Questo lavoro è supportato dal BRC Imperiale, l&#39;Istituto Nazionale per la Ricerca Sanitaria, e lotta contro il cancro, con menzione speciale per Craft Hilary e Sir Douglas Myers.

Le procedure di seguito sono stati eticamente approvati dal Imperial College di Londra. 1. Generazione di mammospheres primarie di Human Breast Cancer Cell Lines NOTA: eseguire le seguenti operazioni sotto una cappa cultura sterile. <ol><li> Preparare Mammosphere media contenente DMEM / F12 integrato con 2 mM L-glutammina, 100 U / ml di penicillina, 100 U / ml di streptomicina. Preparare completo supporto al momento dell&#39;uso con l&#39;aggiunta di 20 ng ml ricombinante fattore / di crescita epidermico umano (EGF, Sigma), 10 ng / ml ricombinante di base del fattore di crescita dei fibroblasti umani (bFGF; Sistemi di R &amp; S) e 1x B27 supplemento. </li><li> Aspirare mezzi di pallone contenente aderenti MCF-7 o MDA-MB-231 cellule (o una linea cellulare di cancro al seno di tua scelta; 70-80% confluenza), lavare due volte in 1x PBS, e trypsinize cellule. </li><li> Cellule centrifugare a 200 xg a temperatura ambiente per 5 min. Surnatante e risospendere le cellule decantare in 1-5 ml di mezzi mammosphere. Pipettasu e giù e se necessario utilizzare un filtro tappo filtro cella 40 micron per ottenere cella singola sospensione. Utilizzare un emocitometro per garantire una sospensione singola cella è formata (se non, utilizzare un ago G 25 alla siringa sospensione cellulare fino a 3 volte). </li><li> Se necessario isolare CD44 + CD24- sottoinsieme cellulare tramite FACS con anti-CD24-ficoeritrina (PE) e isotiocianato (FITC) anticorpi monoclonali anti-CD44 9-fluoresceina. In alternativa, isolare tale popolazione utilizzando una cella magnetica attivato l&#39;ordinamento del sistema (MACS) con anti-CD44 e anti-CD24-biotina microsfere combinati come da istruzioni del produttore. Eseguire selezione positiva utilizzando LS colonne, e selezione negativa utilizzando colonne LD e confermare i fenotipi di tutte le cellule isolate mediante citometria di flusso 17. </li><li> Calcolare il numero di cellule vitali per ml utilizzando trypan blue. NOTA: La vitalità cellulare è calcolato come il numero di cellule vitali diviso per il numero totale di celle all&#39;interno delle griglie sul hemocyTometer. Le cellule che occupano blu trypan sono considerate non vitali. La seguente procedura viene utilizzata per determinare con precisione percentuale di cellule vitali. <ol><li> Preparare una soluzione allo 0,4% di blu trypan in PBS. Aggiungere 0,1 ml di trypan blu stock soluzione a 1 ml di cellule. Caricare un emocitometro ed esaminare immediatamente al microscopio a basso ingrandimento. Contare il numero di cellule totali e il numero di cellule colorazione blu. Cellule vitali = [1.00 - (Numero di celle blu ÷ Numero di cellule totali)] × 100. </li><li> Per calcolare il numero di cellule vitali per ml di cultura, utilizzare la formula seguente. Ricordare di correggere il fattore di diluizione. Numero di cellule vitali × 10 4 × 1,1 = cellule / ml cultura </li></ol></li><li> Risospendere una quantità predeterminata di cellule in 2 ml di mezzi completi mammosphere in ciascun pozzetto di una piastra da 6 pozzetti aderente ultra-bassa. La densità di semina è normalmente 500-4,000 cellule / cm 2 cellule per pozzetto. Uso Opzionalmente loattacco w piastre da 24 pozzetti, mentre la semina di cellule alla stessa densità in 0,5 ml di media. NOTA: si consiglia l&#39;ottimizzazione di densità di semina e il tempo della cultura per ogni linee cellulari di interesse. </li><li> Incubare bassa fissaggio 6 pozzetti ultra a 37 ° C e 5% CO 2 per 5-10 giorni (a seconda della linea cellulare e le dimensioni mammospheres) senza