We thank Dr. Jordi Ochando from the Flow Cytometry Shared Resources at the Mount Sinai Medical Center for their assistance in flow cytometry analysis. We thank Dr. Rumana Huq from the Microscopy Shared Resource Facility at the Mount Sinai Medical Center for their imaging assistance. The authors thank the TJ Martell Foundation for its support in this project.

Questo protocollo è stato condotto presso il nostro istituto con l&#39;approvazione del comitato etico della ricerca istituzionale ed è in conformità con tutte le linee guida istituzionali, nazionali e internazionali per il benessere umano. 1. Raccolta di sangue periferico di pazienti affetti da cancro alla prostata avanzato Nota: Selezionare i pazienti con carcinoma della prostata metastatico. Ottenere il consenso scritto del paziente e registrare le caratteristiche cliniche dei pazienti, tra cui l&#39;età di isolamento e la chemioterapia precedente e trattamenti ormonali. I pazienti con malattia metastatica saranno potenzialmente avere più alta concentrazione di CTC nel sangue periferico. <ol><li> Raccogliere i campioni di sangue dopo consenso informato e sotto un appropriato Institutional Review Consiglio ha approvato il protocollo. Indossare guanti in lattice e un camice da laboratorio durante tutta la procedura. </li><li> Collegare 25 G farfalla ago e tubo alla porta aghi. Utilizzare un tampone imbevuto di alcool per pulire un&#39;area divena periferica tra il bicipite ed avambraccio, quindi applicare laccio emostatico al braccio del paziente. </li><li> Inserire l&#39;ago a farfalla nella vena antecubitale e spingere il tubo di raccolta del sangue in porta aghi per avviare la raccolta. Raccogliere circa 10 ml di sangue in provette per il prelievo che contengono acido etilendiamminotetraacetico (EDTA) per prevenire la coagulazione. </li><li> Rimuovere ago a farfalla da paziente e applicare pressione con garza sterile su sito di puntura della vena per 1 min. Sito Coprire con una benda sterile. </li></ol> 2. Isolamento di cellule mononucleate Nota: Il sangue raccolto dal passaggio 1 conterrà cellule mononucleate del sangue periferico (PBMC) (ad esempio, linfociti e monociti) oltre ai CTC mononucleari. <ol><li> Pipettare sangue intero in 50 ml tubo conico in polistirene con un 5 ml pipetta sierologica e aggiungere Hanks soluzione salina bilanciata (HBSS) in un rapporto 1: 1. Delicatamente miscela pipetta per omogeneizzare. </li><li> Aggiungere 15 ml di una soluzione preparati commercialmente (Ficoll-Paque) per separare componenti del sangue da bassa velocità di centrifugazione in gradiente di densità in una provetta conica da 50 ml in polistirolo vuoto. </li><li> Delicatamente pipetta sangue intero e la miscela di HBSS, dal punto 2.1, nel tubo di polistirolo dal punto 2.2, in modo da formare strato superiore distinta. Set pipetta di valore più basso per ridurre al minimo la miscelazione di soluzioni, questo garantirà la separazione efficiente. </li><li> Centrifugare a 400 xg per 30 min a temperatura ambiente (25 ° C). Impostare la decelerazione a valore più basso per evitare la miscelazione di soluzioni dopo la separazione. L&#39;accelerazione può essere impostato su qualsiasi velocità. </li><li> Dopo centrifugazione, identificare la banda bianco-grigio sottile strato PBMC e CTCs sandwich tra plasma sulla soluzione superiore e la separazione sul fondo della provetta. </li><li> Raccogliere le cellule della striscia bianco-grigio al punto 2.5 in una provetta da 50 ml in polistirolo con una pipetta di plastica trasferimento. </li><li> Aggiungi HBSS a 50 ml tubo conico in polistirene con PBMC e CTC ecentrifuge nuovamente a 400 xg per 10 min a RT lavare. </li><li> Decantare pellet liquido e risospendere in 50 ml di HBSS e centrifugare di nuovo a 400 xg per 3 minuti a temperatura ambiente per lavare. </li><li> Eliminare il surnatante, risospendere il pellet rimanente con 5 ml di globuli rossi Lysis buffer e incubare la soluzione per 5 minuti a temperatura ambiente. </li><li> Rimuovere il globulo rosso tampone di lisi per centrifugazione a 400 xg per 3 minuti a temperatura ambiente. </li><li> Eliminare il surnatante e risospendere il pellet in 1 ml di PBS 1x supplementato con 10% di siero fetale bovino (FBS) per bloccare prima di colorazione degli anticorpi. </li></ol> 3. Cellule mononucleate colorazione