Questo lavoro è stato sostenuto dalla Fondazione Hariri Famiglia e la Fondazione TJ Martell.

1. Raccolta ed elaborazione di Human Prostate Cancer Tissue da campioni chirurgici <ol><li> Preparare due provette coniche da 50 ml in polistirolo contenente 15 ml di Roswell Park Memorial Institute (RPMI) 1.640 terreno di coltura supplementato con 10% di siero fetale bovino (FBS) e 1% di penicillina / streptomicina. </li><li> Personale di servizio Patologia raccolto campioni primari e metastatici della prostata cancro da campioni prostatectomia e la chirurgia palliativa, rispettivamente, sotto un consiglio di Institutional Review approvato il protocollo. Campioni metastatici possono essere ottenuti da dissezioni linfonodali durante chirurgia primaria, le vertebre durante le procedure chirurgiche palliative, come decompressione del midollo spinale e polmoni durante la rimozione diagnostica delle lesioni metastatiche singoli, tra gli altri. <ol><li> Raccolta dei tessuti bulk eccesso non necessario per diagnostica clinica nel primo 50 ml tubo conico da polistirene passo 1.1 e memorizzare immediatamente a 4 ° C. Il Harvested tessuto deve misurare almeno 4-5 cm 2 di superficie e 1-2 cm di spessore. </li></ol></li><li> Elaborano personale di laboratorio tessuto sfuso ottenuto dal servizio patologia in preparazione per l&#39;esame istologico e generazione di sospensioni cellulari. <ol><li> Lavorazione di tessuti rinfusa non deve superare 24 ore da resezione chirurgica per garantire la massima vitalità cellulare. </li><li> Lavorare in un armadio biosicurezza con un bisturi sterile e pinze, prendere 2-4 mm di spessore sezioni di tessuto orizzontali di noduli tumorali macroscopici. Memorizzare immediatamente le sezioni nella seconda provetta da 50 ml conica in polistirolo dal punto 1.1. </li><li> Quando noduli macroscopici non siano visualizzati da campioni prostatectomia, raccogliere casuali spesse sezioni di tessuto orizzontali 2-4 mm dai lobi posteriori e zona di transizione. </li></ol></li><li> Separare una porzione di tessuto chirurgico usando un bisturi sterile e pinza e fissarlo in 10% formalina prima generazionezione di sospensioni cellulari. Filmati e analizzare tale istologicamente materiale per confermare la presenza di tessuto del cancro della prostata. </li></ol> 2. Generazione di Cell Sospensioni da sezioni di tessuto <ol><li> Lavorando in un armadio biosicurezza sterile, trasferire una sezione di tessuto di un piatto 60 mm x 15 coltura contenente 1 ml di soluzione sterile 1x tampone fosfato salino (PBS). <ol><li> Meccanicamente triturare l&#39;esempio utilizzando un bisturi sterile e pinze. </li><li> Non digestioni enzimatiche sono in questo protocollo. </li></ol></li><li> Trasferire la sospensione 1 ml in un tubo da 50 ml sterile. <ol><li> Aggiungere 1 ml al piatto cultura e ripetere l&#39;operazione di triturazione fino alla sezione di tessuto è completamente dissociato, solitamente 3-4x. </li><li> Ripetere questa procedura in serie finché non sono stati dissociate tutte le sezioni di tessuto. </li></ol></li><li> Risospendere il contenuto della provetta conica da 50 ml polistirene con 5 ml pi sierologicapipetta e vortex alla velocità massima (circa 3.200 rpm) per 1 min. </li><li> Filtrare la sospensione risultante attraverso un colino cella 35 micron e raccogliere in un secondo tubo