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.

Dieses Protokoll wurde an unserer Hochschule mit Genehmigung durch die institutionellen Forschungsethikkommission durchgeführt und ist in Übereinstimmung mit allen institutionellen, nationalen und internationalen Richtlinien für das menschliche Wohlergehen. 1. Erhebung der peripheren Blut von Patienten mit fortgeschrittenem Prostatakrebs Hinweis: Wählen Sie Patienten mit metastasierendem Prostatakrebs. Eine schriftliche Einwilligung des Patienten und notieren klinischen Merkmale der Patienten, einschließlich Alter, in Isolation und vorherige Chemotherapie und Hormonbehandlungen. Patientinnen mit metastasiertem Brustkrebs wird potenziell höchste Konzentration von CTCs im peripheren Blut. <ol><li> Sammeln von Blutproben nach der informierten Einwilligung und unter einer geeigneten Institutional Review Board genehmigten Protokoll. Latexhandschuhe und Laborkittel während des gesamten Verfahrens zu tragen. </li><li> Schließen Sie 25 G Butterfly-Nadel und Schlauch mit Nadelhalter. Verwenden Sie einen Alkoholtupfer zur Reinigung BereichAntekubitalvene zwischen Bizeps und Unterarm, dann gelten Tourniquet um Patienten Oberarm. </li><li> Legen Schmetterling Nadel in Antekubitalvene und schieben Blutentnahmeröhrchen in die Nadelhalter zur Sammlung zu beginnen. Sammeln ca. 10 ml Blut in Blutentnahmeröhrchen Ethylendiamintetraessigsäure (EDTA), um ein Gerinnen zu verhindern, enthalten. </li><li> Entfernen Schmetterlingsnadel von Patient und Druck mit steriler Gaze über Ort der Venenpunktion für 1 min. Abdeckung Seite mit einem sterilen Verband. </li></ol> 2. Isolierung von mononukleären Zellen Anmerkung: Die in Schritt 1 gesammelten Blut werden periphere mononukleäre Blutzellen (PBMCs) (zB Lymphozyten und Monozyten) zusätzlich zu den mononukleären CTCs enthalten. <ol><li> Pipette Vollblut in 50 ml konischen Röhrchen Polystyrol mit einer 5 ml serologische Pipette und fügen Hanks Balanced Salt Solution (HBSS) in einem Verhältnis 1: 1. Gently Pipette Mischung zu homogenisieren. </li><li> 15 ml eines kommerziell hergestellten Lösung (Ficoll-Paque), um Blutkomponenten durch niedrige Geschwindigkeit Dichtegradientenzentrifugation getrennt in einen leeren 50 ml konischen Röhrchen Polystyrol. </li><li> Pipette vorsichtig Vollblut und HBSS Mischung aus Schritt 2.1, in die Polystyrol-Röhrchen aus Schritt 2.2, um unterschiedliche obere Schicht zu bilden. Set Pipette auf niedrigste Einstellung zu minimieren Durchmischung von Lösungen, wird dies zu gewährleisten effiziente Trennung. </li><li> Zentrifuge bei 400 × g für 30 min bei RT (25 ° C). Stellen Verzögerung auf niedrigste Einstellung Mischung von Lösungen nach der Trennung zu verhindern. Beschleunigung kann auf jede Geschwindigkeit eingestellt werden. </li><li> Nach dem Zentrifugieren identifizieren den dünnen weißgrauen Streifen von PBMCs und CTCs klemmt Schicht zwischen Plasma auf der Oberseite und Trennlösung am Boden des Röhrchens. </li><li> Sammeln Sie die Zellen von der weiß-grauen Streifen in Schritt 2.5 in ein 50 ml Polystyrolröhrchen mit einem Kunststofftransferpipette. </li><li> In HBSS auf 50 ml konischen Röhrchen Polystyrol mit PBMCs und CTCs und centrifuge wieder bei 400 × g für 10 min RT zu waschen. </li><li> Dekantier-Flüssigkeit und Pellet in 50 ml HBSS und Zentrifuge wieder bei 400 × g für 3 min bei RT waschen. </li><li> Überstand verwerfen, resuspendieren verbleibende Pellet mit 5 ml Lyse der roten Blutkörperchen Puffer und Inkubation der Lösung für 5 min bei RT. </li><li> Entfernen der roten Blutkörperchen Lysepuffer durch Zentrifugation bei 400 × g für 3 min bei RT. </li><li> Der Überstand verworfen und das Pellet in 1 ml PBS 1x mit 10% fötalem Rinderserum (FBS) ergänzt werden, um vor der Antikörperfärbung blockieren. </li></ol> 3. Die Färbung mononukleären Zellen mit CD45-FITC Antikörper für Fluorescence Activated Cell Sorting (FACS) <ol><li> Quantifizierung der Zahl der lebensfähigen (Trypanblau-negative Zellen) (aus Schritt 2.11) mit einer Zählkammer oder