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.

Ce protocole a été réalisée à notre institution avec l&#39;approbation du conseil institutionnel d&#39;éthique de la recherche et est en conformité avec toutes les directives institutionnelles, nationales et internationales pour le bien-être humain. 1. Collecte de sang périphérique de patients atteints de cancer de la prostate avancé Remarque: Sélectionnez les patients atteints de cancer de la prostate métastatique. Obtenir le consentement écrit du patient et enregistrer les caractéristiques cliniques des patients, y compris l&#39;âge à l&#39;isolement et une chimiothérapie antérieure et les traitements hormonaux. Les patients atteints de la maladie métastatique auront potentiellement plus forte concentration de CTC dans le sang périphérique. <ol><li> Prélever des échantillons sanguins après consentement éclairé et sous un Institutional Review Board appropriée protocole approuvé. Porter des gants en latex et une blouse de laboratoire pendant toute la procédure. </li><li> Connectez 25 G aiguille à ailettes et de tubes à porte-aiguille. Utilisez un tampon imbibé d&#39;alcool pour nettoyer la zone deveine antécubitale entre biceps et avant-bras, puis appliquer garrot sur le bras du patient. </li><li> Insérez l&#39;aiguille dans la veine de papillon coude et pousser le tube de collecte de sang dans le support de l&#39;aiguille pour commencer la collecte. Recueillir environ 10 ml de sang dans des tubes de collecte contenant de l&#39;acide éthylènediaminetétraacétique (EDTA) pour empêcher la coagulation. </li><li> Retirer l&#39;aiguille papillon du patient et appliquer une pression avec un tampon de gaze stérile sur le site de ponction de la veine pendant 1 min. Site Couvrir avec un pansement stérile. </li></ol> 2. Isolement des cellules mononucléaires Remarque: Le sang recueilli à partir de l&#39;étape 1 contient des cellules mononucléaires du sang périphérique (PBMC) (par exemple, les lymphocytes et monocytes), en plus des CTC mononucléaires. <ol><li> Pipette sang entier dans 50 ml tube de polystyrène conique avec une pipette sérologique de 5 ml et ajouter une solution de sel équilibrée de Hanks (HBSS) dans un rapport de 1: 1. Doucement mélange de pipette pour homogénéiser. </li><li> Ajouter 15 ml d&#39;une solution préparation du commerce (Ficoll-Paque) pour séparer des composants du sang par centrifugation en gradient de densité faible à une vitesse de 50 ml polystyrène tube conique vide. </li><li> Pipette doucement le sang total et mélange HBSS, de l&#39;étape 2.1, dans le tube de polystyrène de l&#39;étape 2.2, pour former la couche supérieure distincte. Set pipette au plus bas réglage pour minimiser le mélange de solutions, ce qui garantira une séparation efficace. </li><li> Centrifuger à 400 g pendant 30 min à température ambiante (25 ° C). Réglez décélération au plus bas pour empêcher le mélange de solutions après la séparation. Accélération peut être réglé à toute vitesse. </li><li> Après centrifugation, d&#39;identifier la bande blanc-gris de la couche mince CMSP et CTC en sandwich entre le plasma sur la solution supérieure et la séparation au fond du tube. </li><li> Recueillir les cellules de la bande blanc-gris à l&#39;étape 2.5 dans un tube de 50 ml de polystyrène à l&#39;aide d&#39;une pipette de transfert en plastique. </li><li> Ajouter HBSS à 50 ml polystyrène tube conique avec CMSP et CTC et centrifuge de nouveau à 400 g pendant 10 min à RT laver. </li><li> Décanter culot liquide et remettre en suspension dans 50 ml de HBSS et centrifuger à nouveau à 400 g pendant 3 min à température ambiante pour se laver. </li><li> Jeter le surnageant, remettre le culot restant avec 5 ml de tampon de lyse de Red Blood Cell et incuber la solution pendant 5 min à température ambiante. </li><li> Retirer le rouge globules tampon de lyse par centrifugation à 400 g pendant 3 minutes à température ambiante. </li><li> Jeter le surnageant et remettre en suspension le culot dans 1 ml de PBS 1x complété avec 10% de sérum fœtal bovin (FBS) pour bloquer avant la coloration des anticorps. </li></ol> 3. Les cellules mononucléaires coloration avec CD45-FITC anticorps pour cellulaire activé par fluorescence (FACS) <ol><li> Quantifier le nombre de cellules viables (trypan bleu-négative) de l&#39;étape (2.11) à l&#39;aide d&#39;un hémocytomètre ou compteur de cellules