con CD45-FITC anticorpi per fluorescenza cellulare Attivato (FACS) Ordinamento <ol><li> Quantificare il numero di (Trypan Blue-negativo) cellule vitali (dal punto 2.11) utilizzando un emocitometro o contatore di cellule automatizzati. Preparare un 1 x 10 6 cellule / ml sospensione (in PBS 1x con il 10% FBS) e posizionarlo sul ghiaccio per 1 ora. </li><li> Distribuire il quantificatosospensione cellulare dal passo precedente in due tubi. Etichettare le provette come il controllo e CD45 colorazione. </li><li> Nella provetta di controllo, aggiungere IgG1κ-FITC (2,5 mcg / ml) alla diluizione di 1: 250. Nel tubo colorazione CD45, CD45 FITC aggiungere (2,5 mcg / ml) anticorpo primario coniugato alla diluizione di 1: 250 e incubare le sospensioni cellulari in ghiaccio per 30 min. Etichettatura antigene CD45 è utilizzato per escludere le cellule ematopoietiche. </li><li> Centrifugare le cellule a 400 xg per 3 min a 4 ° C e scartare il surnatante. </li><li> Lavare le cellule due volte, sospendendo ogni pellet con 1 x PBS sterile supplementato con 10% FBS seguita da centrifugazione a 400 xg per 3 min a 4 ° C. </li><li> Risospendere le cellule in una soluzione di PBS contenente 1x 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) ad una concentrazione di 10 ug / ml 1 ml. </li><li> Filtrare la soluzione finale attraverso tappi colino 35 micron in 12 millimetri tubi in polistirolo x 75 mm. </li></ol> 4. Isolamento dei prostata CTC perFACS Nota: Utilizzare un citofluorimetro per raccogliere in tempo reale CTC negativi CD45. <ol><li> Controlli di compensazione SET utilizzando cellule dal tubo etichettato, &quot;Control&quot;. </li><li> Creare cancelli che escludono detriti e gruppi di cellule utilizzando opportuni parametri previsionali scatter e side-scatter. </li><li> Stabilire le porte FITC utilizzando le sospensioni di cellule dal tubo etichettato, &quot;CD45-FITC.&quot; </li><li> Vitali (DAPI-negativo), le cellule cancello utilizzando il Pacific Blue-Un canale. </li><li> Raccogliere popolazione negativa CD45 in una sterile tubo da 15 ml in polistirolo contenente 2 ml di Roswell Park Memorial Institute (RPMI) 1640 integrato con il 10% FBS. </li><li> Centrifugare la sospensione di cellule tumorali della prostata filtrate a 400 xg per 3 min, quindi risospendere il pellet in 200 - 500 ml di RPMI supplementato con 10% FBS. </li></ol> 5. L&#39;iniezione di CTC in topi e il monitoraggio dei xenotrapianto crescita Nota:Condurre tutte le procedure animali in conformità con i protocolli approvati dal comitato di cura degli animali istituzionale. Questo protocollo è stato condotto presso la nostra istituzione, in virtù di un uso specifico protocollo animale approvato dal nostro Comitato Animal Care in conformità e il rispetto di tutte le pertinenti normative e istituzionali agenzie, regolamenti e linee guida. <ol><li> Mescolare la prostata sospensione cellulare tumorale filtrate con matrice extracellulare in un rapporto 1: 1 e posto sul ghiaccio. </li><li> Anestetizzare topo prima dell&#39;impianto sottocutaneo in una camera di alimentazione 5% (v / v) isoflurano inalato in 1 L / min di ossigeno. Assicurare anestesia appropriata controllando per la perdita dei riflessi della cornea e punta nel topo. </li><li> Utilizzando un ago G 25 e una siringa da 1 ml, iniettare 250 ml di cellule tumorali della prostata e della matrice extracellulare sospensione cellulare per via sottocutanea in entrambi i fianchi superiori di 8-10 settimane vecchio mouse immunodeficienti maschile. Iniettare 250 microlitri indipendentemente dalla concentrazione di CTC come il volume di iniezione è l&#39;iValore mportante. Amministrazione massima SQ nel topo non è superiore a 1 ml. &#160;</li><li> Monitorare topi eseguendo la palpazione settimanale di siti di iniezione del mouse per la crescita della densità nodulari sottocutanei e misurando topi peso due volte a settimana. </li><li> Euthanize topi di anidride carbonica asfissia seguita da dislocazione cervicale e raccolta sottocutaneo prostata umano xenotrapianti tumorali quando la dimensione del tumore è più di 0,5 cm e meno di 1 cm di diametro per assicurare tessuto tumorale sufficiente per analisi molecolari e di passaggio seriale. Euthanize topi con segni di sofferenza o di perdita di peso del 15%, nonostante nessun tumore è palpabile. </li></ol>