disturbare le piastre, in particolare durante la fase di crescita dei primi 5 giorni . </li></ol> 2. Generazione di mammospheres primarie di cancro al seno umano campioni clinici NOTA: tessuto del cancro del seno umano può essere ottenuto da pazienti sottoposti a chirurgia per la rimozione di tumori al seno. Tessuto Store sul ghiaccio per un massimo di 24 ore in 50 ml provette sterili in coltura contenente 100 U / ml di penicillina e 100 U / ml di streptomicina. Eseguire i seguenti passaggi sotto una cappa cultura sterile. <ol><li> Trasferire il campione in un piatto Petri tessuto 100 mm con un piccolo volume di media. Remove il tessuto adiposo con le forbici sterili, scapel e pinzette. </li><li> Aggiungere 2-3 ml di DMEM / F12 e tritare il campione in 1 mm 3 pezzi con scapel sterile o lama di rasoio fino a quando rimangono senza grandi pezzi. </li><li> Risospendere le parti di tessuto a pre-riscaldato a 10 ml di DMEM contenente enzimi proteolitici (3.000 U / ml di collagenasi e 1000 U / ml ialuronidasi) e incubare a 37 ° C in un agitatore rotativo fino a che tutti i frammenti di tessuto vengono digeriti. Di solito la digestione completa richiede 1-3 ore. Regolare il tempo di digestione secondo caratteri patologici di tumori. (Per esempio adenocarcinoma mammario è generalmente difficile da digerire rispetto al carcinoma mucinoso che è più fragile e richiede tempi più rapidi). Valutare il grado di digestione ogni ora ½ osservando 20 microlitri di sospensione sotto emocitometro. </li><li> Lasciare i frammenti a sedimentare per 5 minuti, poi trasferire il surnatante in una provetta di polipropilene da 15 ml conica e centrifugare a 200 xg per 10 min a temtemperatura. Decantare con cautela le cellule surnatante e risospendere in 1 - 5 ml di media mammosphere. </li><li> Seguire i passaggi 1,1-1,4 sopra descritte per linee cellulari per ottenere e piastra di sospensioni di cellule singole. Passano cellule attraverso il 25 G siringa un massimo di 2 volte, se necessario, per limitare le cellule dannose. </li></ol> 3. Mammosphere Forming Efficiency (%) Calcolo <ol><li> Dopo il periodo di coltura, contare le mammospheres (maggiori di 40 micron di diametro) con un microscopio a 40X di ingrandimento. Immagine 5 campi aleatori utilizzando una fotocamera digitale su un microscopio ottico e determinano la dimensione mammospheres utilizzando il software di acquisizione. Utilizzare qualsiasi immagine software di analisi. </li><li> Calcola Mammosphere Efficienza Forming (MFE%) mediante la seguente equazione: MFE (%) = (° mammospheres per pozzetto) / (° cellule seminate per pozzetto) x 100 </li></ol> 4. Passaging serie di mammospheres per la valutazione di Self-rinnovamento <ol><li&gt; Pipetta i supporti contenenti il ​​mammospheres di ogni bene in un tubo da 15 ml. Lavare i pozzetti con PBS e aggiungere ai media raccolti. Centrifugare a 115 xg per 10 min a temperatura ambiente. </li><li> Gettare il surnatante e risospendere il pellet in 500 ml di pre-riscaldato 0,5% tripsina / 0,2% EDTA. Incubare per 2-3 min. </li><li> Aggiungere 500 ml di FCS per neutralizzare tripsina. Centrifugare a 500 g per 5 min. Gettare il surnatante e risospendere pellet in 100 ml di mammospheres media, pipettando su e giù per disaggregare sfere. </li><li> Contare le cellule con emocitometro e determinare se le cellule sono in sospensione di cellule singole. Se non passare attraverso un 25 G siringa fino a 3 volte per ottenere cellule singole. Seme le cellule in un nuovo minimo attaccamento 6 pozzetti ultra alla stessa densità utilizzata nella generazione primaria. </li><li> Dopo 5-10 giorni, contare sfere&gt; 40 micron e calcolare l&#39;efficienza sfera che forma con la formula riportata prima. </li></ol>