automatisé. Préparer un x 10 6 cellules / ml de suspension 1 (dans PBS 1x avec 10% de FBS) et le placer sur la glace pendant 1 heure. </li><li> Distribuer le quantifiéssuspension cellulaire de l&#39;étape précédente dans deux tubes. Tubes d&#39;étiquetage que le contrôle et CD45 coloration. </li><li> Dans le tube de commande, ajouter IgG1κ-FITC (2,5 pg / ml) à une dilution de 1: 250. Dans le tube de coloration de CD45, CD45 FITC ajouter (2,5 pg / ml) anticorps primaire conjugué à une dilution de 1: 250 et on incube les suspensions de cellules sur de la glace pendant 30 min. CD45 étiquetage d&#39;antigène est utilisée pour exclure les cellules hématopoïétiques. </li><li> Centrifuger les cellules à 400 g pendant 3 min à 4 ° C, et jeter le surnageant. </li><li> Laver les cellules deux fois, en mettant en suspension chaque culot avec 1 x PBS stérile additionné de FBS à 10% suivie d&#39;une centrifugation à 400 g pendant 3 min à 4 ° C. </li><li> Remettre en suspension les cellules dans une solution de 1 ml de PBS 1X contenant 4 &#39;, 6-diamidino-2-phénylindole (DAPI) à une concentration de 10 ug / ml. </li><li> Filtrer la solution finale à 35 um bouchons de tamis 12 mm dans des tubes de polystyrène de 75 x mm. </li></ol> 4. Isolement de la prostate par CTCFACS Remarque: Utiliser un cytomètre de flux pour recueillir en direct CTC CD45 négatives. <ol><li> Définir les contrôles de compensation en utilisant des cellules du tube étiqueté, «contrôle». </li><li> Créer portes qui excluent les débris et amas de cellules en utilisant des paramètres de l&#39;avant-scatter et dispersion latérale appropriées. </li><li> Mettre en place la porte de la FITC en utilisant les suspensions de cellules à partir du tube marqué &quot;CD45-FITC.&quot; </li><li> (Cellules DAPI-négative) Porte viables en utilisant le bleu du Pacifique-un canal. </li><li> Recueillir CD45 démographique négative dans un tube de 15 ml polystyrène conique stérile contenant 2 ml de Roswell Institut Memorial Park (RPMI) 1640 complété avec 10% de FBS. </li><li> Centrifugeuse de la prostate de suspension de cellules tumorales triés à 400 g pendant 3 min, puis remettre en suspension les culots dans 200 - 500 pi de milieu RPMI supplémenté avec 10% de FBS. </li></ol> 5. Injection de CTC dans souris et le suivi de la croissance des xénogreffes Note:Effectuer toutes les procédures animales en conformité avec les protocoles approuvés par le comité de protection des animaux dans les institutions. Ce protocole a été réalisée à notre institution en vertu d&#39;un protocole d&#39;utilisation des animaux spécifiques approuvées par notre Comité de protection des animaux conformément et le respect de toutes les agences réglementaires et institutionnels, règlements et directives pertinentes. <ol><li> Mélanger la prostate suspension de cellules tumorales triés avec la matrice extracellulaire à un ratio de 1: 1 et le placer sur la glace. </li><li> Anesthetize souris avant l&#39;implantation sous-cutanée dans une chambre de fourniture de 5% (v / v) d&#39;isoflurane inhalées dans 1 L / min d&#39;oxygène. Assurer une anesthésie appropriée en vérifiant pour la perte du réflexe cornéen et des orteils chez la souris. </li><li> En utilisant une aiguille de 25 G et une seringue de 1 ml, injecter 250 ul de cellules de tumeur de la prostate et de la matrice extracellulaire suspension cellulaire sous-cutanée dans les deux flancs supérieurs de 8-10 semaines une ancienne souris immunodéficiente mâle. Injecter 250 ul indépendamment de la concentration CTC comme le volume d&#39;injection est le important valeur. Administration SQ maximum chez la souris a pas plus de 1 ml. &#160;</li><li> Surveiller les souris en effectuant palpation hebdomadaire des sites d&#39;injection de la souris sur la croissance de densités nodulaires sous-cutanées et par la mesure de poids des souris deux fois par semaine. </li><li> Euthanasier souris par le dioxyde de carbone asphyxie suivie par dislocation cervicale et les xénogreffes récolte du cancer de la prostate humain sous-cutanés lorsque la taille de la tumeur est supérieure à 0,5 cm et de moins de 1 cm de plus grand diamètre pour assurer un tissu tumoral suffisant pour les analyses moléculaires et des passages en série. Euthanasier les souris présentant des signes de détresse ou de perte de poids de 15% malgré l&#39;absence de tumeur est palpable. </li></ol>

