre süspansiyonu kadar şırınga için bir 25 G iğne kullanımı) oluşmuştur emin olmak için, bir hemasitometre kullanarak. </li><li> FACS ile gerekli izole CD44 + CD24- hücresel alt kümesi, anti-CD24-fikoeritrin (PE) ve bir anti-CD44-floresin izotiyosiyanat (FITC) ile monoklonal antikorlar 9 kullanarak edin. Alternatif olarak, bir manyetik aktive hücre sıralama, anti-CD44 ve üreticinin talimatlarına göre, anti-CD24-biotin kombine mikro ile (MACS) sistemi kullanılarak bu nüfusu izole. LD sütunlarını kullanarak LS sütunları kullanarak pozitif seçim ve negatif seçim yapın ve 17 flow sitometri tüm izole hücrelerin fenotipleri onaylayın. </li><li> Tripan mavisi kullanarak, ml başına canlı hücre sayısını hesaplayın. Not: Hücre canlılığı hemocy ızgaralara içindeki hücrelerin toplam sayısı ile bölünmüş canlı hücre sayısı olarak hesaplanmıştırtometre. Tripan mavisi almak hücreler canlı olmayan olarak kabul edilir. Aşağıdaki prosedür tam olarak yaşayabilir hücrelerin oranını belirlemek için kullanılır. <ol><li> PBS içinde tripan mavisi bir% 0.4 çözelti hazırlayın. Hücrelerin 1 ml tripan mavi stok solüsyonu 0.1 ml ilave edilir. Hemasitometre yükleyin ve düşük büyütmede mikroskop altında incelemek hemen. Toplam hücre sayısı ve mavi boyama hücreleri sayın. Canlı hücreler, = [1,00 - (mavi hücre sayısı ÷ toplam hücre sayısı)] x 100. </li><li> Kültür ml&#39;si başına canlı hücre sayısını hesaplamak için aşağıdaki formül kullanılır. Seyreltme faktörü düzeltmek için unutmayın. 10 4 x 1.1 = hücre / ml kültüre x canlı hücre sayısı </li></ol></li><li> 6-çukurlu ultra-düşük yapışkan bir plakanın her bir tam mammosphere ortam, 2 ml hücre önceden belirlenmiş bir miktarda yeniden süspanse edin. tohum yoğunluğu normalde 500-4,000 hücre / cm oyuk başına 2 hücresidir. İsteğe bağlı kullanım loW ek 24 oyuklu plakalar ortam, 0.5 ml, aynı yoğunlukta tohum hücreleri ise. NOT: Bu ilgi her hücre hatları için ekim yoğunluğu ve kültür zaman optimizasyonu öneririz. </li><li> Özellikle ilk 5 gün büyümesi aşamasında plakalar bozmadan (hücre hattı ve mammospheres boyutuna bağlı olarak), 5-10 gün boyunca 37 ° C&#39;de ve% 5 CO2 seviyesinde ultra düşük bağlanma 6 oyuklu inkübe . </li></ol> İnsan Meme Kanseri Klinik Örneklerden İlköğretim Mammospheres 2. Nesil Not: İnsan göğüs kanser dokusu göğüs tümörlerinin çıkarılması için cerrahi geçiren hastaların elde edilebilir. 100 E / ml penisilin ve 100 U / ml streptomisin ihtiva eden kültür ortamı içinde 50 ml steril tüplerde kadar, 24 saat boyunca buz üzerinde saklayın dokusudur. Bir steril kültür kaputu altında aşağıdaki adımları uygulayın. <ol><li> Küçük bir ortam hacmi ile 100 mm doku Petri kabı içine örnek aktarın. RemovSteril makas, scapel ve cımbız kullanarak e yağ dokusu. </li><li> DMEM / F12 2-3 ml ekleyin ve hiçbir büyük