Diese Arbeit wird von der kaiserlichen BRC, dem Nationalen Institut für Gesundheitsforschung und Krebsbekämpfung mit besonderer Erwähnung Hilary Craft und Sir Douglas Myers unterstützt.

Die Verfahren sind speziell ethisch von Imperial College, London zugelassen. 1. Erzeugung von Primär Mammospheres von menschlichen Brustkrebszelllinien HINWEIS: Führen Sie die folgenden Schritte unter einer Sterilkultur Kapuze. <ol><li> Bereiten Mammosphere Medien DMEM / F12 mit 2 mM L-Glutamin, 100 U / ml Penicillin, 100 U / ml Streptomycin. Bereiten komplettem Medium unmittelbar vor der Verwendung durch Zugabe von 20 ng / ml rekombinanten menschlichen epidermalen Wachstumsfaktor (EGF; Sigma), 10 ng / ml rekombinantem humanem basischem Fibroblastenwachstumsfaktor (bFGF; R &amp; D Systems) und 1x B27 Supplement. </li><li> Absaugen Medien aus Kolben mit adhärenten MCF-7 oder MDA-MB-231-Zellen (oder eine Brustkrebszelllinie Ihrer Wahl; 70-80% Konfluenz), waschen zweimal in 1x PBS und trypsinieren Zellen. </li><li> Zentrifuge Zellen bei 200 · g bei Raumtemperatur für 5 min. Dekantieren und resuspendieren Zellen in 5.1 ml mammosphere Medien. Pipettenach oben und unten und ggf. mit einer 40 um Zellsieb Kappe Filter auf Einzelzellsuspension zu erhalten. Verwenden Sie ein Hämozytometer, um sicherzustellen, eine Einzelzellsuspension gebildet hat (wenn nicht, verwenden Sie eine 25 G Nadel, um die Zellsuspension bis 3 mal spritze). </li><li> Ggf. Isolat CD44 + CD24- Zellmenge über FACS unter Verwendung von Anti-CD24-Phycoerythrin (PE) und Anti-CD44-Fluoresceinisothiocyanat (FITC) -Antikörper 9. Alternativ zu isolieren, wie Bevölkerung mit einem Magnetrührer aktivierte Zellsortierung (MACS) System mit Anti-CD44 und anti-CD24-Biotin kombinierten Mikroperlen nach Herstellerangaben. Führen Sie eine positive Selektion mit LS Spalten und negative Selektion mit LD Spalten und bestätigen Sie die Phänotypen aller isolierten Zellen mittels Durchflusszytometrie 17. </li><li> Berechnung der Anzahl der lebensfähigen Zellen pro ml unter Verwendung von Trypanblau. HINWEIS: Die Lebensfähigkeit der Zellen wird als die Anzahl der lebensfähigen Zellen innerhalb der Gitter geteilt durch die Gesamtzahl der Zellen an der hemocy berechnetenMeter. Zellen, die bis zu Trypanblau gelten als nicht lebensfähig. Das folgende Verfahren wird verwendet, um genau zu bestimmen Anteil der lebensfähigen Zellen. <ol><li> Bereiten Sie eine 0,4% ige Lösung von Trypanblau in PBS. Fügen Sie 0,1 ml Trypanblau-Stammlösung zu 1 ml der Zellen. Legen Sie eine Zählkammer und unverzüglich zu untersuchen unter dem Mikroskop bei geringer Vergrößerung. Zählen die Anzahl der Gesamtzellen und der Anzahl der blauen Färbung Zellen. Lebensfähige Zellen = [1.00 - (Anzahl der blauen Zellen ÷ Anzahl der gesamten Zellen)] × 100. </li><li> Um die Anzahl der lebensfähigen Zellen pro ml Kultur zu berechnen, verwenden Sie die folgende Formel. Denken Sie daran, für den Verdünnungsfaktor zu korrigieren. Anzahl der lebensfähigen Zellen × 10 4 × 1,1 = Zellen / ml Kultur </li></ol></li><li> Resuspendieren eine vorgegebene Menge an Zellen in 2 ml Komplett mammosphere Medien in jede Vertiefung einer 6-Well-Ultra-Low-adhärenten Platte. Die Aussaatdichte beträgt normalerweise 500-4000 Zellen / cm 2 Zelle pro Vertiefung. Optional Verwendung low Aufsatz 24-Well-Platten, während Impfen Zellen mit gleicher Dichte in 0,5 ml Medium. HINWEIS: Wir empfehlen, die Optimierung der Aussaatdichte und Zeit der Kultur für jeden Zelllinien von Interesse. </li><li> Für 5-10 Tage (abhängig von der Zelllinie und der Größe mammospheres) Inkubieren ultra low-Anlage 6-Well-Platten bei 37 ° C und 5% CO 2, ohne die Platten zu stören, insbesondere