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The authors declare that they have no competing financial interests.

Ce manuscrit décrit un procédé pour la génération de modèles PDX cancer de la prostate à partir de CTC. L&#39;utilisation de CTC pour la génération de PDX modèles présente plusieurs avantages potentiels importants par rapport aux méthodes existantes. Tout d&#39;abord, la collecte des CTC accessible à partir de sang périphérique permet de générer des modèles expérimentaux chez le même patient à différents stades de la maladie. D&#39;autre part, la collecte de sang représente un p...

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

Patient xénogreffes dérivés sont des modèles expérimentaux de plus en plus populaires utilisés pour la recherche sur le cancer. Ils peuvent être utilisés pour la caractérisation de biomarqueurs et de voies biologiques, l&#39;évaluation pré-clinique de l&#39;efficacité des médicaments, et la création d&#39;avatars pour les thérapies personnalisés du cancer 1,2. Auparavant, les autres groupes de recherche ont développé des modèles PDX soit par implantation ou injection de suspensions de cellules tumorales uniques ou des explants tumoraux chez des souris immunodéprimées entier 1. Ces PDX modèles nécessitent collection chirurgicale de la tumeur solide frais, ascite maligne ou des épanchements pleuraux provenant d&#39;un patient subissant une intervention chirurgicale qui est à la fois coûteux et expose le patient à un risque accru de morbidité iatrogène. Un développement récent important dans la recherche sur le cancer est la détection, l&#39;isolement et la caractérisation des cellules tumorales circulantes. Ces cellules tumorales échappent à partir de la masse de la tumeur primaire et pénètrent dans la circulationoù ils jouent un rôle essentiel dans les métastases et la rechute, la cause la plus commune de cancer de la mortalité liée 3. L&#39;évaluation et la caractérisation des CTC à partir de plusieurs types de tumeurs solides ont fourni des informations cliniques pour le diagnostic, le pronostic et le suivi de la maladie résiduelle 3. Une variété d&#39;approches actuellement utilisées reposant soit sur ​​les propriétés physiques, l&#39;expression de biomarqueurs, ou des caractéristiques fonctionnelles de CTC peut être utilisé pour isoler efficacement CTC 4. Les méthodes existantes d&#39;isolation CCT macroscopique comprennent la centrifugation en gradient de densité, filtration physique avec des pores de filtrage et de séparation contre des molécules de surface. Les méthodes d&#39;isolement plus largement utilisés CCT sont basés sur la capture à base d&#39;anticorps de la CTC. Tant sélection positive et négative de marqueurs de surface cellulaire peut être utilisée pour isoler les CTC partir de sang périphérique. La sélection positive pour les CTC dans la circulation périphérique utilise couramment marqueurs épithéliaux (par exemple, un EpCAM) quire exprimé sur les cellules, mais pas CTC hématopoïétiques. L&#39;inconvénient de cette méthode est que les CTC avec un potentiel métastatique ont souvent fait l&#39;objet d&#39;épithéliale-mésenchymateuse transition (EMT), qui régule à la baisse des marqueurs de surface 3 épithéliales. Afin d&#39;isoler les CTC avec un potentiel métastatique, une méthode de sélection négative qui utilise le marqueur de surface hématopoïétiques, CD45, pour épuiser la population de cellules de leucocytes normales peut être utilisé 5. Le cancer de la prostate est le cancer le plus fréquemment diagnostiqué et une cause majeure de décès liés au cancer chez les hommes 6. Les mécanismes de la progression de la tumeur