<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.

La valutazione di successo di mammospheres primarie e secondarie si basa su cellule in fase placcato in sufficientemente basse densità che mammospheres forma da singoli cloni, con un minimo di aggregazione sfera. Tuttavia a densità che sono troppo bassi, troppo pochi mammospheres possono formare per distinguere gli effetti dei trattamenti statisticamente. Densità di semina deve essere ottimizzata per ciascuna linea cellulare utilizzata dal momento che possono variare notevolmente in loro efficienza sfera formando (co...

L&#39;esistenza di linee cellulari tumorali guidati da cellule staminali tumorali staminali come ha notevolmente aggiunto alla nostra comprensione del tumore eterogeneità. Mentre alcuni diversità fenotipica nei tumori si pone dalla conseguenza clonale di cloni geneticamente distinte, una componente importante sembra essere il risultato di differenze epigenetiche: le cellule tumorali possono passare (a volte in modo reversibile) tra stelo, progenitore, e gli stati differenziati attraverso l&#39;attivazione o la repressione di specifici geni programmi di espressione 1 - 3. Questo può riflettere cellulari fattori intrinseci o estrinseci, riflettendo il programma genica attualmente espressa in una cella con la sua conseguente Autocrino in combinazione con segnalazione paracrina dalla vicina cancro stromale o cellule immunitarie consegna fattori modulatori e condizioni microambientali quali il grado di ipossia 2,4,5. Anche se gli approcci innovativi lineage tracingstanno avanzando la nostra capacità di studiare le cellule staminali del cancro putativi in loro in vivo nicchia 6-8, saggi-sfera formando rimangono un approccio popolare e conveniente per stimare il potenziale delle cellule del cancro al seno &#39;a comportarsi come cellule staminali, almeno nelle condizioni di analisi utilizzati. E &#39;spesso usato insieme a metodi retrospettivi per le cellule staminali del cancro purificante, per la loro espressione di marcatori di membrana CD44 e CD24 9, e livelli di attività dell&#39;enzima ALDH (aldeide deidrogenasi) 10, marcatori che sono state proposte per corrispondere a più mesenchymal- e epiteliale cellule staminali del cancro -come rispettivamente 11. L&#39;approccio di formazione sfera è stato sviluppato come il saggio neurosfere, permettendo la crescita delle cellule staminali putative da singoli cloni non aderenti, condizioni privo di siero con l&#39;aggiunta di fattore di crescita epiteliale (EGF) 12, dopo essere utilmente applicato alla normalità e cancerose tessuti del seno. <p class = &quot;jove_content&quot;&gt; L&#39;identità della cellula fondatore sfera formatura, ei tipi di cellule miste che compongono la massa sfera, sono rilevanti per le inferenze che possono essere fatte da mammo- o altra sfera formando saggi. Osso quiescente cellule staminali in buona lungo termine, pensato per riposare in fase G0, non sperimenteranno la precisa combinazione di fattori che potrebbero favorire l&#39;attivazione in vivo. Il saggio mammosphere consente invece la crescita delle cellule sia pronta per divisione mitotica o già divisione 13. Questi progenitori, pur non essendo un cellulare veramente riposo, possono essere il palcoscenico delle cellule che prolifera con i mitogeni FEG e il fattore di crescita dei fibroblasti di base (bFGF) utilizzati nel test. Tuttavia, contengono una serie di segnalazione cellulare associata staminali attivato Percorsi di 14. Inoltre, il tasso della loro formazione riguarda la tumorigenicità del tessuto sono stati prelevati da, misurata dalla loro potenza in saggi di diluizione limitate eterotrapianti topo 2,15,16 &lt;/ Sup&gt;. Qui forniamo protocolli dettagliati per isolare singole cellule e generare mammospheres primari da entrambe le linee di cellule di cancro al seno umano e campioni clinici di tumori al seno. Abbiamo anche descrivono come eseguire passaggi seriali di mammospheres primari per valutare auto-rinnovamento, e come calcolare sfera formando efficienza che consente il confronto tra diverse densità di semina (vedi schema in figura 1).

<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

Simile ai tessuti sani, molti di sangue e tumori solidi sono ora pensato di essere organizzati gerarchicamente, con un sottoinsieme di cellule tumorali staminali, come quello auto-rinnovarsi dando origine a una progenie più differenziato. La comprensione e il targeting queste cellule staminali tumorali nel cancro al seno, che può possedere una maggiore chemio e radio-resistenza rispetto alla massa tumorale non-staminali, è diventato un importante settore di ricerca. Marcatori compreso CD44, CD24, e l&#39;attività ALDH possono essere valutati utilizzando la fluorescenza delle cellule attivate (FACS) per isolare in modo prospettico le cellule che mostrano una maggiore tumorigenicità quando impiantati in topi immunocompromessi: il saggio mammosphere è diventato anche ampiamente utilizzato per la sua capacità di identificare in modo retrospettivo Sfera d&#39; formando cellule che si sviluppano da cloni di cellule simil-staminali singolo. Qui descriviamo approcci per la coltura appropriato di mammospheres da linee cellulari o campioni elementari di pazienti, la loro passaging, e calcoli per stimare sfera fefficienza orming (SFE). Prima discutiamo considerazioni chiave e le insidie ​​della pianificazione e l&#39;interpretazione degli esperimenti mammosphere appropriata.

Diversi campioni o quelli sottoposti a trattamenti diversi possono variare nel numero di mammospheres&gt; 40 micron che formano dopo normalizzazione per celle iniziali seminate. Calcola mammosphere formando efficienza (MFE) per ogni trattamento cresciuto in triplice copia. Ciò consente esperimenti con diverse densità di semina da confrontare. I dati sono migliori visualizzata su un grafico a barre, idealmente con controlli positivi e negativi, e la visualizzazione della deviazione standard di tutti i pozzetti in tripl...

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 saggio Formazione di Human Breast Cancer tessuti e linee cellulari

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 Views.  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

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.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

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 ...

Ricerca

Medicina

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.

Elaborazione di mastoplastica riduttiva e tessuti umani mastectomia per coltura cellulare

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.

Time-lapse imaging di primaria preneoplastiche cellule epiteliali mammarie Derivato da modelli murini geneticamente modificati di cancro al seno

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.

L&#39;uso di matrici inverse proteina di fase (RPPA) per esplorare Espressione Variazione proteine ​​all&#39;interno dei singoli tumori a cellule renali

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.

Formazione di epitelio prostatico umano utilizzando tessuti ricombinazione dei roditori urogenitale Mesenchima Sinus e cellule staminali umane

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 di microRNA regolati dagli estrogeni in cellule di carcinoma mammario

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.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

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.

Arricchimento per chemioresistente Ovarian Cancer Stem Cells da linee cellulari umane

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.

Un<em&gt; Ex vivo</em&gt; Modello per studiare Hormone azione in seno umano

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.

Metodi per la coltura di femore umano espianti di tessuto di studio sul carcinoma della mammella cellulare colonizzazione della nicchia metastatico

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