automatisierten Zellzähler. Bereiten Sie eine 1 x 10 6 Zellen / ml Suspension (in PBS 1x mit 10% FBS) und legen Sie sie auf Eis für 1 Stunde. </li><li> Verteilen Sie die quantifiziertZellsuspension aus dem vorherigen Schritt in zwei Röhrchen. Label Rohre als Kontrolle und CD45-Färbung. </li><li> In dem Steuerrohr, fügen IgG1κ-FITC (2,5 ug / ml) in einer Verdünnung von 1: 250. In der CD45-Färbung Rohr, fügen CD45 FITC (2,5 ug / ml) konjugierte primäre Antikörper bei einer Verdünnung von 1: 250 und Inkubation der Zellsuspensionen auf Eis für 30 min. CD45-Antigen Kennzeichnung wird verwendet, um hämatopoetische Zellen auszuschließen. </li><li> Zentrifugieren Sie die Zellen bei 400 × g für 3 min bei 4 ° C, und den Überstand verwerfen. </li><li> Zweimaliges Waschen der Zellen durch Suspendieren jedes Pellet mit sterilem 1 x PBS, ergänzt mit 10% FBS, gefolgt von Zentrifugation bei 400 × g für 3 min bei 4 ° C. </li><li> Resuspendieren der Zellen in einer 1 ml Lösung von PBS 1x enthaltend 4 &#39;, 6-Diamidino-2-phenylindol (DAPI) in einer Konzentration von 10 ug / ml. </li><li> Filtern Sie die endgültige Lösung durch 35 &amp; mgr; m Sieb Kappen in 12 mm x 75 mm Polystyrolröhrchen. </li></ol> 4. Isolierung von Prostata CTCs durchFACS Hinweis: Nutzen Sie eine Durchflusszytometer, um Live-CD45 negativen CTCs zu sammeln. <ol><li> Stellen Kompensationssteuerungen unter Verwendung von Zellen aus dem Röhrchen beschriftet, &quot;Control&quot;. </li><li> Erstellen Tore, die Trümmer und Cluster von Zellen unter Verwendung von geeigneten Vorwärtsstreu und Seitenstreuparameter aus. </li><li> Stellen Sie die FITC-Gates mit den Zellsuspensionen aus dem Röhrchen markiertem &quot;CD45-FITC.&quot; </li><li> Tor lebensfähigen (DAPI-negative) Zellen unter Verwendung des Pacific Blue-A-Kanal. </li><li> Sammeln CD45 negativen Population in einen sterilen 15 ml konischen Röhrchen Polystyrol, das 2 ml Roswell Park Memorial Institute (RPMI) 1640 mit 10% FBS ergänzt. </li><li> Zentrifuge nach der Prostatatumorzellsuspension bei 400 × g für 3 min, dann Resuspendieren des Pellets in 200 bis 500 ul RPMI, supplementiert mit 10% FBS. </li></ol> 5. Die Injektion von CTCs in Mäusen und Überwachung von Xenograft Wachstum Hinweis:Führen Sie alle Tierverfahren in Übereinstimmung mit den Protokollen von der Institutional Animal Care Committee genehmigt. Dieses Protokoll wurde an unserem Institut wurde im Rahmen eines spezifischen Tier Verwenden Protokolls durch unsere Animal Care Committee in Übereinstimmung und Einhaltung aller relevanten rechtlichen und institutionellen Agenturen, Vorschriften und Richtlinien zugelassen durchgeführt. <ol><li> Mischen Sie die sortiert Prostatatumorzellsuspension mit der extrazellulären Matrix in einem Verhältnis 1: 1 und auf Eis. </li><li> Betäuben Maus vor subkutaner Implantation in einer Kammer, Zuführen von 5% (v / v) inhalierten Isofluran in 1 l / min Sauerstoff. Eine angemessene Betäubung, indem für den Verlust der Hornhaut und Zehenreflex in der Maus. </li><li> Verwendung einer 25 G-Nadel und einer 1 ml Spritze, Injizieren 250 ul Prostatatumorzellen und extrazellulärer Matrix Zellsuspension subkutan in beide oberen Flanken einer 8-10 Wochen alten männlichen immundefizienten Maus. Volumina von 250 ul unabhängig von CTC-Konzentration als das Injektionsvolumen ist das important Wert. Maximale SQ Verwaltung in der Maus nicht mehr als 1 ml. &#160;</li><li> Überwachen Mäusen durch Ausführen wöchentliche Palpation der Maus Injektionsstellen für das Wachstum von subkutanen noduläre Dichten und durch Messen Mäusen Gewicht zweimal pro Woche. </li><li> Euthanize Mäuse durch Ersticken mit Kohlendioxid, gefolgt durch Genickbruch und der Ernte subkutanen menschlichen Prostatakrebs-Xenotransplantaten, wenn die Tumorgröße mehr als 0,5 cm und weniger als 1 cm größter Durchmesser ausreichend Tumorgewebe für molekularbiologische Untersuchungen und die serielle Passage zu gewährleisten. Euthanize Mäusen mit Anzeichen von Leiden oder Gewichtsverlust von 15%, obwohl keine Tumor ist spürbar. </li></ol>