parçalar kalmayıncaya kadar steril bir scapel veya jilet ile 1 mm 3 parçaya örnek kıyma. </li><li> Doku parçaları süspanse proteolitik enzimler (3000 U / ml kolajenaz ve 1000 U / ml hiyaluronidaz) ihtiva eden 10 mi DMEM önceden ısıtılmış ve doku parçaları sindirilir kadar bir döner sallayıcı içinde 37 ° C&#39;de inkübe edin. Genellikle tam sindirim 1-3 saat sürer. Tümörlerin patolojik özelliklerine göre sindirim süresini ayarlayın. (Örneğin meme adenokarsinomu daha kırılgan ve daha kısa sindirim zamana ihtiyacı müsinöz karsinom göre sindirimi genellikle zordur). Hemasitometre altında 20 ul süspansiyon gözlemleyerek sindirim her ½ saat derecesini değerlendirin. </li><li> Parçaları daha sonra, 5 dakika boyunca tortu oda tem olarak 10 dakika süreyle 200 x g&#39;de 15 ml konik bir polipropilen tüp ve santrifüj süpernatan transfer etmek içinsıcaklık. Mammosphere medya 5 ml - dikkatlice 1 süpernatant ve tekrar süspansiyon hücrelerini süzün. </li><li> Elde edilir ve tek hücre süspansiyonları plaka hücre hatları için, yukarıda tarif edilen adımları 1.1-1.4 izleyin. Gerekirse 25 G ile hücre hasar hücreleri sınırlamak için 2 kez fazla şırınga geçirin. </li></ol> 3. Mammosphere Verimliliği (%) hesaplanması Şekillendirme <ol><li> Kültür döneminden sonra, 40X büyütmede mikroskop altında mammospheres (fazla 40 um) çapı sayılır. Dijital görüntü 5 rastgele bir ışık mikroskobu dijital kamera kullanarak alanlar ve satın alma yazılımı kullanarak mammospheres boyutunu belirler. Herhangi bir resim Analizi Yazılım kullanın. </li><li> Aşağıdaki denklem kullanılarak Mammosphere Şekillendirme Verimliliği (MFE%) hesaplayın: MFE (%) = (kuyu başına mammospheres içinde) / (oyuk başına tohumlanmış hücrelerin içinde) x 100 </li></ol> Kendini yenileme Değerlendirme Mammospheres 4. Seri Pasajlanması <ol><li&gt; Pipet bir 15 ml tüp içine, her kuyudan mammospheres ihtiva eden ortam. PBS ile kuyu yıkayın ve toplanan medya ekleyin. Oda sıcaklığında 10 dakika boyunca 115 x g&#39;de santrifüjleyin. </li><li> Süpernatant atılır ve önceden ısıtılmış% 0.5 tripsin /% 0.2 EDTA, 500 ul pelletini. 2-3 dakika boyunca inkübe edin. </li><li> Tripsin nötralize etmek için FCS 500 ul ekleyin. 5 dakika boyunca 500 x g&#39;de santrifüjleyin. Küreleri parçalamak için aşağı mammospheres medya, pipet yukarı 100 ul süpernatant ve tekrar süspansiyon pelet atın ve. </li><li> Hemasitometre ile hücre sayımı ve hücreler, tek bir hücre süspansiyonu olup olmadığını belirlemektedir. Bir 25 G geçmesine değilse tek hücre elde etmek için 3 kez şırınga. Birincil oluşturmada kullanılan aynı yoğunlukta bir ultra düşük bağlanma 6 oyuklu bir plakaya hücreleri Tohum. </li><li> 5-10 gün sonra, küreler&gt; 40 um saymak ve daha önce bildirilen formülü kullanarak küre oluşturan verimliliği hesaplamak. </li></ol>

