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 border="0" cellpadding="0" cellspacing="0" width="1049">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Name of Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td style="width:139px;">Lonza</td>
			<td>CC-3151</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:625px;">2mM L-Glutamine</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>G8540</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:625px;">100U/ml Penicillin &#38; Streptomycin</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>P4083</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:625px;">20ng/ml recombinant human epidermal growth factor (EGF)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:625px;">20ng/ml recombinant human basic fibroblast growth factor (bFGF)</td>
			<td style="width:139px;">R&#38;D systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:625px;">1x B27 supplement&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Phosphate buffered saline (PBS);</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>12399902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;0.5% trypsin-0.2%EDTA;</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>59418C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td style="width:139px;">First Link UK</td>
			<td>02-00-850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Trypan Blue</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Low attachment 6 well plates</td>
			<td style="width:139px;">Corning</td>
			<td>CLS3814</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type 1A</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>C9891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile razor blades</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>12443170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile scalpel</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">11758353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile micro-dissecting scissors</td>
			<td style="width:139px;">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...

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.

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 ...

mammosphere-formation-assay-from-human-breast-cancer-tissues-cell

Research

JoVE Journal

Medicine

33.0K 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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Menschliche neuroendokrinen Tumor-Zelllinien als dreidimensionales Modell für das Studium der menschlichen neuroendokrinen Tumor-Therapie

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Forschung

Medizin

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Verarbeitung von Human Reduction Mammoplastik und Mastektomie Gewebe für die Zellkultur

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

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 ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Der Einsatz von Reverse Phase Protein Arrays (RPPA) an Protein Expression Variation innerhalb der einzelnen Renal Cell Cancers erkunden

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Formation of Human Prostate Epithel Mit Tissue Rekombination von Nagetier Urogenitalsinus Mesenchym und menschlichen Stammzellen

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling von Östrogen-regulierte MicroRNAs in Brustkrebszellen

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantifizierung der Brustkrebs-Zelle Invasivität Mit einem Drei-dimensionale (3D) Modell

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

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

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

Bereicherung für chemoresistenten Eierstockkrebs Stammzellen aus menschlichen Zelllinien

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

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Ein<em&gt; Ex-vivo-</em&gt; Modell um die Hormonwirkung in der menschlichen Brust Studieren

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Verfahren zur Kultivierung humaner Femur Gewebeexplantaten an Brustkrebs Zellbesiedlung des metastasierten Niche Studieren

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...