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The authors declare that they have no competing financial interests.

Questo manoscritto descrive un metodo per la generazione di cancro della prostata modelli PDX da CTC. L&#39;uso di CTC per la generazione di PDX modelli ha diversi vantaggi potenziali importanti rispetto ai metodi esistenti. Innanzitutto, raccolta accessibile delle CTC dal sangue periferico consente la generazione di modelli sperimentali dallo stesso paziente a diversi stadi della malattia. In secondo luogo, la raccolta di sangue rappresenta un metodo sicuro ed economico per isolare le cellule tumoral...

A correction was made to <a href="http://www.jove.com/video/53182">Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells</a>. There was a spelling error in one of the authors' surname. The author's name was corrected from:
Veronica Rodriquez-Bravo
to:
Veronica Rodriguez-Bravo

A correction was made to Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells.

Erratum: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Paziente xenotrapianti derivati ​​sono modelli sperimentali sempre più popolari utilizzati per la ricerca sul cancro. Essi possono essere utilizzati per la caratterizzazione di biomarcatori e percorsi biologici, valutazione preclinica di efficacia dei farmaci, e la creazione di avatar per terapie antitumorali personalizzate 1,2. In precedenza, altri gruppi di ricerca hanno sviluppato modelli PDX o impiantando o iniettando sospensioni di cellule tumorali singole o espianti intero tumore in topi immunocompromessi 1. Questi PDX modelli richiedono raccolta chirurgica del tumore solido fresco, ascite maligna o versamento pleurico da un paziente sottoposto ad un intervento chirurgico che è sia costoso e espone il paziente ad un aumentato rischio di morbilità iatrogena. Un recente sviluppo significativo nella ricerca sul cancro è stato il rilevamento, l&#39;isolamento e la caratterizzazione di cellule tumorali circolanti. Queste cellule tumorali sfuggono dalla massa tumorale primaria e in circolodove giocano un ruolo critico nella metastasi e recidive, la causa più comune di cancro legato mortalità 3. La valutazione e la caratterizzazione di CTC da diversi tipi di tumori solidi hanno fornito informazioni cliniche per la diagnosi, la prognosi e il monitoraggio malattia residua 3. Una varietà di approcci attualmente utilizzati basandosi su entrambe le proprietà fisiche, espressione di marcatori o caratteristiche funzionali di CTC può essere utilizzata per isolare efficacemente CTC 4. Esistenti macroscala metodi di isolamento CTC includono gradiente di densità centrifugazione, filtrazione fisica con pori del filtro e la separazione contro molecole di superficie. Le metodologie di isolamento CTC più utilizzati sono basati sulla cattura a base di anticorpi di CTC. Sia selezione positiva e negativa di marcatori di superficie cellulare può essere impiegato per isolare CTC dal sangue periferico. Selezione positiva per CTC nella circolazione periferica utilizza comunemente marcatori epiteliali (ad esempio, EpCAM), che unre espresso sulle CTC, ma le cellule non ematopoietiche. Lo svantaggio di questo metodo è che CTCs con potenziale metastatico sono spesso sottoposti epiteliali-to-mesenchymal transizione (EMT), che downregulates marcatori di superficie epiteliali 3. Per isolare CTC con potenziale metastatico, una metodologia di selezione negativa che impiega il marcatore di superficie ematopoietiche, CD45, per esaurire la popolazione normale di cellule di leucociti può essere usato 5. Il cancro alla prostata è il tumore più comunemente diagnosticato e una delle principali cause di decessi correlati al cancro negli uomini 6. I meccanismi di progressione del tumore e l&#39;aggressività sono state comprese e quindi la generazione e caratterizzazione di modelli sperimentali che ricapitolano l&#39;eterogeneità molecolare del cancro alla prostata sono di notevole interesse. Modelli PDX di carcinoma della prostata sono stati generato in precedenza da attecchimento delle cellule del cancro alla prostata umano in