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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

Birincil ve ikincil mammospheres Başarılı değerlendirme hücreleri az küre agregasyonu ile, tek klonlar formu mammospheres yeterince düşük yoğunluklarda kaplama olan dayanır. Bununla birlikte çok düşük yoğunluklarda, çok az mammospheres istatistiksel tedavisinin etkilerini ayırt oluşturabilir. Ekim yoğunluğu onların küre oluşturan verimlilikleri de önemli ölçüde değişebilir çünkü kullanılan her hücre hattı için optimize edilmelidir (Low-E-kaderin ifade olanlar daha az istikrarlı ve d...

kök benzeri kanser kök hücreleri tarafından başkanlığında tümör hücre soylarının varlığı büyük ölçüde tümör heterojenite anlayışımıza ekledi. Tümörlerde bazı fenotipik çeşitlilik genetik olarak farklı klonların klonal akıbet kaynaklanan yok iken, önemli bir bileşeni epigenetik farklılıklar sonucu görünür: kanser hücrelerinin spesifik genin aktivasyonu veya baskı yoluyla kök, atası ve farklılaşmış devletler arasında (geri dönüşümlü olarak bazen) geçiş yapabilirsiniz ifade programları 1-3. Bu, şu anda modüle faktörleri veren kanser, stromal ve bağışıklık hücrelerinin komşu parakrin sinyal ile bağlantılı olarak elde edilen otokrin sinyalli bir hücrede ifade edilen gen ekspresyon programı yansıtan hücre iç ve dış faktörler yansıtır ve bu tür derecesi gibi microenviromental koşullar hipoksi 2,4,5. Yenilikçi soy izleme yaklaşımlar olsa dain vivo niş 6 varsayılan kanser kök hücrelerini incelemek için yeteneğimizi ilerleyen - 8, küre oluşturan deneyler, en az kullanılan deney koşulları altında, kök hücreleri gibi davranmaya meme kanseri hücrelerinin potansiyelini tahmin etmek popüler ve kullanışlı bir yaklaşım kalır. Genellikle daha mesenchymal- ve epitel karşılık önerilmiştir membran belirteçleri CD44 ve CD24 9 ve enzim ALDH (aldehit dehidrojenaz) aktivitesi seviyeleri, 10, belirteçlerin bunların ifadesi ile, temizleme kanser kök hücreleri için geriye dönük yöntem ile birlikte kullanılır benzeri kanser kök hücreleri sırasıyla 11. Küre oluşumu yaklaşım, ilk olarak epitel büyüme faktörü (EGF), 12 ilavesi ile yapışkan olmayan, serumsuz koşullarda tek klonlardan putatif kök hücrelerinin büyümesini sağlayan neurosphere deneyi geliştirildi, daha sonra yararlı normal ve kanserli uygulanan meme dokularında. <p claP = &quot;jove_content&quot;&gt; küre oluşturucu kurucu hücre kimliği ve küre kitle oluşturan karışık hücre tipleri, meme ya da deneylerde oluşturan diğer küre yapılabilir çıkarımlar için de geçerlidir. Düşünce Uzun vadeli sakin kemik niyetli kök hücreleri, in vivo aktivasyon lehine olacağını faktörlerin kesin kombinasyonu karşılaşmazsınız, G0 fazında dinlenmek için. mammosphere tahlil yerine mitoz bölünme için dengelisin ya da zaten 13 bölünmesi ya hücrelerin büyümesini sağlar. Bu progenitörler, bir gerçek hareketsiz hücre de, testte kullanılan EGF ve bazik fibroblast büyüme faktörü (bFGF), mitojen ile çoğalır hücre aşaması olabilir. Yine de, aktif kök hücre ilişkili sinyal bir dizi 14 yolaklarıyla içerirler. Buna ek olarak, bunların oluşum oranının fare ksenograftları 2,15,16 sınırlı seyreltme deneylerinde gücü ile ölçülen bölgeden alınan doku, tümöre neden olması ile de ilgilidir &lt;/ Sup&gt;. Burada tek hücrelerin izole edilmesi ve insan göğüs kanseri hücre çizgileri ve göğüs tümörlerinin klinik örneklerde her iki primer mammospheres oluşturmak için ayrıntılı protokolleri sağlar. Biz de kendini yenileme değerlendirmek için birincil mammospheres seri pasajlar gerçekleştirmek için nasıl ve küre farklı ekim yoğunlukları arasında karşılaştırma (Şekil 1 şemaya bakınız) izin veren verimliliği oluşturan hesaplamak için nasıl açıklar.