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The authors declare that they have no competing financial interests.

Diese Handschrift beschreibt ein Verfahren zur Erzeugung von Prostatakrebs PDX Modelle CTCs. Die Verwendung von CTCs zur Erzeugung von PDX Modelle hat mehrere wichtige potenzielle Vorteile gegenüber bestehenden Verfahren. Zuerst zugängliche Sammlung von CTCs aus dem peripheren Blut ermöglicht die Erzeugung von experimentellen Modellen des gleichen Patienten in verschiedenen Krankheitsstadien. Zweitens stellt der Blutentnahme eine sicherere und kostengünstiges Verfahren, um Tumorzellen zu isolieren...

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

Patienten abgeleitet Xenotransplantate werden immer beliebter experimentelle Modelle für die Krebsforschung eingesetzt. Sie können für die Charakterisierung von Biomarkern und biologische Pfade, präklinische Bewertung der Wirksamkeit von Medikamenten, und die Schaffung von Avataren für personalisierte Krebstherapien 1,2 verwendet werden. Zuvor haben andere Forschungsgruppen PDX-Modelle entweder durch Implantation oder Injektion von einzelnen Tumorzellsuspensionen oder ganze Tumor Explantate in immungeschwächten Mäusen 1 entwickelt. Diese PDX Modelle erfordern chirurgische Sammlung von frischen soliden Tumor, malignen Aszites oder Pleuraerguss von einem Patienten, der sich einem chirurgischen Eingriff, die sowohl teuer ist und setzt den Patienten einem erhöhten Risiko der iatrogenen Morbidität. Eine signifikante neue Entwicklung in der Krebsforschung ist die Detektion, Lokalisierung und Charakterisierung von zirkulierenden Tumorzellen. Diese Tumorzellen zu entkommen aus dem primären Tumormasse und geben Zirkulationwo sie spielen eine entscheidende Rolle bei der Metastasierung und Rückfall, ist die häufigste Ursache von Krebs bedingte Sterblichkeit 3. Die Auswertung und Charakterisierung von CTCs aus verschiedenen soliden Tumorarten haben klinische Informationen für die Diagnose, Prognose und Überwachung von Resterkrankung 3 zur Verfügung gestellt. Eine Vielzahl von derzeit verwendeten Ansätze sich auf entweder die physikalischen Eigenschaften, die Expression von Biomarkern oder Gebrauchseigenschaften CTCs kann verwendet werden, um effizient zu isolieren CTCs 4 werden. Bestehende Makro CTC Isolationsverfahren beinhalten Dichtegradientenzentrifugation, körperliche Filtration mit Filterporen und Trennung gegen Oberflächenmoleküle. Die am weitesten verbreitete CTC Isolierung Methoden werden auf Antikörper-basierte Erfassung von CTC basiert. Sowohl positive als auch negative Selektion von Zelloberflächenmarkern verwendet werden, um CTCs aus peripherem Blut zu isolieren. Positive Selektion für CTCs im peripheren Kreislauf verwendet allgemein epithelialen Marker (zB EpCAM), die einewieder auf CTCs aber nicht hämatopoetischen Zellen exprimiert. Der Nachteil dieser Methode ist, dass CTCs mit metastatischem Potenzial oft epithelial-to-mesenchymale Transition (EMT), die Epitheloberfläche Marker 3 downregulates zogen. Um CTCs mit metastatischen Potential zu isolieren, eine negative Selektionsmethode, die den blutbildenden Oberflächenmarker CD45 verwendet, um zum Abbau der normalen Zellpopulation von Leukozyten verwendet werden 5. Prostatakrebs ist die am häufigsten diagnostizierte Krebs und eine Hauptursache der durch Krebs verursachten Todesfälle bei Männern 6. Die Mechanismen der Tumorprogression und Aggressivität sind nicht vollständig verstanden, und deshalb die Erzeugung und Charakterisierung von experimentellen Modellen, die die molekulare Heterogenität von Prostatakrebs rekapitulieren sind von großem Interesse. PDX Modelle von Prostatakrebs wurden vorher durch Verpflanzung von menschlichen Prostatakrebszellen erzeugten immunocomversprach Mäusen 7,8. Jedoch ist die Erzeugung solcher Modelle ist durch die geringe Transplantationsrate von Prostatakrebs in immungeschwächten Mäusen, die im Wesentlichen auf die träge Art der Krankheit zurückzuführen ist behindert. Kurzem CTCs wurden verwendet, um Brustkrebs 9, Lungenkrebs und Prostatakrebs 10 11 PDX Modelle zu erzeugen. Diese Proof-of-Concept-Studien wurde die Möglichkeit eingeführt PDX-Modelle unabhängig von der Notwendigkeit eines chirurgischen Probensammlung zu erzeugen. In diesem Artikel beschreiben wir im Detail ein Verfahren zur Erzeugung dieser neuartigen experimentellen Modell.