da 50 ml polistirene sterile. </li><li> Generare un pellet per centrifugazione a 450 xg per 10 min. </li><li> Eliminare il surnatante, risospendere il pellet con 5 ml di tampone di lisi dei globuli rossi, ed incubare la soluzione per 5 minuti a temperatura ambiente. Rimuovere il tampone di sangue rosso lisi cellulare mediante centrifugazione per 5 minuti a 450 xg a temperatura ambiente. </li><li> Eliminare il surnatante, risospendere il pellet in 1x PBS supplementato con 5% FBS, e quantificare il numero di (Tricloroetano-negativo) cellule vitali mediante un emocitometro o contatore di cellule automatizzato. Generare un 1x10 6 cellule / ml sospensione e conservarlo in ghiaccio per non più di 1 ora. <ol><li> La vitalità e la resa delle celle può variare notevolmente tra campioni tumorali. Nell&#39;impostazione prostatectomia, la redditività può variare tra il 45-70%, e five 2-4 mm di spessore sezioni di tessuto orizzontali da un nodulo tumorale macroscopico possono essere tenuti a produrre circa 3x10 6 cellule vitali. </li></ol></li></ol> 3. Antibody colorazione delle cellule per FACS <ol><li> Etichettare cinque provette da 15 ml coniche di polistirene come illustrato di seguito. Utilizzare CD45 e CD31 etichettatura antigene escludere ematopoietiche e elementi endoteliali, rispettivamente. <ol><li> Senza macchia </li><li> IgG 2a κ-PE + IgG 1 κ-FITC </li><li> HLAI-PE + IgG 1 κ-FITC </li><li> IgG 2a κ-PE + CD31-FITC + CD45-FITC </li><li> HLAI-PE + CD45-FITC + CD31-FITC </li></ol></li><li> Distribuire la sospensione cellulare quantificata dalla sezione precedente (passo 2.7) in cinque provette nel passo 3.1. Aggiungere gli anticorpi ad una diluizione di 1:250, e incubare le sospensioni di cellule in ghiaccio per 30 min. </li><li> Centrifugare le cellule a 450 xg per 3 min a 4 ° C, e scartare il surnatante. </li&gt; <li> Lavare le cellule da risospendere ogni pellet con 1x PBS sterile supplementato con 10% FBS seguita da centrifugazione a 450 xg per 3 min a 4 ° C. </li><li> Risospendere le cellule in 1 ml di una soluzione di PBS 1x contenente 4 &#39;,6-diamidino-2-fenilindolo (DAPI) ad una concentrazione di 10 ug / ml. </li><li> Filtrare la soluzione finale attraverso 35 caps setaccio micron in 12 mm x 75 mm tubi di polistirolo. </li></ol> 4. Isolamento di prostata CSC da FACS <ol><li> Utilizzare un citometro di flusso per l&#39;ordinamento delle cellule. </li><li> Impostare i controlli di compensazione utilizzando cellule dai tubi # 1, # 2, # 3 e # 4. </li><li> Creare cancelli che escludono detriti e ammassi di cellule utilizzando i parametri previsionali e side-scatter. </li><li> Stabilire i FITC e PE cancelli con le sospensioni dal tubo # 3 e # 4 il tubo, rispettivamente. </li><li> Vitali (DAPI-negativo) le cellule cancello utilizzando la pacific blue-Un canale. </li><li> Utilizzando tubo # cellule CD31-positive e CD45-positive 5 scartare eraccogliere entrambe le popolazioni HLAI-negativi e HLAI-positivi in ​​provette di polistirene coniche 15 ml sterili contenenti 2 ml di RPMI supplementato con 10% FBS. </li><li> Centrifugare la sospensione cellulare filtrate a 450 xg per 5 min, e poi risospendere il pellet in 200-500 ml di RPMI supplementato con 10% FBS. </li><li> Quantificare (trypan blu-negativo) cellule vitali mediante un emocitometro o contatore di cellule automatizzato. </li></ol>