<table><tbody><tr><td>DMEM/F12</td><td>Lonza</td><td>CC-3151</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Sigma Aldrich</td><td>G8540</td><td></td></tr><tr><td>100 U/ml Penicillin &amp;amp; Streptomycin</td><td>Sigma Aldrich</td><td>P4083</td><td></td></tr><tr><td>20 ng/ml Recombinant human epidermal growth factor (EGF)</td><td>Sigma Aldrich</td><td>E9644</td><td></td></tr><tr><td>20 ng/ml Recombinant human basic fibroblast growth factor (bFGF)</td><td>R&amp;amp;D systems</td><td>233-FB-025</td><td></td></tr><tr><td>1x B27 supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Scientific</td><td>12399902</td><td></td></tr><tr><td>0.5% trypsin/0.2% EDTA</td><td>Sigma Aldrich</td><td>59418C</td><td></td></tr><tr><td>Fetal calf serum</td><td>First Link UK</td><td>02-00-850</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma Aldrich</td><td>93595</td><td></td></tr><tr><td>Low attachment 6-well plates</td><td>Corning</td><td>CLS3814</td><td></td></tr><tr><td>Collagenase type 1A</td><td>Sigma Aldrich</td><td>C9891</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma Aldrich</td><td>H3506</td><td></td></tr><tr><td>Sterile razor blades</td><td>Fisher Scientific</td><td>12443170</td><td></td></tr><tr><td>Sterile scalpel</td><td>Fisher Scientific</td><td>11758353</td><td></td></tr><tr><td>Sterile micro-dissecting scissors</td><td>Sigma Aldrich</td><td>S3146</td><td></td></tr></tbody></table>

mammosphere formation assay from human breast cancer tissues and cell lines

Sağlıklı dokuların, birçok kan ve katı maligniteler benzer şimdi daha farklılaşmış döl yol verirken kendine yenilemek kök benzeri kanser hücrelerinin bir alt kümesi olan, hiyerarşik olarak organize edilmesi düşünülmektedir. Anlama ve gelişmiş kemo ve olmayan kök tümör kitlesi ile karşılaştırıldığında radyo-dirence sahip olabilir meme kanserinde bu kanser kök hücrelerini hedefleyen, önemli bir araştırma alanı haline gelmiştir. Mammosphere deneyi de yaygın olarak geriye dönük sphere- tespit kabiliyeti için kullanılır hale gelmiştir: CD44, CD24, ve ALDH aktivitesi de dahil olmak üzere Marker prospektif, bağışıklık farelere implante edildiğinde gelişmiş tümör oluşum nedenlerinin göstermek hücreleri izole etmek için (FACS), flüoresans ile aktifleştirilen hücre kullanılarak değerlendirilebilir Tek kök hücre benzeri klonlarının gelişir hücreleri oluşturur. Burada hücre kuşakları ya da primer hasta numuneleri, bunların pasajlayarak ve küre f tahmin etmek için hesaplamalar mammospheres uygun kültür için yaklaşımlar anlatılmaktadıroluşturmayan verimlilik (SFE). Önce uygun planlama ve mammosphere deneylerin yorumunda önemli hususları ve tuzaklar tartışmak.

Farklı numuneler ya da farklı muamelelere tabi olan tohumlanmıştır ilk hücreler için normalize sonra meydana mammospheres&gt; 40 um sayı açısından değişiklik gösterebilir. Üç kez yetiştirilen her bir muamele için mammosphere oluşturucu etkinliği (MFE) hesaplayın. Bu durum, farklı tohum yoğunluğu ile deneyler karşılaştırılabilir sağlar. Veri en ideal pozitif ve negatif kontroller ile, bir çubuk grafikte görüntülenir ve üçlü kuyular arasında standart sapma gösteriyor. Sürekli% 60-80...

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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 ...

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Research

JoVE Journal

Medicine

33.2K Görüntüleme.  Imperial College London. 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.

Video: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Kanser hücresel Phenotypes üzerinde Mikroçevresel etki Interrogate için Microdiziler kullanma

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Kanser Araştırmaları

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Fare Meme Epitel HC11 ve EpH4 Hücrelerinin Farklılaşması

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

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 ...

Tıp

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

İnsan Meme Epitel Hücrelerinde Onkojenik Dönüşümün İn Vitro Değerlendirilmesi

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

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 ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Mikrofizyolojik Sistem Kullanarak İnsan Meme Dokusunda Meme Kanseri Modellemesi

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Fare Meme Epitel Hücreleri lactogenic Farklılaşma sırasında Mammospheres formu

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Biyoloji

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Meme Kanseri Araştırmaları için Potansiyel Fonksiyonel In vitro Model Olarak Meme Epitel ve Endotel Hücre Sferoidleri

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

Murine Mammosphere Kültürlerde Kendini Yenileme Nin Niceliği

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

Gelişim Biyolojisi

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Enzimatik olmayan, Mammospheres spontan Üretimi için Ön-invaziv Meme Lezyonların Serum serbest Doku Kültürü

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...