während der Wachstumsphase der ersten 5 Tage . </li></ol> 2. Erzeugung von Primär Mammospheres von Human Breast Cancer klinischen Proben HINWEIS: Menschliche Brustkrebsgewebe von Patienten einer Operation zur Entfernung von Brusttumoren erhalten werden. Speicher Gewebe auf Eis für bis zu 24 h in 50 ml sterile Röhrchen in Kulturmedien, die 100 U / ml Penicillin und 100 U / ml Streptomycin. Führen Sie folgende Schritte im Rahmen einer Sterilkultur Kapuze. <ol><li> Übertragen der Probe in eine 100 mm Gewebepetrischale mit einem kleinen Volumen von Medien. Remove das Fettgewebe mit Hilfe einer sterilen Schere, Skalpell und Pinzette. </li><li> Fügen Sie 2-3 ml DMEM / F12 und Blatt vor der Probe in 1 mm 3 Stück mit einem sterilen Skalpell oder einer Rasierklinge, bis keine großen Stücke bleiben. </li><li> Resuspendieren der Gewebestücke in vorgewärmter 10 ml DMEM mit proteolytischen Enzymen (3.000 U / ml Kollagenase und 1.000 U / ml Hyaluronidase) und bei 37 ° C in einem Rotationsschüttler, bis alle Gewebefragmenten verdaut werden. In der Regel vollständige Verdauung dauert 1-3 Stunden. Stellen Sie die Kochzeit nach pathologischen Zeichen von Tumoren. (Zum Beispiel Brust-Adenokarzinom ist in der Regel schwerer zu verdauen, verglichen mit muzinösen Karzinomen, die empfindlicher ist und benötigt kürzere Aufschlusszeit). Bewerten Sie den Grad der Verdauung jede halbe Stunde durch die Beobachtung 20 ul Suspension unter Zählkammer. </li><li> Ermöglichen die Fragmente für 5 min sedimentieren, dann den Überstand in einem 15 ml konischen Polypropylen-Röhrchen und zentrifugiert bei 200 g für 10 min bei Raum temtur. Dekantieren Sie vorsichtig von den Überstand und resuspendieren Zellen in 1-5 ml mammosphere Medien. </li><li> Folgen Sie den oben für Zelllinien zu erhalten und plattenEinzelZellSuspensionen beschriebenen Schritte 1,1-1,4. Pass-Zellen durch die Spritze 25 G für bis zu 2-mal, wenn notwendig, schädlichen Zellen zu begrenzen. </li></ol> 3. Mammosphere Forming Wirkungsgrad (%) Berechnung <ol><li> Nach der Kulturzeit, zählen die mammospheres (größer als 40 um) Durchmesser unter einem Mikroskop bei 40-facher Vergrößerung. Digital Bild 5 zufälligen Feldern mit einer Digitalkamera auf einem Lichtmikroskop und bestimmen den mammospheres Größe mit der Erfassungssoftware. Verwenden Sie eine Bildanalyse-Software. </li><li> Berechnen Mammosphere Bildungseffizienz (MFE%) mittels der folgenden Gleichung: MFE (%) = (Anzahl der mammospheres pro Vertiefung) / (Anzahl der Zellen pro Vertiefung ausgesät) x 100 </li></ol> 4. Serien Passagieren von Mammospheres zur Bewertung der Selbsterneuerung <ol><li&gt; Pipette die Medien, die die mammospheres aus jeder Vertiefung in eine 15-ml-Tube. Waschen Sie die Vertiefungen mit PBS und fügen Sie den gesammelten Medien. Zentrifuge bei 115 × g für 10 min bei Raumtemperatur. </li><li> Überstand verwerfen und das Pellet in 500 ul vorgewärmtem 0,5% Trypsin / 0,2% EDTA. Inkubationszeit von 2-3 min. </li><li> Fügen Sie 500 ul FCS Trypsin zu neutralisieren. Zentrifuge bei 500 × g für 5 min. Überstand verwerfen und Pellet in 100 ul mammospheres Medien, Auf- und Abpipettieren zu Kugeln zu zerlegen. </li><li> Zählen Sie die Zellen mit Hämozytometer und festzustellen, ob Zellen in Einzelzellsuspension. Wenn nicht durch eine 25 G passieren Spritze bis zu 3 mal, um Einzelzellen zu erhalten. Samen, die Zellen in eine neue Ultra Low-Befestigung 6-Well-Platte mit der gleichen Dichte im Primärerzeugung verwendet. </li><li> Nach 5-10 Tagen rechnen Bereichen&gt; 40 &amp; mgr; m und berechnen Sphäre Bildungseffizienz mit Hilfe der Formel bereits berichtet. </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.