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Magee, J. A., Piskounova, E., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+impact,+heterogeneity,+and+uncertainty.">Cancer stem cells: impact, heterogeneity, and uncertainty.</a> Cancer Cell. 21, 283-296 (2012).</li><li>Nguyen, L. V., Vanner, R., Dirks, P., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+an+evolving+concept.">Cancer stem cells: an evolving concept.</a> Nat. Rev. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+prostate+cancer+stem+cells.">Prospective identification of tumorigenic prostate cancer stem cells.</a> Cancer Res. 65, 10946-10951 (2005).</li><li>Domingo-Domenech, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+acquired+docetaxel+resistance+in+prostate+cancer+through+depletion+of+notch-+and+hedgehog-dependent+tumor-initiating+cells.">Suppression of acquired docetaxel resistance in prostate cancer through depletion of notch- and hedgehog-dependent tumor-initiating cells.</a> Cancer Cell. 22, 373-388 (2012).</li><li>Patrawala, L., Calhoun-Davis, T., Schneider-Broussard, R., Tang, D. 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Nessun conflitto di interessi dichiarati.

Questo protocollo descrive l&#39;isolamento di CSC da tessuti tumorali della prostata umana freschi. Diverse considerazioni importanti influenzano l&#39;esito positivo di questo protocollo. Il recupero di un numero elevato di cellule tumorali della prostata vitali dipende da un&#39;attenta valutazione al lordo dei campioni chirurgici. Nella nostra esperienza, l&#39;isolamento di successo delle cellule prostatiche tumorali è meglio garantita quando i noduli tumorali macroscopici sono osserva...

Eterogeneità intratumorale è considerata una caratteristica del cancro 1. Infatti, sono stati descritti diversi meccanismi di eterogeneità intratumorale, inclusi la mutazione genetica e interazioni con il microambiente. Inoltre, alcuni tipi di cancro possono contenere una gerarchia cellulare con una sottopopolazione di cellule staminali tumorali (CSC), che presenta le caratteristiche di capacità del tumore di avvio e di auto-rinnovamento in saggi di trapianto di serie 2-5. Inizialmente descritto in ematologica 6, seno 7, 8 e cervello neoplasie, CSC sono stati studiati anche nel cancro della prostata 9-12 così come altri tipi di tumore 2-5. CSC sono generalmente considerati una frazione cellulare all&#39;interno di una popolazione eterogenea 2-5. Pertanto, la caratterizzazione funzionale e molecolare di CSC è subordinato al loro arricchimento da popolazioni di massa. Di conseguenza, nel corso degli ultimi due decenni vari soddisfattehodologies di CSC arricchimento sono stati inventati che in genere comportano la separazione delle popolazioni etichettati mediante citometria a flusso. Inoltre, una considerazione importante nello studio di CSC è che l&#39;organizzazione gerarchica dei tessuti umani può essere interrotta da manipolazioni sperimentali come passaging seriale in coltura o immunodeficienti topi. Come risultato, l&#39;isolamento diretto di CSC da tessuti umani è emerso come una metodologia importante nel campo CSC. Il cancro della prostata è una delle principali cause di morbilità e mortalità negli Stati Uniti e in tutto il mondo 13 cancro. Pertanto, l&#39;isolamento e la caratterizzazione biologica di CSC prostata è di notevole interesse. CSC prostata sono stati precedentemente arricchiti da linee cellulari, paziente derivati ​​xenotrapianti e basso passaggio del paziente derivata sospensione culture 9,10,12,14. Recentemente abbiamo riportato l&#39;isolamento di CSC prostata direttamente da s chirurgici umaniamples in virtù della loro HLAI-negativi superficie delle cellule fenotipo 10. Qui abbiamo dettagliato le procedure adottate per l&#39;isolamento di queste cellule. Tumori della prostata vengono raccolte da campioni chirurgici e trasformate in sospensioni cellulari. Le cellule vengono poi colorate con anticorpi contro HLAI nonché stromali e vitalità marcatori, e CSC sono isolati dalla fluorescenza attivato cell sorting (FACS). Isolato CSC possono quindi essere utilizzate per saggi richiedono cellule vitali.