et de l&#39;agressivité sont pas complètement compris et par conséquent la génération et la caractérisation des modèles expérimentaux qui récapitulent l&#39;hétérogénéité moléculaire de cancer de la prostate sont d&#39;un intérêt considérable. PDX modèles de cancer de la prostate ont été précédemment générée par la prise de greffe de cellules de cancer de la prostate humaines dans immunodépriméssouris promis 7,8. Toutefois, la production de ces modèles a été entravée par le taux de prise de greffe bas de cancer de la prostate dans des souris immunodéprimées, qui est principalement attribuable à la nature indolente de la maladie. Récemment, des CTC ont été utilisées pour générer le cancer du sein 9, le cancer du poumon et de la prostate 10 11 modèles PDX. Ces études de preuve de concept introduit la possibilité de générer des modèles PDX indépendante de la nécessité pour la collecte de l&#39;échantillon chirurgicale. Dans cet article, nous décrivons en détail un procédé pour la production de ce nouveau modèle expérimental.

<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.

Ce protocole permettra à la génération de modèles de PDX isolés CD45 prostate négative CTC cancéreuses. Basé sur la méthode de sélection négative utilisée dans notre protocole, il est nécessaire d&#39;exclure les cellules mortes en utilisant DAPI tache. Le pourcentage de cellules CD45 négatives détectées par cytométrie de flux est variable et dépend de la charge tumorale du patient (figure 1A). La coloration par immunofluorescence de cellules non triées à l&#39;aide de CD45 et DAPI (...

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.

Génération de cancer de la prostate des patients Dérivé modèles de xénogreffe de cellules tumorales circulantes

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 vues.  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.

Vidéo: 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.

Imagerie time-lapse de primaires prénéoplasiques cellules épithéliales mammaires issues de modèles de souris génétiquement modifiées de cancer du sein

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

Recherche

Médecine

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).

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

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.

Adaptation des semi-automatique de circulation des cellules tumorales (CTC) Essais pour les applications cliniques et précliniques de recherche

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.

Isolement de cellules souches du cancer à partir d&#39;échantillons cancer de la prostate humaine

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.

Dans la méthode in vitro pour observer les interactions E-sélectine-médiation entre la prostate cellules tumorales circulantes issues de patients et endothéliales cellules humaines

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.

Génération de cellules souches pluripotentes induites à partir de Dystrophie musculaire patients: efficace Reprogrammation libre-intégration de l&#39;urine cellules dérivées

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.

Isolement et immortalisation de lignées cellulaires de patients dérivé de la biopsie musculaire pour la modélisation des maladies

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.

Dans l&#39;Enrichissement vitro du cancer des ovaires cellules initiatrices de tumeurs

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.

Mise en place d&#39;un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l&#39;invasion et l&#39;angiogenèse

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.