<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.

Dieses Protokoll wird auf die Erzeugung von PDX-Modelle von isolierten CD45 negativen Prostatakrebs CTCs führen. Basierend auf der in unserem Protokoll verwendeten negativen Selektionsverfahrens ist es notwendig, um tote Zellen auszuschließen unter Verwendung von DAPI-Färbung. Der Prozentsatz von CD45-negativen Zellen durchflusszytometrisch erfasst ist variabel und hängt von der Tumorbelastung des Patienten (1A). Immunfluoreszenzfärbung von unsortierten Zellen mit CD45 und DAPI (Zellkerne identifiz...

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.

Generation von Prostatakrebs-Patienten gewonnen Xenograft-Modelle von zirkulierenden Tumorzellen

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

generation-prostate-cancer-patient-derived-xenograft-models-from

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14.0K Aufrufe.  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.

Serie: 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.

Zeitraffer-Imaging of Primary Präkanzeröse Brustepithelzellen aus gentechnisch veränderten Mausmodellen der Breast Cancer Abgeleitet

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

Forschung

Medizin

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).

Patienten abgeleitet Zellkultur und Isolierung von CD133<sup&gt; +</sup&gt; Mutmaßliche Cancer Stem Cells von 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.

Halbautomatische Anpassung der Umlauftumorzellen (CTC)-Assays für die klinische und vorklinische Forschungsanwendungen

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.

Isolierung von Krebs Stammzellen aus menschlichen Prostatakrebs-Proben

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.

In-vitro-Methode zur E-Selektin-vermittelte Interaktionen zwischen Prostata zirkulierenden Tumorzellen, abgeleitet von Patienten und humanen Endothelzellen beachten

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.

Erzeugung von induzierten pluripotenten Stammzellen von Muskeldystrophie-Patienten: Effiziente Integration freie Neuprogrammierung der Urin-abgeleiteten Zellen

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.

Isolierung und Immortalisierung von Patienten stammende Zelllinien von Muskelbiopsie 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 ...

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 vitro Anreicherung des Eierstockkrebs Tumor-initiierenden Zellen

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.

Einrichtung eines Menschen Multiple Myeloma Xenograftmodell im Huhn, das Tumorwachstum, Invasion und Angiogenese Studieren

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.