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">11875-093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Penicillin Streptomycin</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">PBS</td>
			<td style="width:195px;">Corning cell gro</td>
			<td style="width:187px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">60 mm-15 mm cell culture dish</td>
			<td style="width:195px;">Corning</td>
			<td style="width:187px;">3295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">35 &#181;m Cell Strainer</td>
			<td style="width:195px;">BD Falcon</td>
			<td style="width:187px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">50 ml conial tube</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">15 ml conical tune</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Red blood cell lysing buffer</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:187px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HLA class I (W6/32) PE antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab43545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD31 FITC antibody</td>
			<td style="width:195px;">ebioscience</td>
			<td style="width:187px;">11-0319-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD45 FITC antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab27287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG2a&#954; PE antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">555574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG1&#954; FITC antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">551954</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DAPI</td>
			<td style="width:195px;">Invitrogen&#160;</td>
			<td style="width:187px;">d3571</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">12 x 75 mm polystyrene tubes with cell strainer cap</td>
			<td style="width:195px;">BD Falcon&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:187px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vortex Mixer</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">CG-BV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">10% Neutral Buffer Formalin</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:187px;">RBBP-0480</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cancer stem cells from human prostate cancer samples

Il modello di cellule staminali cancerose (CSC) è stato notevolmente rivisitato nel corso degli ultimi due decenni. Durante questo periodo CSC sono stati identificati e direttamente isolato da tessuti umani e serialmente propagate in topi immunodeficienti, tipicamente attraverso l&#39;etichettatura degli anticorpi di sottopopolazioni di cellule e frazionamento di citometria a flusso. Tuttavia, le caratteristiche cliniche uniche di cancro alla prostata hanno notevolmente limitato lo studio di CSC prostata da campioni freschi di tumori umani. Recentemente abbiamo riportato l&#39;isolamento di CSC prostata direttamente da tessuti umani in virtù del loro fenotipo HLA di classe I (HLAI)-negativo. Le cellule tumorali della prostata vengono raccolte da campioni chirurgici e dissociate meccanicamente. Una sospensione cellulare viene generato ed etichettato con anticorpi HLAI e stromali coniugati modo fluorescente. Sottopopolazioni di cellule HLAI-negativi sono finalmente isolate utilizzando un citometro a flusso. Il limite principale di questo protocollo è la natura frequentemente microscopica e multifocaletumore primario nei campioni prostatectomia. Tuttavia, isolato CSC prostata dal vivo sono adatti per la caratterizzazione molecolare e validazione funzionale da trapianto in topi immunodeficienti.

<img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51332/51332fig1highres.jpg" src="/files/ftp_upload/51332/51332fig1.jpg" /> Figura 1. Prostata umana raccolta tumore del cancro e citometria a flusso grafico che illustra specifiche popolazioni provenienti da un cancro alla prostata umano. (A) noduli tumorali sono raccolte per generare una sospensione cellulare. Contenuto Tumore del campione processato viene confermata con ematossilina convenzi...

Watch this Scientific Journal Video about Isolation of Cancer Stem Cells From Human Prostate Cancer Samples at JoVE.com

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

L&#39;isolamento di cellule staminali tumorali (CSC) direttamente da tessuti umani è necessaria per la loro caratterizzazione biologica. Questo manoscritto descrive una metodologia per l&#39;isolamento di CSC prostata da tessuti umani, fornendo anche suggerimenti sulla risoluzione dei problemi passi difficili.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

isolation-of-cancer-stem-cells-from-human-prostate-cancer-samples

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Medicine

14.0K Views.  Icahn School of Medicine at Mount Sinai.  L'isolamento di cellule staminali tumorali (CSC) direttamente da tessuti umani è necessaria per la loro caratterizzazione biologica. Questo manoscritto descrive una metodologia per l'isolamento di CSC prostata da tessuti umani, fornendo anche suggerimenti sulla risoluzione dei problemi passi difficili. 

Video: Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Ricerca

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

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 ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un ortotopico Modello murino di Human Prostate Cancer Metastasi

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Isolamento e propagazione di cellule tumorali circolanti da un mouse cancro modello

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).