Erfolgreiche Bewertung der primären und sekundären mammospheres stützt sich auf Zellen, die bei ausreichend niedrigen Dichten, die Form mammospheres von Einzelklonen, mit minimalen Bereich Aggregation überzogen. Jedoch bei Dichten, die zu niedrig sind, können zu wenige mammospheres bilden, um die Auswirkungen der Behandlungen statistisch unterscheiden. Für jede Zelllinie verwendet, da sie erheblich in ihrer Sphäre bildet Effizienz variieren können, sollten Aussaatdichte optimiert werden (die niedrigen E-Cadherin...

Die Existenz von Tumorzelllinien durch stielartigen Krebsstammzellen geleitet hat wesentlich zum Verständnis der Tumorheterogenität aufgenommen. Während einige phänotypische Vielfalt in Tumoren nicht aus der klonalen Auswuchs von genetisch unterschiedlichen Klone entstehen, wird ein wesentlicher Bestandteil von epigenetischen Unterschiede ergeben: Krebszellen zwischen Stammzellen, Vorläufer und differenzierte Staaten über die Aktivierung oder Unterdrückung der spezifischen Gens (manchmal auch reversibel) übergehen kann Ausdruck Programme 1-3. Dies kann Zelle intrinsische oder extrinsische Faktoren widerspiegeln, was die Genexpression Programm, das gerade in einer Zelle mit ihrer resultierenden autokrine Signalisierung in Verbindung mit parakrinen Signalisierung von benachbarten Krebs, Stromazellen oder Immunzellen liefert modulierende Faktoren exprimiert und microenviromental Bedingungen, wie der Grad der Hypoxie 2,4,5. Obwohl innovative Linie Tracing Ansätzeavancieren unsere Fähigkeit, vermeintliche Krebsstammzellen in ihrer in vivo Nische 6 studieren - 8, bleiben Kugel bildenden Tests zu einem beliebten und bequemen Umgang mit potenziellen Brustkrebszellen &#39;schätzen, wie Stammzellen zumindest unter den Testbedingungen verwendet verhalten. Es wird häufig neben retrospektiven Verfahren zur Reinigung von Krebsstammzellen verwendet wird, durch ihre Expression von Membranmarker CD44 und CD24 9 und Aktivität des Enzyms ALDH (Aldehyd Dehydrogenase) 10, Marker, die vorgeschlagen wurden, um entsprechen mehr mesenchymal- und epithelialen -ähnliche Krebsstammzellen bzw. 11. Die Kugelbildung Ansatz wurde zuerst als Neurosphäre Assay entwickelt, um somit das Wachstum von putativen Stammzellen aus einzelnen Klonen in nichthaftenden, serumfreien Bedingungen mit der Zugabe von epithelialen Wachstumsfaktor (EGF) 12, wobei später nutzbringend auf normalen und kanzerösen angewendet Brustgewebe. <p class = &quot;jove_content&quot;&gt; Die Identität der Kugel bildenden Gründerzelle, und die gemischten Zelltypen, aus denen die Kugel Masse, sind für die Folgerungen, die sich aus Mammographie oder andere Kugel bilden Tests gemacht werden können relevant. Langfristige Ruheknochen fide Stammzellen, gedacht, um in G0-Phase ruhen, nicht die genaue Kombination von Faktoren, die Aktivierung in vivo begünstigen würde zu erleben. Die mammosphere Assay ermöglicht stattdessen das Wachstum von Zellen entweder balanciert für mitotische Teilung oder bereits Division 13. Diese Vorläuferzellen, die aber nicht eine wirklich ruhende Zelle kann die Zell-Stadium, die mit den im Test verwendeten EGF und basischen Fibroblasten-Wachstumsfaktor (bFGF) Mitogene vermehrt sein. Trotzdem enthalten sie eine Reihe von aktivierten Stammzellen-assoziierten Signalwege 14. Darüber hinaus ist die Geschwindigkeit ihrer Entstehung betrifft die Tumorigenität der Gewebe sie abgenommen hat, wenn sie durch ihre Wirksamkeit in begrenzter Verdünnung Assays in Maus-Xenotransplantaten 2,15,16 gemessen &lt;/ Sup&gt;. Hier stellen wir ausführliche Protokolle um einzelne Zellen zu isolieren, und zum Generieren von Primär mammospheres sowohl von menschlichen Brustkrebs-Zelllinien und klinischer Proben von Brusttumoren. Wir beschreiben auch, wie serielle Passagen von Primär mammospheres durchführen, um die Selbsterneuerung zu bewerten und wie zu berechnen Sphäre Bildungseffizienz, die einen Vergleich zwischen verschiedenen Einsaatdichten (siehe Schema in Abbildung 1) ermöglicht.