immunocomtopi promesso 7,8. Tuttavia, la generazione di tali modelli è stata ostacolata dal tasso attecchimento basso di cancro alla prostata in topi immunocompromessi, che viene principalmente attribuito alla natura indolente della malattia. Recentemente, CTC sono stati utilizzati per generare il cancro al seno 9, il cancro del polmone 10 e il cancro alla prostata 11 modelli PDX. Questi studi proof-of-concept introdotto la possibilità di generare modelli PDX indipendente dalla necessità di raccolta dei campioni chirurgici. In questo articolo si descrive in dettaglio un metodo per la generazione di questo modello sperimentale romanzo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640</td>
			<td>Gibco Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning Cell Gro</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#181;m Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml polystyrene conical tube</td>
			<td>Crystalgen</td>
			<td>23-2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>d3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm Polystyrene tubes with cell strainer cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Lavender Blood Collection Tubes with EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Winged Blood Collection Set with Push Button Retract Needle 23 gauge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer One Use Needle Holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Latex Tourniquet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Latex or non-latex gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 cotton gauze pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive bandage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of prostate cancer patient derived xenograft models from circulating tumor cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

Questo protocollo porterà alla generazione di modelli PDX da isolati CD45 prostata negativo CTC cancro. Sulla base del metodo di selezione negativa utilizzata nel nostro protocollo è necessario escludere le cellule morte mediante colorazione DAPI. La percentuale di cellule CD45 negative rilevata mediante citometria di flusso è variabile e dipende dal carico tumorale del paziente (Figura 1A). Immunofluorescenza di cellule CD45 e non ordinati utilizzando DAPI (per identificare nuclei cellulari) rivela ...

Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generazione di cancro alla prostata del paziente Derivato modelli di xenotrapianto di cellule tumorali circolanti

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

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14.0K Views.  Icahn School of Medicine at Mount Sinai. This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Video: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

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 ...

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Medicina

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 ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adattamento del semiautomatico circolanti (CTC) Saggi Tumor Cell per applicazioni cliniche e di ricerca preclinica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

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 ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Nel metodo in vitro per osservare le interazioni E-selectina-mediata Tra prostata cellule tumorali circolanti derivate da pazienti e Cellule endoteliali umane

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Generazione di pluripotenti indotte cellule staminali da distrofia muscolare di pazienti: Efficient Riprogrammazione senza integrazione di origine urinaria Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

In Arricchimento vitro di Ovarian Cancer Cells-tumorali avvio

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Istituzione di un mieloma multiplo umano Xenograft modello nel pollo per studiare la crescita del tumore, invasione e l&#39;angiogenesi

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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.