<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

Ähnlich wie bei gesunden Geweben, viele Blut und solide Tumore sind jetzt vermutlich hierarchisch organisiert werden, mit einer Teilmenge der stielartigen Krebszellen, die selbst zu erneuern, während, die zu differenzierteren Nachkommen. Das Verständnis und die Ausrichtung dieser Krebsstammzellen bei Brustkrebs, die verstärkt Chemo- und Strahlenresistenz im Vergleich zu den nicht-Stammtumormasse besitzen kann, ist zu einem wichtigen Forschungsgebiet. Die mammosphere Assay wurde auch bekannt durch ihre Fähigkeit, im Nachhinein zu ermitteln kugel- verwendet: Markierungen einschließlich CD44, CD24 und ALDH-Aktivität kann unter Verwendung von Fluoreszenz-aktivierter Zellsortierung (FACS) prospektiv isolieren Zellen, die eine verbesserte Tumorigenität anzuzeigen, wenn in immunsupprimierten Mäusen implantiert beurteilen bildenden Zellen, die aus einzelnen Stammzellen wie Klone zu entwickeln. Hier beschreiben wir Ansätze für die entsprechende Kultivierung mammospheres von Zelllinien oder primären Patientenproben, ihren Passagieren und Berechnungen zur Sphäre f schätzenorming Effizienz (SFE). Zuerst diskutieren wir wichtige Überlegungen und Fallstricke in der entsprechenden Planung und Auslegung von mammosphere Experimente.

Verschiedene Proben oder Personen unterschiedlichen Behandlungen unterworfen wird, kann die Anzahl der mammospheres&gt; 40 um, die nach der Normalisierung für Ausgangszellen ausgesät bilden variieren. Berechnen mammosphere Bildungseffizienz (MFE) für jede Behandlung in drei Exemplaren gezüchtet. Dies ermöglicht Experimente mit verschiedenen Einsaatdichten verglichen werden. Die Daten werden am besten auf einem Balkendiagramm dargestellt, idealerweise mit Positiv- und Negativkontrollen, und die Anzeige der Standarda...

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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.

Mammosphere Formation Assay von Menschen Brustkrebs Geweben und Zelllinien

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 Aufrufe.  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.

Serie: 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.

Verwendung von Mikroarrays zur Vernehmung von Mikroumweltauswirkungen auf zelluläre Phänotypen bei Krebs

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

Forschung

Krebsforschung

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.

Differenzierung von Mausbrustepithel-HC11- und EpH4-Zellen

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.

Zeitraffer-Imaging of Primary Präkanzeröse Brustepithelzellen aus gentechnisch veränderten Mausmodellen der Breast Cancer Abgeleitet

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

Medizin

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.

In Vitro-Bewertung der onkogenen Transformation in menschlichen Mammaepithelzellen

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.

Isolierung und funktionelle Beurteilung menschlicher Brustkrebsstammzellen aus Zell- und Gewebeproben

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.

Modellierung von Brustkrebs im menschlichen Brustgewebe mit einem mikrophysiologischen 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 ...

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.

Maus Brustepithelzellen Form Mammospheres Während lactogenic Differenzierung

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

Biologie

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.

Brustepithel- und Endothelzell-Sphäroide als potenzielles funktionelles In-vitro-Modell für die Brustkrebsforschung

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.

Quantifizierung der Selbsterneuerung in Murine Mammosphärenkulturen

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

Entwicklungsbiologie

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.

Nicht-enzymatische, Serumfreie Zellkultur von Pre-invasive Brustläsionen für Spontane Erzeugung Mammospheres

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